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1 June 2005 SYSTEMATIC REVIEW OF THE FROG FAMILY HYLIDAE, WITH SPECIAL REFERENCE TO HYLINAE: PHYLOGENETIC ANALYSIS AND TAXONOMIC REVISION
JULIÁN FAIVOVICH, CÉLIO F.B. HADDAD, PAULO C.A. GARCIA, DARREL R. FROST, JONATHAN A. CAMPBELL, WARD C. WHEELER
Author Affiliations +
Abstract

Hylidae is a large family of American, Australopapuan, and temperate Eurasian treefrogs of approximately 870 known species, divided among four subfamilies. Although some groups of Hylidae have been addressed phylogenetically, a comprehensive phylogenetic analysis has never been presented.

The first goal of this paper is to review the current state of hylid systematics. We focus on the very large subfamily Hylinae (590 species), evaluate the monophyly of named taxa, and examine the evidential basis of the existing taxonomy. The second objective is to perform a phylogenetic analysis using mostly DNA sequence data in order to (1) test the monophyly of the Hylidae; (2) determine its constituent taxa, with special attention to the genera and species groups which form the subfamily Hylinae, and c) propose a new, monophyletic taxonomy consistent with the hypothesized relationships.

We present a phylogenetic analysis of hylid frogs based on 276 terminals, including 228 hylids and 48 outgroup taxa. Included are exemplars of all but 1 of the 41 genera of Hylidae (of all four nominal subfamilies) and 39 of the 41 currently recognized species groups of the species-rich genus Hyla. The included taxa allowed us to test the monophyly of 24 of the 35 nonmonotypic genera and 25 species groups of Hyla. The phylogenetic analysis includes approximately 5100 base pairs from four mitochondrial (12S, tRNA valine, 16S, and cytochrome b) and five nuclear genes (rhodopsin, tyrosinase, RAG-1, seventh in absentia, and 28S), and a small data set from foot musculature.

Concurring with previous studies, the present analysis indicates that Hemiphractinae are not related to the other three hylid subfamilies. It is therefore removed from the family and tentatively considered a subfamily of the paraphyletic Leptodactylidae. Hylidae is now restricted to Hylinae, Pelodryadinae, and Phyllomedusinae. Our results support a sister-group relationship between Pelodryadinae and Phyllomedusinae, which together form the sister taxon of Hylinae. Agalychnis, Phyllomedusa, Litoria, Hyla, Osteocephalus, Phrynohyas, Ptychohyla, Scinax, Smilisca, and Trachycephalus are not monophyletic. Within Hyla, the H. albomarginata, H. albopunctata, H. arborea, H. boans, H. cinerea, H. eximia, H. geographica, H. granosa, H. microcephala, H. miotympanum, H. tuberculosa, and H. versicolor groups are also demonstrably nonmonophyletic. Hylinae is composed of four major clades. The first of these includes the Andean stream-breeding Hyla, Aplastodiscus, all Gladiator Frogs, and a Tepuian clade. The second clade is composed of the 30-chromosome Hyla, Lysapsus, Pseudis, Scarthyla, Scinax (including the H. uruguaya group), Sphaenorhynchus, and

INTRODUCTION

Hylidae is a large family of American, Australopapuan, and temperate Eurasian treefrogs of approximately 870 known species, composed of four subfamilies (Duellman, 2001; Darst and Cannatella, 2004; Frost, 2004). Although some groups of Hylidae have been addressed phylogenetically (e.g., Campbell and Smith, 1992; Duellman and Campbell, 1992; Mendelson et al., 2000; Faivovich, 2002; Haas, 2003; Moriarty and Cannatella, 2004; Faivovich et al., 2004), a comprehensive phylogenetic analysis has never been presented.

The first goal of this paper is to review the state of hylid systematics. We focus on the very large subfamily Hylinae, evaluate the monophyly of named taxa, and examine the evidential basis of the existing taxonomy. The second objective is to perform a phylogenetic analysis using four mitochondrial and five nuclear genes in order to (1) test the monophyly of the family Hylidae; (2) determine its constituent taxa, with special attention to the genera and species groups which form the subfamily Hylinae; and (3) propose a new, monophyletic taxonomy consistent with the hypothesized relationships.

MATERIALS AND METHODS

In most revisionary studies involving major taxonomic rearrangements and phylogenetic analyses it is normal to have a section on the history of taxonomic changes. The scale of this particular study makes that goal impractical. A discussion of the state of the taxonomy of hylid frogs is dealt with simultaneously with discussions of the taxa chosen for the purpose of phylogenetic analysis.

Taxon Sampling

Any phylogenetic analysis has an important component of experimental design associated with the selection of the terminal taxa. In an ideal phylogenetic study, all terminal descendants of a given hypothetical ancestor should to be included in order to avoid “problems” due to taxon sampling. This ideal condition is unattainable, and all notions of relationships among organisms are affected to an unknown degree by incomplete taxon sampling. Because there is no way of ever knowing all the hylid species that have become extinct, we concentrate on the diversity that we do know. Furthermore, due to the unavailability of samples we cannot include sequences of all of the nearly 860 currently described species of Hylidae. What, therefore, is the best taxon sampling for this study? Because our primary goal is to test the monophyly of all available genera and species groups of Hylidae, the most appropriate terminals to include are those that provide the strongest test of previously hypothesized relations. By considering morphological divergence as a rough guide to DNA sequence diversity, maximally diverse taxa within a given group are likely to pose a stronger test of its monophyly than do morphologically similar taxa (Prendini, 2001). Groups for which no apparent synapomorphies are known are a priori more likely to be nonmonophyletic, and therefore good representations of the morphological diversity of these groups are especially appropriate. Our success varied in securing multiple representatives of these groups.

The following discussion deals in part with the state of knowledge of frog phylogenetics. Included within the discussion is a list of terminals used in this analysis along with a justification and explanation for our choices. A summary of the species included is presented in table 1. To conserve space, species authorships are not mentioned in the text but are given in the section “Taxonomic Conclusions: A New Taxonomy of Hylinae and Phyllomedusinae” and in appendix 1. For museum abbreviations used throughout this paper see appendix 2.

Outgroup Selection

Recent studies (Haas, 2003; Darst and Cannatella, 2004) have suggested that the Hylidae as traditionally understood is not monophyletic, with the Hemiphractinae displaced phylogenetically from the Hylinae, Phyllomedusinae, and Pelodryadinae. The aforementioned studies did not provide extensive outgroup comparisons. In order to avail ourselves of a strong test of hylid monophyly, we included 48 nonhylid outgroup taxa representing 14 neobatrachian families.

Basal Neobatrachians

Heleophrynidae, Sooglossidae, Limnodynastinae and Myobatrachinae1 have been related to each other by several authors (Lynch, 1973; Duellman and Trueb, 1986; Ford and Cannatella, 1993; Hay et al., 1995; Ruvinsky and Maxson, 1996; Biju and Bossuyt, 2003; Darst and Cannatella, 2004). While in the past they were considered part of Hyloidea, they were recently excluded by Darst and Cannatella (2004). The evidence indicates that they are basal neobatrachians distantly related to the apparently monophyletic Hyloidea; however, their exact positions and interrelationships are still unclear (Darst and Cannatella, 2004; Haas, 2003). We include one heleophrynid (Heleophryne purcelli), one myobatrachine (Pseudophryne bibroni), and two limnodynastines (Limnodynastes salmini and Neobatrachus sudelli) in our study. Furthermore, because some members of the Australopapuan hylids have been posited to be related to the Myobatrachidae (Lynch, 1971; Savage, 1973), their inclusion provides a strong test of hylid monophyly.

Ranoidea

Recent papers dealing with ranoid groups (Emerson et al., 2000; Vences et al., 2003a) or at least ranoid exemplars (Biju and Bossuyt, 2003; Darst and Cannatella, 2004) have suggested the existence of three major clades, although this remains to be elucidated. The three major clades are composed of (1) Arthroleptidae, Astylosternidae, Hemisotidae, and Hyperoliidae; (2) Microhylidae; and (3) the remaining ranoids (including Petropedetidae, Mantellidae, Rhacophoridae, and the paraphyletic Ranidae). We include exemplars of Astylosternidae, Hyperoliidae, Hemisotidae, Microhylidae, Ranidae, Mantellidae, and Rhacophoridae.

Hyloidea

The Hylidae has long been considered to be embedded within a poorly defined major group of neobatrachians (Nicholls, 1916; Noble, 1922; Lynch, 1971, 1973; Ford and Cannatella, 1993) for which no morphological evidence of monophyly exists, although molecular evidence (Hay et al., 1995; Ruvinsky and Maxson, 1996; Darst and Cannatella, 2004) does support its monophyly. As redefined by Darst and Cannatella (2004) Hyloidea includes the nonmonophyletic Leptodactylidae (Ford and Cannatella, 1993; Haas, 2003), Dendrobatidae, Hylidae, Bufonidae, Brachycephalidae, Centrolenidae, Rhinodermatidae, and the monotypic Allophrynidae. In order to have a strong test of the monophyly of hylids, we include representatives of most of the nonhylid hyloid families.

The monophyly of Dendrobatidae is not controversial (see Grant et al., 1997), although recent phylogenetic analyses using ribosomal mitochondrial sequences (Vences et al., 2000; Santos et al., 2003; Vences et al., 2003b) show that there is a serious need to redefine most of the currently recognized genera. According to the results of Vences et al. (2003b), there are two major clades of dendrobatids: (1) one composed of Dendrobates, Phyllobates, Cryptophyllobates, Epipedobates (paraphyletic), Minyobates, and several groups of the rampantly polyphyletic Colostethus; and (2) another clade composed of Mannophryne, Nephelobates, Allobates, and two separate clades of Colostethus (none of the aforementioned analyses included the apparently primitive genus Aromobates; see Myers et al. 1991). We include as exemplars of the first major clade Dendrobates auratus and Phyllobates bicolor, and as exemplar of the second clade, Colostethus talamancae.

Bufonidae is a monophyletic group (for a list of morphological synapomorphies, see Ford and Cannatella, 1993; Haas, 2003) for which no study addressing comprehensively its internal relationships has been published. Partial studies (Graybeal, 1997; Darst and Cannatella, 2004; Haas, 2003) support the idea that Melanophryniscus is one of its most basal clades in the family. As a rough sample of bufonid diversity we include representatives of Melanophryniscus, Dendropryniscus, Atelopus, Didynamipus, Schismaderma, Bufo, Pedostibes, and Osornophryne.

The notion that the presumably monophyletic Centrolenidae (Ruiz-Carranza and Lynch, 1991) is closely related to hylids (Lynch, 1973; Duellman and Trueb, 1986; Ford and Cannatella, 1993) was recently challenged by the phylogenetic analyses of Haas (2003), who used larval morphology, and Darst and Cannatella (2004), who used mitochondrial ribosomal genes. Austin et al. (2002) recently provided molecular evidence supporting a relationship between Centrolenidae and the monotypic Allophrynidae. The internal relationships of Centrolenidae remain virtually unstudied. As a rough representation of centrolenid diversity, in this study we include Centrolene prosoblepon, Cochranella bejaranoi, and Hyalinobatrachium eurygnathum. We also include Allophryne ruthveni.

Leptodactylid nonmonophyly has been accepted for some time (Lynch, 1971) and was confirmed in an explicit cladistic framework by analyses using morphology (Haas, 2003) and DNA sequences (Ruvinsky and Maxson, 1996; Darst and Cannatella, 2004; Vences et al., 2003b). In the analysis by Darst and Cannatella (2004), Leptodactylidae is rampantly paraphyletic because all the other hyloid exemplars are nested within it.

From the five currently recognized subfamilies of leptodactylids (Laurent, 1986) there is more or less convincing evidence of monophyly for two of them: Eleutherodactylinae (direct development, eggs relatively large, few in number; Lynch, 19712) and Leptodactylinae (presence of a bony element in the sternum; Lynch, 1971). The analysis of Darst and Cannatella (2004) corroborated the monophyly of these two groups, albeit with limited taxon sampling. In the analysis by Haas (2003), the exemplars of Leptodactylinae were not monophyletic. No demonstrable synapomorphies are known for Ceratophryinae, Cycloramphinae or Telmatobiinae. Considering this situation, we include several leptodactylid exemplars (see table 1); our poorest sampling is within Cycloramphinae, where we only have representation for one genus, Crossodactylus.

The single representative of Brachycephalidae included by Darst and Cannatella (2004) in their analysis was nested within the exemplars of the leptodactylid subfamily Eleutherodactylinae, as suggested earlier by Izecksohn (1988). We include Brachycephalus ephippium in our analysis.

Of the hyloid families, only Rhinodermatidae is not represented in our analysis. This group was suggested to be nested in the subfamily Telmatobiinae by Barrio and Rinaldi de Chieri (1971) based on a similar karyotype and by Manzano and Lavilla (1995a) based on the presence of the m. pelvocutaneus in Rhinoderma and Eupsophus. In the DNA sequences analyses by Ruvinsky and Maxson (1996) and Biju and Bossuyt (2003) Rhinoderma appears in different positions within Hyloidea.

The Ingroup: Hylidae

Inasmuch as the hylids are the primary focus of this study, our sampling is most dense for this taxon and requires substantially more detailed discussion than does our outgroup selection.

Duellman (1970) arranged the family in four subfamilies: Amphignathodontinae, Hemiphractinae, Hylinae (including both the Australian and American groups), and Phyllomedusinae. Trueb (1974) subsequently synonymized the Amphignathodontinae and Hemiphractinae. On the basis of evidence presented by Tyler (1971), Duellman (1977) placed the Australian hylids in their own subfamily, Pelodryadinae, although Savage (1973) had previously regarded it as a different family and suggested that it was derived from Myobatrachidae.

Duellman (2001), based mostly on da Silva's results (1998), presented a phylogenetic analysis where hylids, including the subfamily Pseudinae, were considered to be monophyletic. Synapomorphies suggested by Duellman (2001) as being common to his three most parsimonious trees are the possession of claw-shaped terminal phalanges and the three articular surfaces on metacarpal III. The possibility of hylid polyphyly has not been seriously considered by most frog systematists, even after Ruvinsky and Maxson's (1996) results (which showed their single Hemiphractinae exemplar not closely related with the other hylid exemplars), until the idea was suggested on morphological grounds by Haas (2003), followed by Darst and Cannatella (2004) on the basis of molecular evidence. These authors found no evidence of a relationship between Hemiphractinae and the other hylid subfamilies, which were thought to form a monophyletic group. Beyond this result, Haas (2003) presented evidence from larval morphology that suggested that Pelodryadinae is paraphyletic with respect to Hylinae (Hylinae not being demonstrably monophyletic), with Pseudinae and Phyllomedusinae possibly being imbedded within it.3

Hemiphractinae

Mendelson et al. (2000) and Duellman (2001) presented brief taxonomic histories of this taxon. The monophyly of Hemiphractinae is supported by the presence of bell-shaped gills in larvae and by female transport of eggs in a specialized depression or sac in the dorsum (Noble, 1927). Burton (2004) added the broad m. abductor brevis plantae hallucis. In Duellman's (2001) cladogram, Hemiphractinae is considered to be the sister group of Phyllomedusinae, with the evidence of this relationship being the proximal head of metacarpal II not between prepollex and distal prepollex, and the larval spiracle sinistral and ventrolateral.

Haas's (2003) exemplar4 of the Hemiphractinae was Gastrotheca riobambae. His results suggested that Hemiphractinae are unrelated to other hylids, although the position of Hemiphractinae within Neobatrachia is still unresolved. A similar result regarding Hemiphractinae as being unrelated to hylids was advanced by Ruvinsky and Maxson (1996) and corroborated by Darst and Cannatella (2004). These authors also did not recover the exemplars of Hemiphractinae that they used (Gastrotheca pseustes and Cryptobatrachus sp.) as forming a monophyletic group.5

Hemiphractinae includes five genera: Cryptobatrachus, Flectonotus, Gastrotheca, Hemiphractus, and Stefania. Mendelson et al. (2000) studied the relationships among these genera, performing a phylogenetic analysis using morphological and life-history characters, arriving at the topology (Cryptobatrachus Flectonotus (Stefania (“Gastrotheca” + Hemiphractus))). This analysis included five outgroups, all of which were representatives of the other hylid subfamilies. The results suggested that Cryptobatrachus and Flectonotus are each monophyletic, and that Gastrotheca is paraphyletic with Hemiphractus nested within it. As Hass (2003) noted, however, Mendelson et al.'s (2000) outgroup structure could not test the proposition of hylid diphyly.

Cryptobatrachus: In the analysis performed by Mendelson et al. (2000), the monophyly of the two representatives of Cryptobatrachus is supported by several osteological characters, among them the presence of an anteromedial process in the neopalatine.6 In their analysis, the relationship between Cryptobatrachus and the other Hemiphractinae is unresolved. This genus comprises three described species; in this study we include sequences of an unidentified species available from GenBank.

Gastrotheca: Mendelson et al. (2000) suggested that Hemiphractus is nested within Gastrotheca, a result that contrasts with the opinions of previous workers (Noble, 1927; Trueb, 1974; Duellman and Hoogmoed, 1984) who considered Hemiphractus basal among marsupial frogs because they lack a brooding pouch. However, Mendelson et al. (2000) continued to recognize Hemiphractus (the older of both names) and Gastrotheca pending a more complete phylogenetic study. Synapomorphies of the clade composed of these two genera are: cultriform process becoming distinctly narrow anteriorly; anterior process of vomer articulating only with maxilla; pre- and postchoanal process bifurcating at the level of the dentigerous process; nature of occipital artery pathway (a groove); brooding pouch with posterior opening; and bell-shaped gills fused distally.

Most of the 49 currently recognized species of Gastrotheca are placed in four species groups, the G. marsupiata, G. nicefori, G. plumbea, and G. ovifera groups (Duellman et al., 1988a). These groups are generally defined on the basis of overall similarity. The only character-based test of their monophyly, the analysis of Mendelson et al. (2000), included 17 exemplars and suggested that none of the three nonmonotypic groups is monophyletic.

Dubois (1987) placed the species within three subgenera: Gastrotheca, Duellmania (part of the G. plumbea group), and Opisthodelphys (G. ovifera as well as parts of the G. marsupiata and G. plumbea groups). Duellmania and Opisthodelphys were shown to be paraphyletic by Mendelson et al. (2000), although they did not test the monophyly of the subgenus Gastrotheca. In this study we include two species of the Gastrotheca marsupiata group (G. cf. marsupiata and G. pseustes) and two of the G. ovifera group (G. cornuta and G. fissipes).

Hemiphractus: Relationships of this genus were recently reviewed by Sheil et al. (2001), who provided a number of unambiguous synapomorphies to support its monophyly (such as the cultriform process of the parasphenoid that becomes distinctly narrow anteriorly, the presence of a zygomatic ridge, and the presence of a supraorbital ridge). Hemiphractus has six described species; we include Hemiphractus helioi in our study.

Stefania: Duellman and Hoogmoed (1984), Señaris et al. (“1996” [1997]), and MacCulloch and Lathrop (2002) reviewed this genus. Señaris et al. (“1996” [1997]) suggested that the zygomatic ramus of the squamosal being close to or in contact with the maxilla was a diagnostic character state of Stefania, “at least for the Venezuelan species” (translated from the Spanish). Mendelson et al. (2000) did not test the monophyly of Stefania since they included only one exemplar (S. evansi). It is unclear if any of the autapomorphies of S. evansi in that study are actually synapomorphies of Stefania.

Stefania was divided by Rivero (1970) into two species groups based on head shape (“as broad as long or longer than broad” in the S. evansi group; “much broader than long” in the S. ginesi group). The only test of the monophyly of these two groups is the phylogenetic analysis of the seven species then known, performed by Duellman and Hoogmoed (1984). In that analysis, the S. ginesi group was monophyletic and nested within the paraphyletic S. evansi group. Although Señaris et al. (“1996” [1997]) suggested the origin of the S. ginesi group from the S. evansi group, they continued to recognize of both groups.

Since the revision of Duellman and Hoogmoed (1984), another 11 species assigned to both species groups of Stefania have been named (see Barrio Amorós and Fuentes, 2003; MacCullogh and Lathrop, 2002). In our analysis we include one exemplar of the Stefania evansi group (S. evansi) and one of the S. ginesi group (S. schuberti).

Flectonotus: Duellman and Gray (1983) reviewed these frogs as two genera, Flectonotus and Fritziana, even though their phylogenetic analysis indicated that Flectonotus was paraphyletic with respect to Fritziana. Subsequently, Weygoldt and Carvalho e Silva (1991) placed Fritziana in the synonymy of Flectonotus to render a monophyletic taxonomy. Flectonotus is composed of five described species.

In the analysis by Mendelson et al. (2000), the position of Flectonotus remained unresolved with respect to Cryptobatrachus and the clade composed of Stefania plus Gastrotheca. Synapomorphies of Flectonotus in that analysis are: quadratojugal that does not articulate with maxilla; brooding pouch formed by dorsolateral folds of skin; overlap between m. intermandibularis and m. submentalis; and absence of supplementary elements of m. intermandibularis. Because of very limited availability of species of Flectonotus, in this study we include only Flectonotus sp., an unidentified species from southeastern Brazil, whose female has a brooding pouch with a middorsal slit.

Pelodryadinae

The monophyly of this Australopapuan group is supported by a single possible synapomorphy: presence of supplementary apical elements of m. intermandibularis (Tyler, 1971). Although relationships between Australian and New World hylids were recognized very early (most species of Litoria were named as Hyla), hypotheses regarding the relationships of Pelodryadinae with other groups have been rarely advanced. Trewavas (1933), Duellman (1970), and Bagnara and Ferris (1975) suggested a relationship between Pelodryadinae with Phyllomedusinae. Trewavas (1933) observed similarities in laryngeal structures in the limited data set at her disposal (only three pelodryadines and two phyllomedusines). Duellman (1970) referred to “similarities in vertebral characters” without further details and to identical number of chromosomes. Bagnara and Ferris (1975) noticed the presence in some species of Litoria of large melanosomes containing the red pigment rhodomelanochrome (later identified as pterorhodin; Misuraca et al., 1977), a character state previously known only for some species of Phyllomedusinae. Specifically, Bagnara and Ferris (1975) suggested that some species of Litoria could be related to the Phyllomedusinae, an implicit suggestion of Pelodryadinae paraphyly. This idea was discussed by Tyler and Davies (1978a), who rejected the possibility of pelodryadine paraphyly but did not address a possible sister-taxon relationship of Pelodryadinae and Phyllomedusinae. This alternative was suggested again, based on chromosome morphology, by Kuramoto and Allison (1991). In the phylogenetic analyses presented by Duellman (2001), Pelodryadinae was placed as the sister of a clade composed of the remaining subfamilies, which are united in having a distally bifid tendo superficialis. In this same cladogram, the only synapomorphy of Pelodryadinae is the anterior differentiation of the m. intermandibularis, although the monophyly of Pelodryadinae was assumed and not tested in that analysis.

The phylogenetic analysis performed by Haas (2003) presents the most extensive test of Pelodryadinae monophyly so far published; his Pelodryadinae exemplars form a paraphyletic series with respect to his Neotropical hylid exemplars. More recently, Darst and Cannatella (2004) and Hoegg et al. (2004) presented evidence from the ribosomal mitochondrial and nuclear genes supporting the monophyly of their Pelodryadinae sample and the monophyly of Pelodryadinae + Phyllomedusinae.

Litoria: The diagnosis and contents of Litoria were reviewed by Tyler and Davies (1978b). It is unclear whether any of the character states included in their extensive diagnosis are synapomorphic. However, considering subsequent comments by several authors (King et al., 1979; Tyler, 1979; Tyler and Davies, 1979; Maxson et al., 1985; Haas and Richards, 1998), the available evidence suggests that Litoria is paraphyletic with respect to the other genera of Pelodryadinae, Nyctimystes and Cyclorana, with the latter being the sister taxon of the L. aurea group.

The 132 currently recognized species (updated from Frost, 2004) placed in 37 species groups (Tyler and Davies, 1978b) make an exhaustive sampling of the group a goal beyond the present analysis.7 Relationships among some species groups of Litoria were addressed by means of albumin immunological distances as determined through microcomplement fixation (Maxson et al., 1982; Hutchinson and Maxson, 1986, 1987). From a character-based (as opposed to a distance-based) perspective, relationships among the species groups of Litoria remain unknown, and the monophyly of most of those groups with more than single species remains untested.

Tyler and Davies (1978b) tentatively divided the 37 species groups into three “Categories”, A (8 species groups), B (14 species groups), and C (7 species groups). Tyler (1982) added a fourth category (D), where he included the Litoria nannotis group. These groupings were criticized by King (1980) on karyotypic grounds. Besides, the monophyly of these four categories remain largely untested, with the only possible exception being the work by Cunningham (2002) on the L. nannotis group; however, his lack of sufficient outgroup sampling precluded a rigorous test.

In our analyses we include representatives of two species groups of category A, Litoria aurea (the L. aurea group) and L. freycineti (the L. freycineti group); three representatives of category B, L. caerulea (the L. caerulea group), L. infrafrenata (the L. infrafrenata group), and L. meiriana (the L. meiriana group); and one representative of category C, L. arfakiana (the L. arfakiana group).

Nyctimystes: This genus was rediagnosed by Tyler and Davies (1979). Among the list of characters provided by them, the synapomorphies of Nyctimystes seem to be the vertical pupil and the presence of palpebral venation. Tyler and Davies (1979) suggested that Nyctimystes was most closely related to some species groups of Litoria from New Guinea, implying that Nyctimystes is nested within Litoria. Specifically, they referred to the L. angiana, L. arfakiana, L. becki, L. dorsivena, L. eucnemis, and L. infrafrenata groups as the most likely to be related to Nyctimystes, because they share with Nyctimystes similarities in cranial structure (the L. infrafrenata and L. eucnemis groups) or the presence of large unpigmented ova and lotic tadpoles bearing large, ventral, suctorial mouths (the other groups). Nyctimystes currently comprises 24 described species, 5 of which (N. disruptus, N. oktediensis, N. trachydermis, N. tyleri, and N. papua) were included in the N. papua species group by Zweifel (1983) and Richards and Johnston (1993). We could not locate tissues of any members of the N. papua group, so we cannot test its monophyly. Nevertheless, we include the available species N. kubori, N. narinosus, and N. pulcher.

Cyclorana: This genus was thought to be related to the Australian leptodactylids (now Myobatrachidae) by Parker (1940), and was considered as such by Lynch (1971). Tyler (1972) first proposed its relationship to Australian hylids on the basis of the presence of a differentiated apical element of the m. intermandibularis. Subsequently, Tyler (1978) transferred Cyclorana to Hylidae. Tyler (1979), King et al. (1979), and Tyler et al. (1981) considered it to be related to the Litoria aurea group, a result that was coincident with the analyses of albumin immunological distances generated by microcomplement fixation (Hutchinson and Maxson, 1987). Burton (1996) suggested that having the m. palmaris longus divided into two segments (as opposed to three) is a synapomorphy supporting the monophyly of Cyclorana, L. dahlii, and L. alboguttata (two species of the L. aurea group). Based on sperm morphology, Meyer et al. (1997) transferred L. alboguttata to Cyclorana. The only morphological synapomorphy suggested for Cyclorana is the anterior ossification of the sphenethmoid to incorporate a portion of the tectum nasi (Tyler and Davies, 1993).

The 13 species of Cyclorana have been separated into different groups based on karyotypes, sperm morphology, and immunological distances (King et al., 1979; Maxson et al., 1982; Maxson et al., 1985; Meyer et al., 1997). These are the C. brevipes, C. australis, and C. platicephala “lineages”. In the present study, we include only C. australis.

Phyllomedusinae

Cruz (1990) and Duellman (2001) provided taxonomic histories of this group, mostly at the generic level. The monophyly of Phyllomedusinae has not been controversial; several character states have been advanced to support it. Some of the muscular character states include the supplementary posterolateral elements of the m. intermandibularis (Tyler, 1971); tendo superficialis pro digiti II (pes) arising from a deep, triangular muscle, which originates on the distal tarsal 2–3; tendo superficialis pro digiti III arising entirely from the aponeurosis plantaris; and m. extensor brevis superficialis digiti IV with a single slip (Burton, 2004). There are also several larval character states that support the monophyly of this group; for example, the arcus subocularis of larval chondrocranium with distinct lateral processes (Fabrezi and Lavilla; 1992, Haas, 2003); ultralow suspensorium (Haas, 2003); secondary fenestrae parietales (Haas, 2003); and absence of a passage between ceratohyal and ceratobranchial I (Haas, 2003).

The subfamily is comprised of six nominal genera: Agalychnis (8 species), Hylomantis (2 species), Pachymedusa (1 species), Phasmahyla (4 species), Phrynomedusa (5 species), and Phyllomedusa (32 species). Cruz (1990) discussed the taxonomic distribution of several character states shared by subsets of these genera. A cladistic analysis testing the monophyly of each of these and their interrelationships remains to be completed.

Agalychnis: Duellman (2001) presented a phylogenetic analysis of Agalychnis and Pachymedusa, using a vector of character states present in the Phyllomedusa buckleyi group as an outgroup (data taken from Cannatella, 1980). His analysis suggested no synapomorphies for Agalychnis. In our analysis we include all species available to us: A. calcarifer, A. callidryas, A. litodryas, and A. saltator (species not included are A. annae, A. craspedopus, A. moreletii, and A. spurrelli).

Hylomantis: This genus was resurrected by Cruz (1990) for the species formerly placed in the Phyllomedusa aspera group (Cruz, “1988” [1989]). From the extensive diagnosis presented by Cruz (1990), the only apparent synapomorphy of Hylomantis seems to be the lanceolate discs of fingers and toes. Cruz (1990: 725), however, considered likely that Hylomantis was paraphyletic with respect to Phasmahyla. Hylomantis has two described species, H. aspera and H. granulosa. Only H. granulosa was available for this study.

Pachymedusa: This monotypic genus was recognized by Duellman (1968a) to reflect his view that a remnant of the ancestral stock gave rise to the other Phyllomedusinae. However, Duellman (2001) found no evidence supporting the monophyly of Agalychnis independent of Pachymedusa. The single species Pachymedusa dacnicolor is included in our analysis.

Phasmahyla: This genus was erected by Cruz (1990) for the species formerly contained in the Phyllomedusa guttata group (Bokermann and Sazima, 1978; Cruz, 1982). Cruz (1990) provided an extensive definition of the genus based on adult and larval morphology. Probable synapomorphies of Phasmahyla are the lack of a vocal sac in adult males and larval modifications presumably associated with surface film feeding, such as the anterodorsal position of the oral disc, reduction in number and size of labial tooth rows, distribution and shape of submarginal papillae, and the upper jaw sheath with a medial projection (see Cruz, 1982, 1990). The genus is composed of four species, P. cochranae, P. exilis, P. guttata, and P. jandaia. We include Phasmahyla cochranae and P. guttata in our study.

Phrynomedusa: This genus was resurrected by Cruz (1990) for the species formerly placed in the Phyllomedusa fimbriata group (Izecksohn and Cruz, 1976; Cruz, 1982). From the extensive definition provided by Cruz (1990), possible synapomorphies of Phrynomedusa appear to be the presence of a bicolored iris and the complete marginal papillae in the oral disc of the larva. Phrynomedusa contains five described species; unfortunately, we could not secure any representatives of this taxon for the present study.

Phyllomedusa: No synapomorphies are known to support the monophyly of the 32 species of Phyllomedusa. This genus includes simply those species that are not included in the other five genera of Phyllomedusinae. There are currently five species groups recognized within Phyllomedusa: the P. buckleyi group (Cannatella, 1980), P. burmeisteri group (Lutz, 1950; Pombal and Haddad, 1992), P. hypochondrialis group (Bokermann, 1965a), P. perinesos group (Cannatella, 1982), and P. tarsius group (De la Riva, 1999). The monophyly of these groups had not been tested, and relationships among them remain unstudied. Furthermore, several species (e.g., P. bicolor, P. palliata, P. tomopterna, and P. vaillanti) have not been assigned to any species group. Some authors (Funkhouser, 1957; Duellman, 1968a, 1969; Cannatella, 1980; Jungfer and Weygoldt, 1994) have suggested that the Phyllomedusa buckleyi group deserves generic recognition.

Duellman et al. (1988b) posited the existence of a clade composed of most species of Phyllomedusa (excluding the P. buckleyi group and the P. guttata group, now Phasmahyla, but see below), implicitly including the species now placed in Hylomantis. Apparent synapomorphies of this clade are the well-developed parotoid glands; the presence of the slip of the m. depressor mandibulae that originates from the dorsal fascia at the level of the m. dorsalis scapulae; first toe longer than second; and the eggs wrapped in leaves. Duellman et al. (1988b) explicitly excluded the species then included in the P. guttata group (now Phasmahyla) from this apparent clade. However, Lutz (1954), Bokermann and Sazima (1978), Weygoldt (1984), and Haddad (personal obs.) reported P. guttata, P. jandaia, P. exilis, and P. cochranae, respectively, to oviposit in folded leaves, and Cruz (1990) reported in Hylomantis the presence of the slip of the m. depressor mandibulae that originates from the dorsal fascia at the level of the m. dorsalis scapulae. In this study we include representatives of the Phyllomedusa buckleyi group (P. lemur), P. burmeisteri group (P. tetraploidea), P. hypochondrialis group (P. hypochondrialis), and P. tarsius group (P. tarsius). We include also P. bicolor, P. palliata, P. tomopterna, and P. vaillanti, four species unassigned to groups.

Hylinae

This taxon is the primary focus of this study. Hyline monophyly is supported by two synapomorphies: the tendo superficialis digiti V with an additional tendon that arises ventrally from m. palmaris longus (da Silva, 1998, as cited by Duellman, 2001), and karyotype with 24 (or more) chromosomes (Duellman, 2001). The published molecular evidence is ambiguous regarding the issue.8

No comprehensive study of hyline systematics has been published, although the results of one unpublished dissertation (da Silva, 1998) have been widely circulated (e.g., Duellman, 2001). Although the problems of hylid systematics have been recognized for some time, almost all work has been done at the level of satellite genera (e.g., Duellmanohyla, Plectrohyla, Ptychohyla, Scinax) or species groups of Hyla.

For the purpose of our analysis we included representatives of all 27 genera of Hylinae. Within the genus Hyla, we included exemplars of 39 of the 41 species groups that had been recognized (we lack exemplars for the H. claresignata and H. garagoensis groups). We are not recognizing monotypic species group because (1) they do not represent testable hypothesis, and (2) they are not a rank in the Linnean taxonomy and therefore are not required for consistency and stability purposes.

Hyla is the most species-rich genus of hylid frogs. It is currently placed in 41 species groups, plus other species that had not been associated with any group. In turn, major clades composed of several of these species groups have been suggested. Because no study has ever suggested Hyla to be monophyletic and several have suggested that it is paraphyletic with respect to other hyline genera (Duellman and Campbell, 1992; Cocroft, 1994; Faivovich, 2002; Haas, 2003; Darst and Cannatella, 2004; Faivovich et al., 2004), we refer further discussion to the headings for the various genera and species groups. Comments about apparent major clades and proposed relationships among species groups are mostly reserved for the discussion section of this paper.

Gladiator Frogs

Kluge (1979: 1) referred to the frogs then placed in the Hyla boans group as “gladiator frogs” “in view of their extremely pugnacious behavior and the well developed pre-pollical spines that they use when fighting.” Following Duellman (1970, 1977), Kluge (1979) included in the group H. boans, H. circumdata, H. crepitans, H. faber, H. pardalis, and H. pugnax. Nevertheless, a prepollical spine has been reported for several species groups. Furthermore, territorial fighting has been reported or suspected to occur in several of these species.9 Because of this, and due to the lack of a better term, we prefer to use the term “Gladiator Frogs” to refer collectively to all the mostly South American species having a prepollical spine, as was done by Faivovich et al. (2004), instead of restricting its use to the H. boans group. The species groups that are currently referred to the Gladiator Frog clade are the H. albomarginata, H. albopunctata, H. boans, H. circumdata, H. claresignata, H. geographica, H. granosa, H. martinsi, H. pseudopseudis, H. pulchella, and H. punctata10 groups (Bokermann, 1972, Hoogmoed, 1979, Duellman et al., 1997, Eterovick and Brandão, 2001, Duellman, 2001, Faivovich et al., 2004).

Hyla albomarginata Group: The H. albomarginata group was first recognized formally by Cochran (1955) for a group of species (H. albomarginata, H. albosignata, H. albofrenata, H. musica) that Lutz (“1948” [1949]) referred to as the green species of Hyla of southeastern Brazil. Cochran (1955) included H. prasina (latter included in the H. pulchella group by Lutz, 1973) in this group. Duellman (1970) presented a definition of the group and included, besides the species considered by Cochran (1955), H. rufitela, H. pellucens, H. albopunctulata (now considered a member of the H. bogotensis group; see below), and H. albolineata (now considered a species of Gastrotheca; see Sachsse et al., 1999).

Cruz and Peixoto (“1985” [1987]) divided the Hyla albomarginata group into three “complexes”: the H. albomarginata complex, containing H. albomarginata and H. rufitela; the H. albofrenata complex, containing H. albofrenata, H. arildae, H. ehrhardti (as H. arianae; see Faivovich et al., 2002), H. musica, and H. weygoldti; and the H. albosignata complex, containing H. albosignata, H. callipygia, H. cavicola, H. fluminea, and H. leucopygia. Hyla pellucens should also be included in the albomarginata complex, because this species was included by Duellman (1970) but was overlooked by Cruz and Peixoto (“1985” [1987]); very likely the same applies for H. rubracyla, a species that was resurrected from the synonymy of H. pellucens by Duellman (1974) and included in the H. albomarginata group by Duellman in Frost (1985). The only possible synapomorphies that were proposed for these complexes were the red coloration of the webbing in the two species of the H. albomarginata complex studied by the authors, and presence of cloacal ornamentation in the H. albosignata complex (several instances of homoplasy within hylids). Haddad and Sawaya (2000) and Hartmann et al. (2004) further suggested that the H. albofrenata and H. albosignata complexes share a reproductive mode where the male constructs a subterranean nest in the muddy side of streams and pools that is completely concealed after spawning, a nest where larvae spend early stages of development; subsequent to flooding, the exotrophic larvae live in ponds or streams.

Cruz et al. (2003) added Hyla ibirapitanga and H. sibilata to the H. albosignata complex. Note that species included in both the H. albofrenata and H. albosignata complexes do not posses a prepollical spine, as do species in the H. albomarginata complex. On recent occasions, some authors (Haddad and Sawaya, 2000; Garcia et al., 2001a) referred directly to the H. albofrenata and H. albosignata groups without further comment. We include in the present analysis representatives of the three complexes: H. albosignata, H. callipygia, H. cavicola, and H. leucopygia as representatives of the H. albosignata complex; H. arildae, H. weygoldti, and a Hyla sp. 1, a new species similar to H. ehrhardti, as representatives of the H. albofrenata complex; and H. albomarginata, H. pellucens, and H. rufitela as exemplars of the H. albomarginata complex.

Hyla albopunctata Group: Cochran (1955) recognized the H. albopunctata group on the basis of the “more streamlined body shape, by lacking an outer metatarsal tubercle, and by having the fingers webbed only at the base …”. She included in the group H. albopunctata, H. raniceps, and several species from southeastern Brazil that are now in the H. claresignata and H. pulchella groups. Cochran and Goin (1970) recognized a H. lanciformis group (on the basis of large size, a white margin on the upper lip, pointed heads, and reduced webbing between the fingers) in which they included H. lanciformis, H. multifasciata, and H. boans (name applied incorrectly to H. albopunctata; see Duellman, 1971a). Duellman (1971a) implicitly united these two groups and considered the larger H. albopunctata group to be composed of H. albopunctata, H. lanciformis, H. multifasciata, and H. raniceps. De Sá (1995, 1996) stated that there was no evidence supporting the monophyly of the group. Caramaschi and Niemeyer (2003) added H. leucocheila to the group and suggested that it was monophyletic, but they presented no evidence to this effect. We include all species except H. leucocheila in our analysis.

Hyla boans Group: The constitution of the H. boans group as well as the definition of the group present a rather confusing history. Affinities between species of what is currently called the H. boans group were first recognized by Cochran (1955), who included in what she called the H. faber group the species H. crepitans, H. faber, H. langsdorffii (now a species of Osteocephalus, see Duellman, 1974), and H. pardalis. Some of the diagnostic characters of this group were large size and the presence of what she called a prominent spurlike prepollex in males. Cochran and Goin (1970) included H. faber, H. pardalis, H. rosenbergi, and H. maxima (now a junior synonym of H. boans; see Duellman, 1971a) in the H. maxima group, and they excluded H. crepitans, placing it in its own group. Duellman (1970) presented a formal definition of the H. boans group, in which he included H. boans, H. circumdata (now in the H. circumdata group), H. crepitans, H. faber, H. langsdorffii, H. pardalis, and H. rosenbergi. Lutz (1973) included the species in three different groups,11 in one of which she also included several species now included in the H. circumdata, H. pseudopseudis, and H. martinsi groups (the “species with long, sharp pollex rudiment”). Kluge (1979) resurrected H. pugnax from the synonymy of H. crepitans, including it also in the H. boans group. Martins and Haddad (1988) included in the group H. lundii (using the name H. biobeba, a junior synonym, see Caramaschi and Napoli, 2004), based on observations of nest construction done by Jim (1980). Implicitly, they also removed H. circumdata from the group. Hoogmoed (1990) resurrected H. wavrini from the synonymy of H. boans and retained it in the group. Duellman (2001) omitted H. lundii from the group without comment. Caramaschi and Rodrigues (2003) added H. exastis, suggesting that it was related to H. lundii and H. pardalis on the basis of its lichenous color pattern and the rugose skin texture. Caramaschi and Napoli (2004) presented a formal definition of the group. In summary, and following Caramaschi and Napoli (2004), we regard the H. boans group to be composed of H. boans, H. crepitans, H. exastis, H. faber, H. lundii, H. pardalis, H. pugnax, H. rosenbergi, and H. wavrini. The only synapomorphy that has ever been proposed for this group is the nest-building behavior of males, which has been observed in most species (see Martins and Moreira, 1991 for a review). Early reports of H. crepitans indicated that males do not construct nests; this was shown to be facultative by Caldwell (1992). This behavior is still unknown in H. pugnax. From this group we include in our analysis H. boans, H. crepitans, H. faber, H. lundii, and H. pardalis.

Hyla circumdata Group: This group was first mentioned by Bokermann (1967a, 1972), without providing any diagnosis. Heyer (1985) provided the first formal definition, diagnosing the group by the combination of a well-developed prepollex and the posterior face of the thigh having dark vertical stripes. The group was further discussed and expanded by Caramaschi and Feio (1990), Pombal and Haddad (1993), Napoli (2000), Caramaschi et al. (2001), and Napoli and Pimenta (2003). Three other species groups, the H. claresignata, H. martinsi, and H. pseudopseudis groups, as well as H. alvarengai, historically had been satellites of the H. circumdata group, with these species being alternatively included or excluded from the group. These groups and H. alvarengai are treated separately. With the recognition of these three groups being separate from the H. circumdata group, it is unclear which synapomorphies support its monophyly as currently defined.

Duellman et al. (1997) suggested that all species of the Hyla circumdata group should be transferred to the H. pulchella group. Faivovich et al. (2004) demonstrated by using DNA sequences from four mitochondrial genes that the two groups are not closely related. In the analysis of Faivovich et al. (2004), the three exemplars of the H. circumdata group then available (H. astartea, H. circumdata, and H. hylax) formed a monophyletic group that is the sister taxon of the remaining Gladiator Frogs they included in their analysis. Napoli and Pimenta (2003), Napoli and Caramaschi (2004), and Napoli (2005) recognized 15 species in the group: H. ahenea, H. astartea, H. caramaschii, H. carvalhoi, H. circumdata, H. feioi, H. gouveai, H. hylax, H. ibitipoca, H. izecksohni, H. lucianae, H. luctuosa, H. nanuzae, H. ravida, and H. sazimai. We include five species in our analysis: H. astartea, H. circumdata, H. hylax, as well as Hyla sp. 3 and Hyla sp. 4, two undescribed species from littoral areas of northern São Paulo (state) and southrn Rio de Janeiro (state), Brazil, respectively.

Hyla claresignata Group: A close relationship between H. clepsydra and H. claresignata was suggested by Bokermann (1972), who noticed striking similarities in larval and adult morphology. Bokermann (1972) suggested a possible relationship of these species with the H. circumdata group; following him, Jim and Caramaschi (1979) included H. clepsydra and H. claresignata in the H. circumdata group. However, subsequent workers (Caramaschi and Feio, 1990; Pombal and Haddad, 1993) who referred to the H. circumdata group did not include them in the group. The H. claresignata group was recognized in the restricted form by Duellman et al. (1997). Possible synapomorphies of the H. claresignata group are character states associated with the torrent-dwelling larvae of these species: oral disc completely surrounded by marginal papillae, and 7/12–8/13 labial tooth rows. We were not able to secure samples of either of the two species of this group.

Hyla geographica Group: This group was delimited by Cochran (1955: 180) as being characterized by its “extremely attenuate limbs”. Cochran and Goin (1970) characterized the species of this group as “moderate-sized tree frogs with elongate dermal appendages on the heels and reduced webbing between the fingers.” Duellman (1973a) presented an extensive characterization of the group (including vomers large with angular dentigerous processes, each bearing as many as 20 teeth; nuptial excrescences present in breeding males; projecting prepollices absent in both sexes; calcars present; palpebral membrane clear or reticulated). However, it is unclear from his account if any of these character states could be considered synapomorphic of the group.

Duellman (1973a) included in this group Hyla calcarata, H. fasciata, and H. geographica. Later, Pyburn (1977, 1984) added H. microderma and H. hutchinsi. Goin and Woodley (1969) considered H. kanaima related to the H. geographica group, and Pyburn (1984) included H. kanaima in the group. Lutz (1963, 1973) and Bokermann (1966a) stressed similarities between H. secedens and H. semilineata (as H. geographica), but Caramaschi et al. (2004a) suggested that actually this species is closer to H. bischoffi (of the H. pulchella group). Duellman (in Frost, 1985) and Duellman and Hoogmoed (1992), respectively, included H. picturata and H. roraima in the H. geographica group. D'Heursel and de Sá (1999) argued for the recognition of H. semilineata, a species that had previously been placed in the synonymy of H. geographica. Lescure and Marty (2000) included H. dentei, a species that Bokermann (1967b) considered to have character states of both H. raniceps (H. albopunctata group) and the H. geographica group. Caramaschi et al. (2004a) added H. pombali to the group. In summary, the H. geographica group is currently composed of 11 species: H. calcarata, H. dentei, H. fasciata, H. geographica, H. hutchinsi, H. kanaima, H. microderma, H. picturata, H. pombali, H. roraima, and H. semilineata. We include H. fasciata, H. calcarata, H. kanaima, H. microderma, H. picturata, H. roraima, and H. semilineata in our study.

Hyla granosa Group: This group was first defined by Cochran and Goin (1970) as green frogs that share the vomerine teeth being in rather heavy, arched series, and with males having a “protruding spine in the prepollex”. These authors included H. granosa, H. rubracyla (now in the H. albomarginata complex of the H. albomarginata group, see above), and H. guibei (now a junior synonym of H. pellucens; see Duellman, 1974). Previously, Rivero (1964) stated that H. alemani was allied with H. granosa. Rivero (“1971” [1972]) considered H. sibleszi to be related to H. granosa. Hoogmoed (1979) mentioned, without any diagnosis, the H. granosa group in the Guayanas, in which he included H. ornatissima. Mijares-Urrutia (1992a) considered H. alemani, H. granosa, H. ornatissima, and H. sibleszi to be the members of this group, and he provided a characterization based on larval features. We are not aware of any synapomorphies for this group. In this analysis we include H. granosa and H. sibleszi.

Hyla martinsi Group: This group was recognized by Bokermann (1965b) for two species, H. langei and H. martinsi, characterized by the presence of an extensive hooklike humeral crest and by a bifid prepollex. Bokermann (1964a) noticed “superficial similarities” of H. martinsi with H. circumdata. Caramaschi and Feio (1990) and Cardoso (1983) included H. martinsi in the H. circumdata group for having the diagnostic characters established by Heyer (1985). However, based on the presence of bifid prepollex and a humeral spine, Caramaschi et al. (2001) preferred to keep it as a separate species group. As a representative of this group we include H. martinsi in the analysis.

Hyla pseudopseudis Group: This group was recognized by Pombal and Caramaschi (1995) as closely related to the H. circumdata group, from which it was differentiated mostly by its color pattern. Eterovick and Brandão (2001) further differentiated both groups based on the presence of short, lateral irregular tooth rows and for having additional posterior tooth rows (6–8 rows) in the oral discs of the larvae of the H. pseudopseudis group (a maximum of 5 posterior rows in the H. circumdata group). Caramaschi et al. (2001) transferred H. ibitiguara from the H. circumdata group to the H. pseudopseudis group on the basis of its similar external morphology, color pattern, and habits. The group currently comprises three species, H. ibitiguara, H. pseudopseudis and H. saxicola, plus Hyla sp. 6 (aff. H. pseudopseudis), a new species from Bahia, Brazil, that is being described by Lugli and Haddad (in prep.). Only tissues of this new species were available for this study.

Hyla pulchella Group: The history of this group was recently reviewed by Faivovich et al. (2004). These authors also presented a phylogenetic analysis based on mitochondrial DNA sequences of four genes, and included 10 of the then 14 species included in the group, plus exemplars of the former H. polytaenia group and several outgroups. Their results indicate that the H. polytaenia group, as defined by Cruz and Caramaschi (1998), is nested within the H. pulchella group. Consequently, species included in the H. polytaenia group were transferred to the H. pulchella group, where they are recognized as the polytaenia clade. Considering this action and the species status given to H. cordobae and H. riojana, Faivovich et al. (2004) raised the number of species included in the H. pulchella group to 25. Carnaval and Peixoto (2004) recently added H. freicanecae to the group. Caramaschi et al. (2004a) suggested that H. secedens is related to H. bischoffi, therefore adding implicitly the species to the H. pulchella group. Caramaschi and Cruz (2004) added H. beckeri and H. latistriata to the H. polytaenia clade, adding two more species to the H. pulchella group. Faivovich et al. (2004) had doubts regarding the recognition of H. callipleura. Duellman et al. (1997) included this name as a junior synonym of H. balzani, but Köhler (2000) resurrected it using the combination H. cf. callipleura for some populations in Bolivia. We tentatively recognize H. callipleura as valid, but stress the necessity of further studies to clarify its status.

While the monophyly of this redefined Hyla pulchella group is supported by molecular evidence, no morphological synapomorphies have been proposed so far (see also comments for the H. circumdata and H. larinopygion groups). In summary, there are 30 species included in this group: H. albonigra; H. andina; H. balzani; H. beckeri; H. bischoffi; H. buriti; H. caingua; H. callipleura; H. cipoensis; H. cordobae; H. cymbalum; H. ericae; H. freicanecae; H. goiana; H. guentheri; H. joaquini; H. latistriata; H. leptolineata; H. marginata; H. marianitae; H. melanopleura; H. palaestes; H. phaeopleura; H. polytaenia; H. prasina; H. pulchella; H. riojana; H. secedens; H. semiguttata; and H. stenocephala. In this analysis we include the same species that were available to Faivovich et al. (2004) (H. andina; H. balzani; H. bischoffi; H. caingua; H. cordobae; H. ericae; H. guentheri; H. joaquini; H. leptolineata; H. marginata; H. marianitae; H. prasina; H. pulchella; H. riojana; H. semiguttata, and an undescribed species), plus H. polytaenia. The species that Faivovich et al. (2004) called Hyla sp. 2 corresponds to what Caramaschi and Cruz (2004) recently described as H. latistriata, and so is included under that name.

Hyla punctata Group: This group was first recognized by Cochran and Goin (1970), who included H. punctata, H. rhodoporus, and H. rubeola (these last two were subsequently considered to be synonyms of H. punctata by Duellman, 1974). They characterized the group as “moderately small green tree frogs with small vomerine tooth patches, reduced webbing between the fingers, without spines on the pollex, and without ulnar or tarsal ridges.” Hoogmoed (1979) mentioned this group without defining it. No synapomorphies have been proposed for this group. Besides H. punctata, two other species could be included on this poorly defined group: H. hobbsi, a species resurrected from the synonymy of H. punctata by Pyburn (1978), and H. atlantica, a name recently applied by Caramaschi and Velosa (1996) for the populations on eastern Brazil previously considered as H. punctata. In this analysis we include H. punctata.

Species of Probable Gladiator Frogs Unassigned to Species Group: Hyla alvarengai: This bizarre species was said by Bokermann (1964a) to share some character states with H. martinsi and H. saxicola (now placed in the H. martinsi and H. pseudopseudis groups, respectively), such as the notable development of the prepollex and the shape of the sacral diapophyses. Lutz (1973) included it in the group of the “species with long, sharp pollex rudiment”, together with H. crepitans, H. faber, and species now included in the H. circumdata and H. martinsi groups. She referred to it as Hyla (Plectrohyla?) alvarengai and stated that it was “devoid of affinities with the very large species of Hyla”, suggesting instead a possible relationship with Plectrohyla. A similar opinion was presented by Sazima and Bokermann (1977), who noticed “superficial similarities” with Plectrohyla, but they argued that they differed in larval morphology, spawn, and vocalizations. Duellman et al. (1997) included H. alvarengai in the H. circumdata group, presumably because it shares the diagnostic characters of the group. Eterovick and Brandão (2001) and Caramaschi et al. (2001) did not consider it as a member of the H. circumdata group. Unfortunately, we could not secure this species for our analysis, although we include a new species similar to H. alvarengai that is in the process of being described by Lugli and Haddad (in prep.).

Hyla fuentei: This species was described by Goin and Goin (1968) based on a single adult female from Surinam. Hoogmoed (1979) mentioned the existence of two additional specimens collected close to the type locality. Since then, no author has referred to this species. From the original description, there are few characters that allow the association of this species with any other group of Hyla. The angulate dentigerous process of the vomer suggests that this species could be associated with certain Gladiator Frogs, as some species currently placed in the H. albopunctata, H. boans, and H. geographica groups have this character state. A study of the holotype and discovery of male specimens should clarify the matter.

Hyla heilprini: This West Indian hylid was associated with the H. albomarginata group by Duellman (1970) based on the presence of a “green” peritoneum (actually it is white parietal peritoneum, like in species of the H. albomarginata, H. bogotensis, H. granosa, and H. punctata groups; Lynch and Ruiz-Carranza [1991: 4]; Faivovich, personal obs.) and external pigmentation. This was followed by Trueb and Tyler (1974), who noticed that its morphology was “highly reminiscent” of those from that species group. While it seems clear that H. heilprini is a Gladiator Frog, we are not aware of any synapomorphy relating it to the H. albomarginata group. This species was included in the analysis.

Three species of Hyla from the Venezuelan Tepuis: There are three species of Hyla from the Venezuelan Tepuis that have not been posited to be related to any other species or group of species: H. benitezi, H. lemai, and H. rhythmicus. The presence of a prepollical spine (Rivero, “1971” [1972]; Donnelly and Myers, 1991; Señaris and Ayarzagüena, 2002) associates these species with Gladiator Frogs. Hyla benitezi and H. lemai were included in the analysis.

Hyla varelae: Carrizo (1992) described this species based on a single adult male. It was suggested to be related to H. raniceps in the description. No additional specimens have been collected since the description, and it was not included in this analysis.

Andean Stream-Breeding Hyla

Duellman et al. (1997) presented a phylogenetic analysis restricted to wholly or partially Andean species groups of Hyla. On their most parsimonious tree, the H. armata, H. bogotensis, and H. larinopygion groups together form a monophyletic group supported by three transformations in larval morphology: the enlarged, ventrally oriented oral disc; the complete marginal papillae; and labial tooth rows formula 4/6 or more.

Hyla armata Group: The H. armata group was first recognized by Duellman et al. (1997) for its single species, H. armata. Köhler (2000) and De la Riva et al. (2000) subsequently reported that H. charazani was a second member of the H. armata group. Duellman et al. (1997) described four synapomorphies for the H. armata group: the presence in males of keratin-covered bony spines on the proximal ventral surface of the humerus, on the expanded distal element of the prepollex, and on the first metacarpal; tadpole tail long with low fins and bluntly rounded tip; forearms hypertrophied; and the presence of a “shelf” on the larval upper jaw sheath. We include both species in our analysis.

Hyla bogotensis Group: This group was reviewed by Duellman (1970, 1972b, 1989) and Duellman et al. (1997). The only synapomorphy that has been suggested for this group is the presence in males of a mental gland.12 Hyla albopunctulata was redescribed by Duellman and Mendelson (1995), who rejected a possible relationship with the H. bogotensis group, as suggested by Goin in Rivero (1969) and Duellman (in Frost, 1985), and they simply stated that its relationships were unclear. Faivovich et al. (in prep.) studied two male syntypes (BMNH 1880.12.5159 and 1880.12.5160), which posses a noticeable mental gland. For this reason, we associate this species with the H. bogotensis group. The Hyla bogotensis group is then composed of 15 species: H. albopunctulata, H. alytolylax, H. bogotensis, H. callipeza, H. colymba, H. denticulenta, H. jahni, H. lascinia, H. lynchi, H. palmeri, H. phyllognata, H. piceigularis, H. platydactyla, H. simmonsi, and H. torrenticola. In this analysis we were able to include only H. colymba and H. palmeri.

Hyla larinopygion Group: Duellman and Hillis (1990) and Duellman and Coloma (1993) reviewed this group, and Duellman and Hillis (1990) and Duellman et al. (1997) provided a formal definition, although it is unclear whether any of the morphological character states employed in these characterizations is synapomorphic for the group. Duellman and Hillis (1990) performed a phylogenetic analysis using isozymes of five species of the group. In the phylogenetic analysis of Duellman et al. (1997), the authors did not identify any synapomorphy for the H. larinopygion group; it merely lacks the apparent synapomorphies of the H. armata and H. bogotensis groups. Because of problems with the limits of the H. larinopygion group, Kizirian et al. (2003) were uncertain about the placement of H. tapichalaca, a species that they considered most similar to the H. larinopygion, H. armata, and H. pulchella groups.13 Faivovich et al. (2004) showed that H. tapichalaca and H. armata (the only exemplars of Andean stream-breeding Hyla they included) were sister taxa, and only very distantly related to the H. pulchella group. The H. larinopygion group currently comprises nine species: H. caucana, H. larinopygion, H. lindae, H. pacha, H. pantosticta, H. psarolaima, H. ptychodactyla, H. sarampiona, and H. staufferorum. In this analysis, we include H. pacha, H. pantosticta, and H. tapichalaca.

The 30-Chromosome Hyla

Only the species of the Hyla microcephala group were initially reported to have 30 chromosomes (Duellman and Cole, 1965; Duellman, 1967). However, as species of other groups were reported to have 30 chromosomes (Duellman, 1970; Bogart, 1973), it became evident that this was a characteristic of several species groups. Currently, the H. columbiana, H. decipiens, H. garagoensis, H. labialis, H. leucophyllata, H. marmorata, H. microcephala, H. minima, H. minuta, H. parviceps, and H. rubicundula groups, plus several species unassigned to any group are believed to conform to a monophyletic group supported by this character state (Duellman, 1970; Duellman and Trueb; 1983; Duellman et al., 1997; Napoli and Caramaschi, 1998; Carvalho e Silva et al., 2003).

Hyla columbiana Group: This group was first proposed by Duellman and Trueb (1983) for three species: H. carnifex, H. columbiana, and H. praestans. Kaplan (1991, 1999) found no evidence of monophyly for the group. Kaplan (1997) resurrected H. bogerti from the synonymy of H. carnifex, adding a fourth species to the group. Kaplan (1999) suggested that “the presence of two close, triangular lateral spaces between the cricoid and arytenoids at the posterior part of the larynx” is a synapomorphy of the H. columbiana group excluding H. praestans, which he considered to be closely related to the H. garagoensis group. The group therefore is composed of H. bogerti, H. carnifex, and H. columbiana. In the present analysis, we include H. carnifex.

Hyla decipiens Group: While describing the tadpoles of H. oliveirai and H. decipiens, Pugliese et al. (2000) noticed that they have marginal papillae (unlike other known larvae of the H. microcephala group), and they pointed out that they may not be members of the H. microcephala group as considered by Bastos and Pombal (1996). Pugliese et al. (2000) noticed similarities in tadpole morphology with H. berthalutzae, with which these two species also share oviposition on leaves outside the water. Pugliese et al. (2000) also associated H. haddadi with these three species based on external similarity.

Carvalho e Silva et al. (2003) suggested the recognition of the Hyla decipiens group for these species. The group was defined by larval features that include one row of marginal papillae, an ovoid body in lateral view, eyes in the anterior third of the body, dorsal fin arising at the end of the body, tail with transverse dark stripes on a light background, and pointed tip without a flagellum. It is unclear which of these character states could be considered to be possible synapomorphies. Perhaps one apparent synapomorphy of the group, not mentioned by the authors, could be the oviposition on leaves outside the water. The group is composed of H. berthalutzae, H. decipiens, H. haddadi, and H. oliveirai. In our analysis, we include H. berthalutzae.

Hyla garagoensis Group: This species group was first recognized by Kaplan and Ruiz-Carranza (1997), who diagnosed it by the presence of alternated pigmented and unpigmented longitudinal stripes on the hindlimbs of larvae. The H. garagoensis group is currently composed of three species, H. garagoensis, H. padreluna, and H. virolinensis. Unfortunately, no species of this group was available for our analysis.

Hyla labialis Group: This group was first recognized by Cochran and Goin (1970) for H. labialis (including what is now H. platydactyla, a species of the H. bogotensis group). They characterized the group by the vomerine teeth being in two rounded patches and by the presence of a well-developed axillary membrane (referred to as a patagium) that is bright blue in life. Duellman (1989) presented a more extensive definition of the group, noting that the axillary membrane was absent, a point with which we agree. A similar definition was presented by Duellman et al. (1997). No synapomorphies were suggested for this species group. The group currently comprises three species, H. labialis, H. meridensis, and H. pelidna. In this study we include H. labialis.

Hyla leucophyllata Group: This group was defined and later partly reviewed by Duellman (1970, 1974). From Duellman's (1970) extensive definition, the only possible synapomorphy seems to be the presence of two glandular patches in the pectoral region (this character state, however, was ignored in every subsequent paper dealing with phylogenetic relationships of 30-chromosome Hyla). In the phylogenetic analysis presented by Duellman and Trueb (1983), the only synapomorphy they proposed for the group was violin larval body shape. This character state seems problematic in that it is present in larvae of some species associated with the H. microcephala and H. rubicundula groups (see Lavilla, 1990; Pugliese et al., 2001), and therefore the level of inclusiveness of the clade that is supported by this transformation is unclear. From the perspective of adult morphology, we suggest that glandular patches in the pectoral region is a synapomorphy of the H. leucophyllata group; we observed it clearly on specimens of both sexes in all species of the group. Another character state that is either a likely synapomorphy of the H. leucophyllata group or of a more inclusive clade is the oviposition on leaves hanging over water (this oviposition mode occurs also in other 30-chromosome species; see comments in the H. microcephala and H. parviceps groups).

The Hyla leucophyllata group is composed of seven species: H. bifurca, H. ebraccata, H. elegans, H. leucophyllata, H. triangulum, H. rossalleni, and H. sarayacuensis. In our analysis, we include H. ebraccata, H. sarayacuensis, and H. triangulum.

Hyla marmorata Group: This group was recognized by Cochran (1955) for H. marmorata, H. microps, and H. giesleri based on the presence of an axillary membrane, warty skin around the margin of the lower lip, short snout, crenulated margin of limbs, short hindlimbs, developed finger and toe webbing, dorsal marbled pattern, and orange coloration in thighs and webbings. Bokermann (1964b) further diagnosed the group by its possession of a very large vocal sac. Several of these character states are possible synapomorphies for the group (such as the warty skin around the margin of the lower lip, the crenulated margin of limbs, the dorsal marbled pattern). Duellman and Trueb (1983) suggested that this group could be diagnosed by the presence of a row of small marginal papillae in the larval oral disc.

Bokermann (1964b), like Cochran (1955), included in the Hyla marmorata group other species as well: H. parviceps, H. microps, H. schubarti, and H. moraviensis (now considered a synonym of H. lancasteri; see Duellman, 1966). Subsequent authors transferred these species to other groups. This group is now composed of eight species: H. acreana, H. dutrai, H. marmorata, H. melanargyrea, H. nahdereri, H. novaisi, H. senicula, and H. soaresi. In our analysis we include H. marmorata and H. senicula.

Hyla microcephala Group: This group was defined by Duellman and Fouquette (1968) and Duellman (1970). Despite their extensive characterization, the only character state mentioned by these authors that subsequently has been considered a possible synapomorphy of this group is the lack of marginal papillae in the oral disc. Later, Duellman and Trueb (1983) added the depressed body shape of the larvae, another likely synapomorphy.

While the study of Duellman and Fouquette (1968) was focused on Middle American species, Duellman (1970) referred a number of South American species to the Hyla microcephala group. Overall, he included in the group H. elongata (a junior synonym of H. rubicundula; see Napoli and Caramaschi, 1999), H. microcephala, H. minuta, H. nana, H. phlebodes, H. robertmertensi, H. sartori, and H. werneri. Duellman (1972a) also included H. mathiassoni and H. rhodopepla in the group, and he excluded H. minuta. Cochran and Goin (1970) also had excluded H. minuta by placing it in their H. minuta group. Duellman (1973b) included H. gryllata, and Langone and Basso (1987) added H. minuscula, H. sanborni, and H. walfordi.

Cruz and Dias (1991) placed Hyla bipunctata in the group on the basis of larval characters.14 Márquez et al. (1993) included H. leali in the H. microcephala group, without mentioning that it was included by Duellman (1982) in the H. minima group. Furthermore, Márquez et al. (1993) noticed that H. leali and H. rhodopepla share vocalizations with short, pulsatile notes with extremely low dominant frequencies. Because of these similarities in vocalizations of H. leali with a species of the H. microcephala group, in the absence of other evidence we prefer to keep H. leali in this group.

Bastos and Pombal (1996) suggested that Hyla branneri, H. decipiens, H. haddadi, and H. oliveirai were closely related species that could be tentatively associated with the H. microcephala group based on overall similarity of adult morphology, but this was questioned by Pugliese et al. (2000) and Carvalho e Silva et al. (2003), who excluded these species from the group (see comments for the H. decipiens group). Pombal and Bastos (1998) also added H. berthalutzae, H. cruzi, and H. meridiana. Cruz et al. (2000) added H. pseudomeridiana. Köhler and Lötters (2001a) tentatively included H. joannae, based on its similarities in vocalization and adult morphology with H. leali (but see comments regarding H. leali above). Carvalho e Silva et al. (2003) added H. studerae and excluded H. berthalutzae (see comments for the H. decipiens group).

Of the 20 species currently included in the Hyla microcephala group, tadpoles are only known for 9 species: H. bipunctata, H. meridiana, H. microcephala, H. nana, H. phlebodes, H. pseudomeridiana, H. rhodopepla, H. sanborni, and H. studerae (Bokermann, 1963, Duellman, 1970, 1972a, Lavilla, 1990, Cruz and Dias, 1991, Cruz et al., 2000, Pugliese et al., 2000, Carvalho e Silva et al., 2003). All of these species have the two apparent synapomorphies of the H. microcephala group (see comments for the H. rubicundula group).

The 20 species currently included in the Hyla microcephala group are H. bipunctata, H. branneri, H. cruzi, H. gryllata, H. joannae, H. leali, H. mathiassoni, H. meridiana, H. microcephala, H. minuscula, H. nana, H. phlebodes, H. pseudomeridiana, H. rhodopepla, H. robertmertensi, H. sanborni, H. sartori, H. studerae, H. walfordi, and H. werneri. In this analysis we include H. bipunctata, H. microcephala, H. nana, H. rhodopepla, H. sanborni, and H. walfordi.

Hyla minima Group: Duellman (1982) tentatively grouped together five species from the Upper and Middle Amazon Basin and eastern Andes for which data on larval morphology, vocalizations, and osteology were mostly absent. He based the grouping of these species on small body size and distribution. The species he included were H. aperomea, H. leali, H. minima, H. riveroi, and H. rossalleni. The group as such was not named until Duellman in Frost (1985) referred to it as the H. minima group. Vigle and Goberdhan-Vigle (1990) added H. miyatai to this group; Márquez et al. (1993) implicitly transferred H. leali to the H. microcephala group (see comments for the H. microcephala group above); De la Riva and Duellman (1997) redescribed H. rossalleni and placed it in the H. leucophyllata group.

Small size is a difficult criterion to apply for the Hyla minima group, considering that its constituent species are not smaller than several species of the H. microcephala group. Duellman (2001) suggested that the H. minima group should be associated with the H. parviceps group. Unfortunately, this does not improve the systematics of these frogs, because no synapomorphies are known for either of these two groups (see the H. parviceps group for more comments). In the present analysis, we could include only H. miyatai.

Hyla minuta Group: This group was first defined by Cochran (1955); most of the species then included subsequently were transferred by several authors to the H. leucophyllata, H. microcephala, or H. rubicundula groups. The character state Cochran (1955) used to distinguish the H. minuta group was the immaculate anterior and posterior surfaces of the thigh, a character state that is shared by several 30-chromosome Hyla.

Martins and Cardoso (1987) described Hyla xapuriensis and referred it to the H. minuta group without providing any definition. Köhler and Lötters (2001b) described H. delarivai and tentatively suggested that it is close to H. minuta. Duellman and Trueb (1983) stated that the H. minuta group contained two species, but they did not state which one was the second species, and they provided no evidence for its monophyly. The group is currently composed of H. minuta and H. xapuriensis, and, following Köhler and Lötters (2001b), we tentatively include H. delarivai. For the purpose of our analysis, only H. minuta was available.

Hyla parviceps Group: This group was first defined by Duellman (1970), and by Duellman and Crump (1974), who also reviewed it. According to Duellman and Crump (1974), the species in the H. parviceps group differ from other 30-chromosome Hyla species by having: (1) a pronounced sexual dimorphism in size; (2) shorter snout; (3) tympanic ring indistinct or absent; (5) small (or reduced) axillary membrane; (9) sexual dimorphism in width of dorsolateral stripes; (10) suborbital bars; (11) thighs marked with spots; (13) iris pale gray with red ring around pupil; (15) more perichondral ossification in the tectum nasi and solum nasi; and (17) squamosal articulating with prootics. Duellman and Crump (1974) characterized the tadpoles as having ovoid bodies and xiphicercal tails with moderately deep fins not extending into body; anteroventral oral disc, large marginal papillae, robust serrated jaw sheaths, and no more than one row of labial teeth. It is unclear which of these character states could be synapomorphies of the group. Duellman and Trueb (1983) did not provide any synapomorphy for the group.

Duellman and Crump (1974) included in the Hyla parviceps group H. bokermanni, H. brevifrons, H. luteoocellata, H. microps, H. parviceps, and H. subocularis. Heyer (1977) added H. pauiniensis. Heyer (1980) resurrected H. giesleri from the synonymy of H. microps. Weygoldt and Peixoto (1987) tentatively included H. ruschii in the group. Martins and Cardoso (1987) added H. timbeba. Duellman and Trueb (1989) added H. allenorum and H. koechlini. Duellman (2001) added H. grandisonae and H. schubarti. Lescure and Marty (2000) referred H. luteoocellata to a separate group, the H. luteoocellata group, which they characterized by the presence of a cream-colored suborbital stripe. Because they did not discuss differences with the H. parviceps group, we still consider H. luteoocellata a member of the H. parviceps group. However, the species they described, H. gaucheri, does not seem to have this stripe.

The Hyla parviceps group is currently composed of 15 species: H. allenorum, H. bokermanni, H. brevifrons, H. gaucheri, H. giesleri, H. grandisonae, H. koechlini, H. luteoocellata, H. microps, H. parviceps, H. pauiniensis, H. ruschii, H. schubarti, H. subocularis, and H. timbeba. In our analysis we include H. brevifrons, H. giesleri, and H. parviceps.

Hyla rubicundula Group: This group was recently defined by Napoli and Caramaschi (1998) and was diagnosed by small body size, patternless thighs, and green dorsum in life that changes to pinkish or violet when preserved. Bogart (1970), Rabello (1970) and Gruber (2002) reported a 30-chromosome karyotype in H. rubicundula and H. elianeae. Historically, species of this group were associated with species now placed in the H. microcephala group, such as H. nana and H. sanborni (Lutz, 1973). The group is currently composed of H. anataliasiasi, H. araguaya, H. cachimbo, H. cerradensis, H. elianeae, H. jimi, H. rhea, H. rubicundula, and H. tritaeniata. In our analysis, we include H. rubicundula.

Species Known or Presumed to Have 30 Chromosomes Not Assigned to Any Group: Hyla anceps: Lutz (1948, 1973) associated H. anceps with H. leucophyllata based on it having an axillary membrane, tadpoles with xiphicercal tail, and vivid flash colors. Cochran (1955) considered this species to have no known close relatives and placed it on its own species group. Bogart (1973) reported a 30-chromosome karyotype for this species, and he tentatively associated it with H. microcephala, H. bipunctata, H. elongata (a junior synonym of H. rubicundula), and H. rhodopepla for sharing a single pair of telocentric chromosomes. Regardless of having been reported to have 30 chromosomes, Hyla anceps has been ignored in all subsequent literature dealing with phylogenetic relations of 30-chromosome species of Hyla. Wogel et al. (2000) redescribed the tadpole of H. anceps and stated that its morphology supported the idea of this species belonging to its own species group, without giving further details. We include this species in the analysis.

Hyla amicorum: This species was considered to be similar to H. battersbyi and H. minuta (Mijares-Urrutia, 1998). On this basis we consider it a 30-chromosome species. We could not include this species in the analysis.

Hyla battersbyi: Since its original description (Rivero, 1961) this species has been scarcely mentioned in the literature. We consider it tentatively as a 30-chromosome species based on the association made by Mijares-Urrutia (1998) of this species with H. minuta based on overall similarity. We could not include this species in the analysis.

Hyla haraldschultzi: Bokermann (1962) stated that the relationships of this species were uncertain. Lutz (1973), while treating the “small to minute forms”, where she included most of the 30-chromosome Hyla, considered H. haraldschultzi to be insufficiently known. We could not include this species in the analysis.

Hyla limai: In the original description, Bokermann (1962) associated this species with H. minuta and H. werneri. Apart from a brief comment by Lutz (1973), it has not been referred to again in the literature. Haddad (unpubl. data), based on morphological variation in populations of H. minuta, finds that H. limai could be a junior synonym of this species. We could not include this nominal species in the analysis.

Hyla praestans: Duellman and Trueb (1983) originally placed this species in the H. columbiana group. Kaplan (1999), based on the presence of a small medial depression in the internal surface of each arytenoid, suggested that H. praestans was related to the H. garagoensis group and possibly its sister taxon. We could not secure samples for the analysis.

Hyla stingi: This species, externally similar to H. minuta, has been suggested to be the sister group of a clade composed of the H. minuta, H. marmorata, H. parviceps, H. leucophyllata, and H. microcephala groups (this being supported by the reduction in the labial tooth row formula from 1/2 to 0/1; Kaplan, 1994). The apparent synapomorphy uniting H. stingi with this clade is the anterior position of the oral disc. This species could not be included in the analysis.

Hyla tintinnabulum: This species was associated with H. branneri and H. rubicundula by Lutz (1973). This fact and our study of one of the syntypes (NHMG 473; adult male) suggest that it is a 30-chromosome Hyla. We could not include this species in the analysis.

Hyla yaracuyana: This species was associated with the 30-chromosome Hyla by Mijares-Urrutia and Rivero (2000), but it was not included in any species group. We could not include this species in the analysis.

Middle American/Holarctic Clade

The monophyly of most Middle American and Holarctic Hylinae was suggested by Duellman (1970, 2001) based on biogeographic grounds. This clade would include most Middle American and Holarctic species groups of Hyla plus the genera Acris, Anotheca, Duellmanohyla, Plectrohyla, Pseudacris, Pternohyla, Ptychohyla, Smilisca, and Triprion.

Acris: This very distinctive taxon was reviewed by Duellman (1970) and was included in several studies of Holarctic hylids (Gaudin, 1974; Hedges, 1986; Cocroft, 1994; da Silva, 1997). In the strict consensus of the analysis by Cocroft (1994), the position of Acris was unresolved with respect of the other Holarctic hylids. In the reanalysis done by da Silva (1997), it is sister to a clade composed of the species of the Hyla cinerea and H. versicolor groups. The two species A. crepitans and A. gryllus are included in our analysis.

Anotheca: This monotypic genus was reviewed by Duellman (1970, 2001). Autapomorphies of this taxon include the unique skull ornamentation composed of sharp, dorsally pointed spines in the margins of frontoparietal, maxilla, nasal (including canthal ridge), and squamosal, and character states that result in its reproductive mode, including the female feeding tadpoles with trophic eggs (see Jungfer, 1996). We include the single species Anotheca spinosa in this analysis.

Duellmanohyla: This genus was reviewed by Duellman (2001). Duellmanohyla was proposed by Campbell and Smith (1992), based on four suggested synapomorphies involving larval morphology: a greatly enlarged, pendant oral disc (referred to as funnel-shaped mouth by Duellman, 2001); long and pointed serrations on the jaw sheaths; upper jaw sheath lacking lateral processes; and greatly shortened tooth rows. Duellman (2001) added the bright red iris and the labial stripe expanded below the orbit. Duellmanohyla contains the following eight species: D. chamulae, D. ignicolor, D. lythrodes, D. rufioculis, D. salvavida, D. schmidtorum, D. soralia, and D. uranochroa. In our analysis, we include D. rufioculis and D. soralia.

Hyla arborea Group: All of the species of Hyla of Europe, North Africa, and Asia have long been recognized as composing a single species group (Stejneger, 1907; Pope, 1931). Probably because species of this group have generally been discussed in isolation of other hylids, we are not aware of any author having provided a diagnosis of this group that could differentiate them, at least phenotypically, from the Nearctic species placed in the H. cinerea and H. eximia groups. No synapomorphy has ever been suggested; the monophyly of the group has apparently been assumed based on geography and the external similarity of most species (9 of the 16 currently recognized species have been considered at some point of it taxonomic history to be subspecies or varieties of H. arborea).

Various authors considered the Hyla arborea group to be closely related with some North American species. Based on similarities in advertisement calls, Kuramoto (1980) suggested that the H. arborea group is closely related to the H. eximia group of temperate Mexico and southwestern United States.

The assumed monophyly of the Hyla arborea group was challenged by Anderson (1991) on karyotypic grounds, and this was supported by Borkin (1999). These authors suggested the presence of at least two lineages resulting from two independent invasions to Eurasia. According to Anderson (1991), at least H. japonica and H. suweonensis (she did not include other eastern Asian species) share the presence of a NOR in chromosome 6 with the representatives she studied of the H. eximia group (H. arenicolor, H. euphorbiacea, H. eximia), some of the H. versicolor group (H. avivoca, H. chrysoscelis, H. versicolor; H. femoralis), and with H. andersonii. Hyla arborea, H. chinensis, H. meridionalis, and H. savignyi share with most Nearctic species of Hyla, as well as several of the outgroups that Anderson (1991) included, the NOR located in chromosomes 10/11 (she considered the chromosome pairs to be homologous in these species, with the shift in NOR position presumably corresponding to modulation of heterochromatin material and not to a translocation event; Anderson 1991: 323).

The Hyla arborea group as currently understood is composed of the following 16 species: H. annectans, H. arborea, H. chinensis, H. hallowellii, H. immaculata, H. intermedia, H. japonica, H. meridionalis, H. sanchiangensis, H. sarda, H. savignyi, H. simplex, H. suweonensis, H. tsinlingensis, H. ussuriensis, and H. zhaopingensis. In this analysis we include H. arborea, H. annectans, H. japonica, and H. savignyi.

Hyla bistincta Group: This group was reviewed by Duellman (2001). The lack of evidence supporting the monophyly of the H. bistincta group, together with the possibility that Plectrohyla is nested within it, has been repeatedly noted (Duellman and Campbell: 1992; Toal, 1994; Wilson et al., 1994a; Mendelson and Toal, 1995; Ustach et al., 2000; Canseco-Márquez et al., 2002).

Duellman (2001) presented a cladistic analysis of the Hyla bistincta group that included the 17 species known at that time, using a vector of character states of Plectrohyla and H. miotympanum as the root. He reported that the analysis resulted in 89 most parsimonious trees, from which he chose one that is presented in his figure 400 (Duellman 2001: 952). In this tree, the H. bistincta group is monophyletic, being supported by a single transformation, the loss of vocal slits. A reanalysis of his data set15 indicates that Plectrohyla is nested within the H. bistincta group on several of the most parsimonious trees, and that the strict consensus tree is almost entirely collapsed. Therefore, Duellman's analysis provides no evidence for the monophyly of the H. bistincta group.

The Hyla bistincta group currently comprises the following 18 described species: H. ameibothalame, H. bistincta, H, calthula, H. calvicollina, H. celata, H. cembra, H. charadricola, H. chryses, H. crassa, H. cyanomma, H. labedactyla, H. mykter, H. pachyderma, H. pentheter, H. psarosema, H. robertsorum, H. sabrina, and H. siopela. In this analysis we include H. bistincta and H. calthula.

Hyla bromeliacia Group: The taxonomy, composition, and history of this group were reviewed by Duellman (1970, 2001). Possible synapomorphies suggested by Duellman (2001) are those modifications of the phytotelmic larvae, mostly the depressed body and the elongated tail. The other characters he used to separate this group from the bromeliad-dwelling species of the H. pictipes group are likely plesiomorphic, such as the lack of massive temporal musculature (present only in H. zeteki and H. picadoi), the presence of more than one tooth row (only one in H. picadoi and H. zeteki), and the oral disc not entirely bordered by a single row of papillae (as in H. zeteki and H. picadoi). The group comprises two species, H. bromeliacia and H. dendroscarta. In this study we include only H. bromeliacia.

Hyla cinerea Group: This group was first recognized by Blair (1958) based on advertisement call structure. For a review of its history see Anderson (1991). The group is currently composed of four species, H. cinerea, H. femoralis, H. gratiosa, and H. squirella, and the group is monophyletic in the analyses of Hedges (1986), Cocroft (1994), and da Silva (1998), being supported by allozyme data (taken in the last two studies from Hedges, 1986). We included all four species of the group in the present analysis.

Hyla eximia Group: The taxonomy, composition, and history of this group were thoroughly reviewed by Duellman (1970, 2001). Duellman (2001) presented an extensive definition; however, it is unclear which of the character states could be taken as evidence of monophyly of the group. Hedges (1986) and Cocroft (1994) included two species, H. arenicolor and H. eximia, in their analyses. While in Hedges's (1986) results these two species form a monophyletic group, on the strict consensus of Cocroft's (1994) most parsimonious trees, both species are not monophyletic and form a basal polytomy with several other species of Hyla. da Silva's (1997) reanalysis of Cocroft's (1994) data set yielded on its strict consensus H. arenicolor and H. eximia as a monophyletic group, which appears to be supported by the allozyme data of Hedges (1986). Besides the seven species included by Duellman (2001) in the group, Eliosa León (2002) resurrected H. arboricola from the synonymy of H. eximia, adding an eigth species to the group. See the H. arborea group for additional comments. The H. eximia group is currently composed of eight species: Hyla arboricola, H. arenicolor, H. bocourti, H. euphorbiacea, H. eximia, H. plicata, H. walkeri, and H. wrightorum. In this analysis we include H. arenicolor, H. euphorbiacea, H. eximia, and H. walkeri.

Hyla godmani Group: The taxonomy, composition, and history of this group were reviewed by Duellman (1970, 2001). Duellman (2001) included in this group the only lowland pond-breeders from Middle America that do not have 30 chromosomes. He suggested as tentative evidence of monophyly the weakly ossified skulls and the presence of an axillary membrane. This group currently comprises four species: H. godmani, H. loquax, H. picta, and H. smithii. We include H. picta and H. smithii in our analysis.

Hyla miotympanum Group: The taxonomy, composition, and history of this group were thoroughly reviewed by Duellman (1970, 2001). Duellman (2001), using a hypothetical outgroup, suggested eight synapomorphies for the group16: abbreviated axillary membrane; indistinct fold on wrist; fingers less than one-half webbed; tarsal fold present through the entire length of the tarsus; cloacal opening directed posteroventrally; nuptial excrescences present; medial ramus of pterygoid short, not in contact with prootic; and larval oral disc in ventral position. It seems unlikely that any of these character transformations will hold as synapomorphies of the group in the context of a more inclusive analysis. The group currently contains 11 species: H. abdivita, H. arborescandens, H. bivocata, H. catracha, H. cyclada, H. hazelae, H. juanitae, H. melanomma, H. miotympanum, H. perkinsi, and H. pinorum. Our analysis includes H. arborescandens, H. cyclada, H. melanomma, H. miotympanum, and H. perkinsi.

Hyla mixomaculata Group: Duellman (1970) reviewed the group and listed several character states that define it. Some of these are probably synapomorphies, such as (known) larvae with an enlarged oral disc with 7/10 or 11 labial tooth rows (the largest number of posterior tooth rows known for a Middle American hylid group); the taxonomic distribution of other character states indicate that they are likely synapomorphies of a more inclusive clade, such as the large frontoparietal fontanelle and the absence (or reduction, unclear on fig. 211 of Duellman, 1970) of the quadratojugal. The group is currently composed of four species, H. mixe, H. mixomaculata, H. nubicola, and H. pellita. Only H. mixe was available for this analysis.

Hyla pictipes Group: The taxonomy, composition, and history of this group were reviewed by Duellman (1970, 2001). Duellman (2001) provided a phylogenetic analysis17 of the montane species of Hyla of lower Central America, where he included the H. pseudopuma and H. pictipes groups.

Duellman (2001) suggested six synapomorphies for the group: slender nasals in adults; tadpoles with stream-dwelling habits; oral disc ventral; complete marginal papillae; only one row of marginal papillae; and presence of submarginal papillae. Note that with the possible exception of slender nasals in adults, the other three character states as defined by Duellman (2001) are also present in several other groups of Middle American stream-breeding frogs (i.e., the Hyla bistincta group, H. mixomaculata group, H. sumichrasti group, and Plectrohyla).

The Hyla pictipes group includes 11 species: H. calypsa, H. debilis, H. insolita, H. lancasteri, H. picadoi, H. pictipes, H. rivularis, H. thorectes, H. tica, H. xanthosticta, and H. zeteki. Considering the uncertainties regarding the monophyly of this group, an appropriate taxon sampling should ideally include representatives of the four former species groups currently combined into the H. pictipes group. Unfortunately, the only representatives available are H. rivularis and Hyla sp. 5 (aff. H. thorectes), an undescribed species from Mexico similar to H. thorectes.

Hyla pseudopuma Group: The taxonomy, composition, and history of this group were reviewed by Duellman (1970, 2001), who could not provide a synapomorphy for it. In his phylogenetic analysis of the H. pseudopuma and H. pictipes groups, the H. pseudopuma group appears as a basal unresolved grade, although this is a consequence of constraining the nonmonophyly of the H. pseudopuma group by using H. angustilineata as the root. The H. pseudopuma group includes four species: H. angustilineata, H. graceae, H. infucata, and H. pseudopuma. In this study we include only H. pseudopuma.

Hyla sumichrasti Group: The taxonomy, composition, and history of this group were reviewed by Duellman (1970, 2001). Possible synapomorphies for the group (Duellman, 2001) are the presence of massive nasals, and tadpoles with immense oral discs, with 3/6 to 3/7 labial tooth rows instead of the 2/3 to 2/6 of the H. miotympanum group. The group currently includes four species: H. chimalapa, H. smaragdina, H. sumichrasti, and H. xera. In this study we include H. chimalapa and H. xera.

Hyla taeniopus Group: This group was reviewed by Duellman (1970, 2001), who defined it as having well-ossified quadratojugals in contact with maxillaries, and tadpoles that have ventral mouths with two or three anterior rows of teeth and three or four posterior rows. While the presence of enlarged testes is a possible synapomorphy of a subgroup composed of H. altipotens, H. trux, and H. taeniopus, there is no evidence for the monophyly of the entire group (Mendelson and Campbell, 1999; Duellman, 2001). Five species are currently included in this group: H. altipotens, H. chaneque, H. nephila, H. taeniopus, and H. trux. In this analysis, we include H. nephila and H. taeniopus.

Hyla tuberculosa Group: Affinities among species of fringe-limbed treefrogs were first suggested by Dunn (1943) and by Taylor (1948, 1952) based on their overall appearance. Firschein and Smith (1956) suggested that the presence of a “prepollex” (presumably referring to an enlarged prepollex) and similarity of external habitus, size, skin texture, and fringed limbs were indicative of a common origin. Duellman (1970, 2001) presented a formal definition of the group, asserting that these frogs be placed in the same group based on their large size, presence of dermal fringes on the limbs (although absent in H. dendrophasma), extensive webbing on hand and feet, and modified prepollices. (Importantly, note that apparently the modified prepollices involve three different morphologies: modification into a projecting spine or a spadelike blade or a clump of spines; Duellman, 1970.) Duellman (2001) added that there is no compelling evidence that the group is monophyletic, an opinion that we share. Furthermore, he tentatively suggested that this group could be related with the Gladiator Frogs rather than with the Middle American/ Holarctic clade. The Hyla tuberculosa group has been referred to as the H. miliaria group (Campbell et al., 2000; Duellman, 1970, 2001; Savage and Heyer, “1968” [1969]) and is composed of H. dendrophasma, H. echinata, H. fimbrimembra, H. miliaria, H. minera, H. phantasmagoria, H. salvaje, H. thysanota, H. tuberculosa, and H. valancifer. In this analysis, we include H. dendrophasma and H. miliaria.

Hyla versicolor Group: This group was first recognized by Blair (1958) on the basis of advertisement call structure. See Duellman (1970) and Anderson (1991) for a brief taxonomic history. Both Blair (1958) and Duellman (1970) included H. arenicolor in the group until Hedges (1986), Cocroft (1994), and da Silva (1997) showed on successive analyses that this species was more closely related to H. eximia (see the H. eximia group for further comments), always on the basis of the allozyme data collected by Hedges (1986).

Although placed originally in the Hyla cinerea group by Blair (1958), H. andersonii was transferred to the H. versicolor group by Wiley (1982), with this being supported by Hedges (1986), Cocroft (1994), and da Silva (1997) on the basis of the allozyme data collected by Hedges (1986). Similarly, H. femoralis was considered a member of the H. versicolor group until Hedges (1986) transferred it to the H. cinerea group, an assignment also corroborated by da Silva (1997). The group currently comprises four species: H. andersonii, Hyla avivoca, H. chrysoscelis, and H. versicolor. In this study we include all but H. chrysoscelis.

Pseudacris: The phylogenetic relationships of this genus were reviewed by Hedges (1986), Cocroft (1994), da Silva (1997), and Moriarty and Cannatella (2004). Hedges (1986) presented an electrophoretic analysis of 33 presumed genetic loci, where all species of Pseudacris were monophyletic, and which provided evidence to include the former Hyla cadaverina, H. crucifer, and H. regilla in Pseudacris. Cocroft (1994) performed a phylogenetic analysis of Pseudacris, including several Holarctic hylids as outgroups, with characters from various sources (osteology, vocalizations, karyotypes, allozymes, sperm morphology) and previous analyses (Hedges, 1986). The strict consensus of his most parsimonious trees shows P. crucifer as the sister taxon of the remaining species of Pseudacris; the two classically recognized species groups (the P. ornata and the P. nigrita groups) each being monophyletic; and P. ocularis being the sister taxon of the P. nigrita group. However, Hedges (1986) found no evidence supporting the inclusion of P. cadaverina and P. regilla in Pseudacris, and for this reason he treated them as Hyla. Furthermore, the relationships of the remaining, monophyletic species of Pseudacris with the other Holarctic hyline are unresolved. In the modified reanalysis performed by da Silva (1997), the strict consensus shows that the clade composed of H. regilla and H. cadaverina is sister to Pseudacris and is supported apparently by allozyme data. Because of this, these two species are considered again to be within Pseudacris (da Silva, 1997).

Moriarty and Cannatella (2004) presented a phylogenetic analysis of the mitochondrial ribosomal genes 12S, tRNA valine, and 16S that included all species of Pseudacris and three outgroups, Hyla andersonii, H. chrysoscelis, and H. eximia. The analysis identified four major clades: (1) the P. regilla clade (P. cadaverina and P. regilla; Moriarty and Cannatella referred to it as the West Coast clade); (2) the Pseudacris ornata clade (P. ornata, P. streckeri, and P. illinoiensis; Moriarty and Cannatella called it fat frogs clade); (3) the P. crucifer clade (P. crucifer and P. ocularis); and (4) the P. nigrita clade (including P. brimleyi, P. brachyphona, P. clarkii, P. feriarum, P. maculata, and P. triseriata; Moriarty and Cannatella called it Trilling Frogs clade). In our study we include Pseudacris cadaverina, P. crucifer, P. ocularis, P. regilla, and P. triseriata.

Pternohyla: This Mexican casque-headed frog genus was reviewed by Trueb (1969) and Duellman (1970, 2001). In Duellman's (2001) phylogenetic analysis of Pternohyla, Smilisca, and Triprion, the monophyly of Pternohyla is supported by four synapomorphies: small discs on fingers; supernumerary tubercles diffuse or absent; large inner metatarsal tubercle18; and large marginal papillae in the larval oral disc. Unfortunately, small discs on fingers are a synapomorphy only under an accelerated optimization (ACCTRAN), and therefore they have no evidential value for the clade.

In Duellman's (2001) analysis, Triprion plus Pternohyla forms a monophyletic group nested within Smilisca. The synapomorphies supporting Triprion plus Pternohyla are19: nasals with broad medial contact; median ramus of pterygoid not in contact with prootic; maxilla moderately expanded laterally; cranial-integumentary co-ossification present; webbing between fingers absent; and inner metatarsal tubercle small. Pternohyla is composed of two similar species, P. dentata, and P. fodiens. In this analysis we include P. fodiens.

Plectrohyla: This genus was reviewed by Duellman (2001) and discussed by McCranie and Wilson (2002). Its phylogenetic relationships were addressed by Duellman and Campbell (1992), Wilson et al. (1994a), and Duellman (2001). The monophyly of Plectrohyla does not appear to be controversial. Duellman and Campbell (1992) listed six synapomorphies: bifurcated alary process of premaxilla; sphenethmoid ossified anteriorly, incorporating the septum nasi and projecting forward to the leading margins of the nasals; frontoparietals abutting broadly anteriorly and posteriorly, exposing a small area of the frontoparietal fontanelle; hypertrophied forearms; and absence of lateral folds in the oral disc. Wilson et al. (1994a) added “prepollex enlarged, elongated, ossified, flat, terminally blunt.” Duellman (2001) interpreted that this definition corresponded to more than one character, and so he divided it into two characters, the derived state of the first one being “enlarged and ossified prepollex in both sexes”, and the derived state of the second one being “enlarged and truncate prepollex.” See comments under the Hyla bistincta group.

Plectrohyla currently contains 18 species: P. acanthodes, P. avia, P. chrysopleura, P. dasypus, P. exquisitia, P. glandulosa, P. guatemalensis, P. hartwegi, P. ixil, P. lacertosa, P. matudai, P. pokomchi, P. psiloderma, P. pycnochila, P. quecchi, P. sagorum, P. tecunumani, and P. teuchestes. In this analysis we include P. guatemalensis, P. glandulosa, and P. matudai.

Ptychohyla: This group was reviewed by Duellman (2001). Campbell and Smith (1992) suggested three synapomorphies for Ptychohyla: the presence of two rows of marginal papillae, an increased number of tooth rows in larvae (from 3/5 to 6/9), and a strongly developed lingual flange of the pars palatina of the premaxilla. Duellman (2001) also suggested as synapomorphies the presence of ventrolateral glands in breeding males, and the coalescence of tubercles to form a distinct ridge on the ventrolateral edge of the forearm. The increase in the number of tooth rows could actually be a synapomorphy not of Ptychohyla but for a more inclusive clade containing Ptychohyla plus other species of stream-breeding hylids that also have a tooth row formula larger than 2/3. Similarly, ventrolateral glands are present also in some species of Duellmanohyla (Campbell and Smith, 1992; Duellman, 2001).

For an unstated reason, Savage (2002a) excluded Ptychohyla legleri and P. salvadorensis from Ptychohyla, placing them back in Hyla. These two species were originally in Hyla (former H. salvadorensis group; see Duellman, 1970) until Campbell and Smith (1992) transferred them to Ptychohyla. Because we are not aware of any evidence supporting Savage's action, we consider them to be members of Ptychohyla.

Ptychohyla is composed of 12 species: P. acrochorda, P. erythromma, P. euthysanota, P. hypomykter, P. legleri, P. leonhardschultzei, P. macrotympanum, P. panchoi, P. salvadorensis, P. sanctaecrucis, P. spinipollex, and P. zophodes. In this analysis we include P. euthysanota, P. hypomykter, P. leonhardschultzei, P. spinipollex, P. zophodes, and Ptychohyla sp., an undescribed species from Oaxaca, Mexico.

Smilisca: This genus was reviewed by Duellman and Trueb (1966) and Duellman (1970, 2001). Duellman (2001) could advance no evidence for the monophyly of Smilisca. He presented a phylogenetic analysis rooted with a hypothetical ancestor, whose strict consensus showed Pternohyla plus Triprion nested within Smilisca, being more closely related to S. baudinii and S. phaeota. The synapomorphies supporting Pternohyla + Triprion + “Smilisca” are the presence of lateral flanges on the frontoparietals, and the unexposed frontoparietal fontanelle. The species of Smilisca have been divided (Duellman and Trueb, 1966) into the S. sordida group (S. puma and S. sordida), the S. baudinii group (S. baudinii, S. cyanosticta, and S. phaeota), and S. sila, a form considered intermediate between these two groups. In Duellman's (2001) phylogenetic analysis, S. sila plus the S. sordida group is monophyletic, with its synapomorphy being the short maxillary process of the nasal. Smilisca contains six species: S. baudinii, S. cyanosticta, S. phaeota, S. puma, S. sila, and S. sordida. In this analysis we include the three species in the S. baudinii group, S. baudinii, S. cyanosticta, S. phaeota, and one species of the S. sordida group, S. puma.

Triprion: This genus was reviewed by Trueb (1969) and Duellman (1970, 2001). In Duellman's (2001) phylogenetic analysis of Pternohyla, Smilisca, and Triprion, the monophyly of Triprion is supported by three synapomorphies20: maxilla greatly expanded laterally, prenasal bone present, and presence of parasphenoid odontoids. See comments for Smilisca and Pternohyla. Triprion is composed of two species, T. petasatus and T. spatulatus. In the analysis we include T. petasatus.

Casque-Headed Frogs and Related Genera

Duellman's (2001) suggestion of Middle American/Holarctic frogs being monophyletic clearly separates the Middle American casque-headed frogs (Triprion, Pternohyla) from the South American and West Indian casque-headed frogs. This is not surprising considering that traditionally the group known as the casque-headed frogs was considered to be nonmonophyletic (Trueb, 1970a, 1970b). However, the position of the South American and West Indian casque-headed frogs remains controversial, and no author has presented evidence indicating whether they form a monophyletic group.

Aparasphenodon: This genus of casque-headed frogs was reviewed and characterized by Trueb (1970a) and Pombal (1993). The presence of a prenasal bone is a likely synapomorphy of Aparasphenodon (with a known homoplastic occurrence in Triprion, as reported by Trueb, 1970a). This genus currently comprises three species, A. bokermanni, A. brunoi, and A. venezolanus. We include A. brunoi in the analysis.

Argenteohyla: This monotypic genus was described and reviewed by Trueb (1970b), who segregated it from Trachycephalus, where it had been placed by Klappenbach (1961). Motives for this segregation were the absence in Argenteohyla of several character states of Trachycephalus as redefined by Trueb (1970a), such as the dermal sphenethmoid, the poorer development of ossification and cranial sculpturing, and vocal sacs that when inflated protrude posteroventrally to the angles of the jaw. Possible autapomorphies of this taxon include the fusion of the zygomatic ramus of the squamosal with the pars facialis of the maxilla. The genus comprises a single species, A. siemersi, for which a northern subspecies, A. s. pederseni, was described by Williams and Bosso (1994). In this analysis we included a specimen that corresponds to the northern form.

Corythomantis: This monotypic genus was reviewed by Trueb (1970a). Autapomorphies of this genus include the absence of palatines, and nasals that conceal the allary processes of premaxillaries (Trueb, 1970a). We include the single species Corythomantis greeningi in this analysis.

Osteocephalus: This genus was diagnosed by Goin (1961) and Trueb (1970a) and studied in detail by Trueb and Duellman (1971). These authors recognized five species: Osteocephalus verruciger, O. taurinus, O. buckleyi, O. leprieurii, and O. pearsoni. In the last 20 years, several new species were described, adding to a total of 18 currently recognized species (see Jungfer and Hödl, 2002; Lynch, 2002). Trueb and Duellman (1971) employed 20 character states to characterize Osteocephalus. Jungfer and Hödl (2002) modified some of these characters to take into account subsequently discovered species. As stated by Ron and Pramuk (1999), referring to the diagnostic states employed earlier by Trueb and Duellman (1971), it is unclear which, if any, of the character states are synapomorphic for the genus. Trueb (1970a) and Trueb and Duellman (1971) suggested, based on the presence of paired lateral vocal sacs in the five species then recognized, that Osteocephalus was related to a group composed of Argenteohyla, Trachycephalus, and Phrynohyas.

Martins and Cardoso (1987) described Ostecephalus subtilis that, unlike the other species known at that time, is characterized by a single, subgular vocal sac that expands laterally; a similar morphology was described by Smith and Noonan (2001) in O. exophthalmus. Jungfer and Schiesari (1995) described O. oophagus, a species with a single, median vocal sac, a reproductive mode involving oviposition in bromeliads, and phytotelmous oophagous larvae. Jungfer et al. (2000), Jungfer and Lehr (2001), and Lynch (2002) described four species, O. deridens, O. fuscifacies, O. leoniae, and O. heyeri, which also have a single, median vocal sac. According to Lynch (2002), O. cabrerai also shares this characteristic. Reproductive modes are unknown for O. cabrerai, O. exophthalmus, O. heyeri, O. leoniae, and O. subtilis; spawning in bromeliads is suspected for O. deridens and O. fuscifacies (Jungfer et al., 2000). Note that Lynch (2002) doubted a possible relationship between O. heyeri and what he called the “presumed clade of oophagous species” (where he included O. deridens, O. fuscifacies, and O. oophagus), suggesting instead that it could be related to what he called O. rodriguezi (at that time already transferred to the new genus Tepuihyla by Ayarzagüena et al., “1992” [1993b]). While the species known or suspected to spawn in bromeliads could be monophyletic, we are not aware of any synapomorphy supporting the monophyly of all remaining species of Osteocephalus.

The species currently included in Osteocephalus are O. buckleyi, O. cabrerai, O. deridens, O. elkejungingerae, O. exophthalmus, O. fuscifacies, O. heyeri, O. langsdorffii, O. leoniae, O. leprieurii, O. mutabor, O. oophagus, O. pearsoni, O. planiceps, O. subtilis, O. taurinus, O. verruciger, and O. yasuni. Considering the uncertainties regarding Osteocephalus, we attempted to include representatives of the morphological and reproductive diversity within the genus: O. cabrerai, O. langsdorffii, O. leprieurii, O. oophagus, and O. taurinus.

Osteopilus: The genus Osteopilus was resurrected by Trueb and Tyler (1974) for three apparently related species that were often referred to collectively as the Hyla septentrionalis group (see Dunn, 1926; Trueb, 1970a). Trueb and Tyler (1974) provided a diagnostic definition of the genus; a possible synapomorphy is the differentiation of the m. intermandibularis to form supplementary apical elements. Trueb and Tyler (1974) also maintained, due to the impressive morphological divergence, that Osteopilus, the other Antillean groups then considered to be in Hyla (H. heilprini, H. marianae, H. pulchrilineata, H. vasta, H. wilderi), and the new genus they erected, Calyptahyla, represented several independent invasions from the mainland.

Maxson (1992) and Hass et al. (2001), using albumin immunological distances, suggested that Osteopilus is paraphyletic with respect to most other West Indian hylids (with the exception of Hyla heilprini, a Gladiator Frog). Hedges (1996) mentioned that unpublished DNA sequence data confirmed these findings. Anderson (1996) presented a karyological study of the three species of Osteopilus, indicating that her data were compatible with a monophyletic Osteopilus. Based on the comments by Hedges (1996), and immunological results of Hass et al. (2001), Franz (2003), Powell and Henderson (2003a, 2003b), and Stewart (2003) transferred Calyptahyla crucialis, H. marianae, H. pulchrilineata, H. vasta, and H. wilderi to Osteopilus that now includes eight species. Osteopilus is grouped together only on the basis of the immunological distance results, as no discrete character data set supporting its monophyly has yet been published. The species of Osteopilus available for our study were O. crucialis, O. dominicensis, O. septentrionalis, and O. vastus.

Phrynohyas: This genus was reviewed by Duellman (1971b). Although very distinctive externally, the only seeming synapomorphy in the diagnostic definition of Phrynohyas provided by Duellman (1971b) is the extensively developed parotoid glands in the occipital and scapular regions. Likely related to this character state, the viscous, milky secretions of the species of this genus could also be considered synapomorphic. Lescure and Marty (2000) transferred Hyla hadroceps to this genus; this was confirmed in a phylogenetic analysis using the mitochondrial ribosomal gene 12S by Guillaume et al. (2001). Pombal et al. (2003) described P. lepida. See Osteocephalus for further comments. Phrynohyas currently contains seven species: P. coriacea, P. hadroceps, P. imitatrix, P. lepida, P. mesophaea, P. resinifictrix, and P. venulosa. In our analysis we include P. hadroceps, P. mesophaea, P. resinifictrix, and P. venulosa.

Tepuihyla: This genus was defined by Ayarzagüena et al. (“1992” [1993b]) for five species of Osteocephalus previously considered to constitute the O. rodriguezi species group (Duellman and Hoogmoed, 1992; Ayarzagüena et al., “1992” [1993a]). Ayarzagüena et al. (“1992” [1993b]) differentiated Tepuihyla from Osteocephalus using the following character states present in Tepuihyla: subgular vocal sac, absence or extreme reduction of hand webbing, more reduced toe webbing, smaller size, absence of cranial co-ossification, large frontoparietal fontanelle, shorter nasals, and shorter frontoparietals. It is unclear which, if any, of these character states are apparent synapomorphies of Tepuihyla. There are eight species currently included in this genus: T. aecii, T. celsae, T. edelcae, T. galani, T. luteolabris, T. rimarum, T. talbergae, and T. rodriguezi. In this analysis we include only T. edelcae.

Trachycephalus: The relationships of this casque-headed taxon were discussed by Trueb (1970a) and Trueb and Duellman (1971). They diagnosed Trachycephalus from Argenteohyla, Osteocephalus, and Phrynohyas for having heavily casqued and co-ossified skulls, a medial ramus of pterygoid that does not articulate with the prootic, and a parasphenoid having odontoids. A likely synapomorphy of Trachycephalus is the presence of exostosis on the alary process of the premaxillae (Trueb, 1970a). Trachycephalus contains three species: T. atlas, T. jordani, and T. nigromaculatus. In this analysis we include T. jordani and T. nigromaculatus.

Species and Species Groups of Hyla Not Associated with Any Major Clade

Hyla aromatica Group: This group was proposed by Ayarzagüena and Señaris (“1993” [1994]) for two species from the Venezuelan Tepuis, H. aromatica and H. inparquesi, which they could not associate with any of the species groups known from the Guayanas. Ayarzagüena and Señaris (“1993” [1994]) noticed that the H. aromatica group shares some characters with the H. larinopygion group; however, they preferred to retain it as a separate group. They justified this decision based on the smaller size of members of the H. aromatica group, different coloration pattern, supraorbital cartilaginous process, vomerine odontophores smoothly S-shaped and with more odontophores, small nasals, and large prepollex. They included as well other character states, that actually, like some of these just mentioned, are either shared with several neotropical groups (supraorbital cartilaginous process; Faivovich, personal obs.), or some species of the H. larinopygion group (vomerine odontophores smoothly S-shaped; Duellman and Hillis, 1990: 5), or with the H. armata group (labial tooth row formula; see Cadle and Altig, 1991), or they support the monophyly of the H. aromatica group (adults with strong odor). Considering the lack of evidence of monophyly for the H. larinopygion group, Ayarzagüena and Señaris (“1993” [1994]) cannot be questioned for recognizing a separate species group.

Ongoing research by Faivovich and McDiarmid suggests that Hyla loveridgei should also be considered part of the H. aromatica group. For this analysis, we include H. inparquesi.

Hyla uruguaya Group: This group has never been mentioned as such in the literature. However, clear similarities had been shown by Langone (1990) between H. uruguaya and H. pinima (these species being almost undistinguishable). Possible synapomorphies of the H. uruguaya group are the bicolored iris (also shared with Aplastodiscus; see Garcia et al., 2001a), the presence in tadpoles of two small, keratinized plates below the lower jaw sheath, and a reduction in the size of the marginal papillae of the posterior margin of the oral disc relative to the other papillae (Kolenc et al., “2003” [2004]). From this apparent clade we include H. uruguaya in our analysis.

Hyla chlorostea: Duellman at al. (1997) proposed the recognition of a species group to include the enigmatic Hyla chlorostea, a species known only from its holotype (a subadult male), which could not be associated with any known group of Hyla after its description (Reynolds and Foster, 1992). Unfortunately, we were unable to include this taxon in our analysis.

Hyla vigilans: Different perspectives concerning this enigmatic species were summarized by Suarez-Mayorga and Lynch (2001a). These authors rejected the possibility of a relationship with Scinax (as suggested by La Marca in Frost, 1985). Instead, they asserted that they suspected a possible relationship with Sphaenorhynchus or with H. picta (from the H. godmani species group) based on “oral disc and mouth position” of the tadpoles. We could not obtain samples of this species for our analysis.

Hyla warreni: This species, known only from two adult females, was described by Duellman and Hoogmoed (1992), who did not associate it with any other species or species group. Unfortunately, we could not obtain samples of this species for our analysis.

Other Genera

Aplastodiscus: The taxonomy and history of this genus was recently reviewed thoroughly by Garcia et al. (2001a). According to these authors the monophyly of the genus is supported by four putative synapomorphies: (1) the absence of webbing between toes I and II and basal webbing between the other toes; (2) bicolored iris; (3) females with unpigmented eggs; and (4) great development of internal metacarpal and metatarsal tubercles. Based on overall morphological and advertisement call similarities B. Lutz (1950) suggested a close relationship of this genus with Hyla albosignata. Garcia et al. (2001a) suggest that Aplastodiscus could be related with the H. albofrenata and H. albosignata complexes of the H. albomarginata group, as defined by Cruz and Peixoto (1984), based on the presence of enlarged internal metacarpal and metatarsal tubercles, and unpigmented eggs. Haddad et al. (2005) described the reproductive mode of A. perviridis and noticed that it was the same as that described by Haddad and Sawaya (2000) and Hartmann et al. (2004) in species included in H. albofrenata and H. albosignata complexes. Based on this, Haddad et al. (2005) suggested a possible relationship between these two species complexes and Aplastodiscus. Aplastodiscus is composed of two species, Aplastodiscus cochranae and A. perviridis; we include both in our analysis.

Nyctimantis: This monotypic Neotropical genus was considered a member of the Hemiphractinae by Duellman (1970) and Trueb (1974). Duellman and Trueb (1976) reviewed the taxon and placed it in Hylinae. Duellman and Trueb (1976) considered Nyctimantis to be related with Anotheca spinosa because both share the medial ramus of the pterygoid that is juxtaposed squarely against the anterolateral corner of the ventral ledge of the otic capsule. Also, frogs of both genera are known (Anotheca; Taylor, 1954; Jungfer, 1996) or suspected (Nyctimantis; Duellman and Trueb, 1976) to deposit their eggs in water-filled tree cavities. However, Duellman (2001) latter placed Anotheca in the Middle American/Holarctic clade, implicitly suggesting no relationship with Nyctimantis. Considering the uncertainty of the position of Nyctimantis within hylines, at this stage it is difficult to interpret which character states are autapomorphic. We include the single species Nyctimantis rugiceps in this analysis.

Phyllodytes: The history of this genus was reviewed by Bokermann (1966b). Possible synapomorphies of the taxon are the presence of odontoids on the mandible and on the cultriform process of the parasphenoid (Peters, “1872” [1873]), something unique within the Hylinae. Peixoto and Cruz (1988) noticed that among the six species recognized at that time, four species (P. acuminatus, P. brevirostris, P. luteolus, and P. tuberculosus) share the presence of series of enlarged tubercles on the venter and an enlarged tubercle on each side at the origin of the thigh (Bokermann, 1966b: fig. 6). The other two species, P. auratus and P. kautskyi, have uniform granulation on the venter and lack enlarged tubercles on the thighs, as also seems to be the case in P. melanomystax, a species described later (see Caramaschi et al., 1992). Peixoto et al. (2003) described two additional species, P. edelmoi and P. gyrinaethes; both have a tubercle on each side at the origin of the thigh. Phyllodytes edelmoi has a series of indistinct tubercles on the venter; in P. gyrinaethes they do not form series. Caramaschi and Peixoto (2004) added P. punctatus, which has two medial, poorly distinct rows of tubercles. Caramaschi et al. (2004a) resurrected P. wuchereri. Peixoto et al. (2003) suggested three different species groups based on color pattern, and Caramaschi et al. (2004) further expanded the definitions. The P. luteolus group is characterized by a plain pattern with a variably defined dorsolateral dark brown to black line on canthus rostralis and/or behind the corner of eye. This group includes P. acuminatus, P. brevirostris, P. edelmoi, P. kautskyi, P. luteolus, and P. melanomystax. The P. tuberculosus group has a pale brown dorsum with scattered dark brown dots and includes P. punctatus and P. tuberculosus. The P. auratus group has a dorsal pattern of two dorsolateral, longitudinal white or yellowish stripes, with each stripe being bordered by a dark brown or black line from posterior corner of eye to groin. This group includes P. auratus and P. wuchereri. Finally, P. gyrinaethes is placed in its own group for having red color on hidden surfaces of thighs and a highly modified tadpole. It is unclear if any of these groups is monophyletic. Tissues were available for P. luteolus and an unidentified species, Phyllodytes sp., from Bahia, Brazil.

Lysapsus and Pseudis: The monophyly of the former subfamily Pseudinae has not been historically controversial; it is supported by the presence of a long, ossified intercalary element between the ultimate and penultimate phalanges. Haas (2003) added several synapomorphies from larval morphology, based on the study of larvae of two species of Pseudis. The limits and definitions of Pseudis and Lysapsus were reviewed by Savage and Carvalho (1953) and by Klappenbach (1985). From their observations it is unclear which character states support the monophyly of either genus. Savage and Carvalho (1953: 199) implicitly proposed the paraphyly of Pseudis, when they suggested that Lysapsus “seems to have arisen from Pseudis.” Garda et al. (2004) recently distinguished both genera on the basis of sperm morphology. In Lysapsus laevis (the only species of Lysapsus available to them) the subacrosomal cone is nearly absent, but it is clearly present in the four species of Pseudis they studied. Regardless, the monophyly of either genus has not been satisfactorily documented.

Morphological diversity within Pseudis includes large species, several of which were included in the past in the synonymy of P. paradoxa and were recently resurrected (Caramaschi and Cruz, 1998), and smaller species with a double vocal sac, P. cardosoi and P. minuta (Klappenbach, 1985; Kwet, 2000). Lysapsus includes three species, L. caraya, L. laevis and L. limellum; we include in our analysis the last two. Pseudis is composed of six species: P. bolbodactyla, P. cardosoi, P. fusca, P. minuta, P. paradoxa, and P. tocantins, of which we include in our analysis P. minuta and P. paradoxa.

Scarthyla: Duellman and de Sá (1988) and Duellman and Wiens (1992) suggested that this monotypic genus was sister to Scinax, but more recently, Darst and Cannatella (2003) presented evidence supporting a sister group relationship between Scarthyla and “pseudids”. The single species, Scarthyla goinorum, is included in our analysis.

Scinax: With roughly 86 recognized species, Scinax is the second largest genus within Hylinae. This genus includes the species formerly placed in the Hyla catharinae and H. rubra groups; a taxonomic history was presented by Faivovich (2002). The relationships among the species of Scinax were recently addressed by Faivovich (2002), who performed a phylogenetic analysis using 38 species representing the five species groups then recognized. Although he employed eight outgroups, the analysis is not a strong test of the monophyly of Scinax nor of the relationships of Scinax with other hylines. Duellman and Wiens (1992) suggested that Scinax is the sister group of Scarthyla and that this clade is sister to Sphaenorhynchus. Faivovich (2002) did not test this assertion because his selection of outgroups was heavily influenced by da Silva's results (1998), which did not suggest a close relationship between Scinax and these two genera. Taxon choice in the present study will test more appropriately the hypothesis of (Scinax + Scarthyla) + Sphaenorhynchus.

Faivovich's (2002) results suggested that Scinax contains two major clades: (1) a S. ruber clade composed of species that had been previously grouped into the S. rostratus, S. ruber, and S. staufferi groups; and (2) a S. catharinae clade composed of the species that were included in the S. catharinae and S. perpusillus groups. Faivovich (2002) continued recognition of these two species groups within the S. catharinae clade, as well as the S. rostratus group within the S. ruber clade, as the individual monophyly of the S. catharinae and S. rostratus groups were corroborated by his analysis. The S. perpusillus group is recognized because its monophyly could not be tested, and it still awaits a rigorous test. All species previously included in the nonmonophyletic groups of S. ruber and S. staufferi are included in the larger S. ruber clade, without being assigned to any group. For a list of the species currently included in Scinax, see page 95.

In anticipation of a forthcoming study of the phylogeny of Scinax by Faivovich and associates, we include only S. berthae and S. catharinae as exemplars of the S. catharinae clade, and S. acuminatus, S. boulengeri, S. elaeochrous, S. staufferi, S. fuscovarius, S. ruber, S. squalirostris, and S. nasicus as exemplars of the S. ruber clade.

Sphaenorhynchus: More has been written about nomenclatural confusion surrounding Sphaenorhynchus than about its systematics (see Frost, 2004). This genus has been reviewed by Caramaschi (1989). Duellman and Wiens (1992) proposed the following synapomorphies for Sphaenorhynchus: posterior ramus of pterygoid absent; zygomatic ramus of squamosal absent or reduced to a small knob; pars facialis of maxilla and alary process of premaxilla reduced; postorbital process of maxilla reduced, not in contact with quadratojugal; neopalatine reduced to a sliver or absent; pars externa plectri entering tympanic ring posteriorly (rather than dorsally); pars externa plectri round; hyale curved medially; coracoids and clavicle elongated; transverse process of presacral vertebra IV elongate, oriented posteriorly; and prepollex ossified, bladelike. The genus is composed of 11 species: S. bromelicola, S. carneus, S. dorisae, S. lacteus, S. orophilus, S. palustris, S. pauloalvini, S. planicola, S. prasinus, S. platycephalus, and S. surdus. In our analysis we include S. dorisae and S. lacteus.

Xenohyla: This genus was named by Izecksohn (1996) for the bizarre frog Hyla truncata, which had previously been suggested to be related to Sphaenorhynchus by Izecksohn (1959, 1996) and Lutz (1973). According to Izecksohn (1996), Xenohyla shares with Sphaenorhynchus the reduced number of maxillary teeth, a relatively short urostyle, and the development of the transverse processes of presacral vertebra IV; furthermore, Xenohyla shares with Sphaenorhynchus the quadratojugal not in contact with the maxilla. Izecksohn (1996) suggested also a close relationship with Scinax based on the presence in Xenohyla of a coracoid ridge and an internal, subgular vocal sac. While the coracoid ridge is present in Scinax, it is also present in several other hylines (e.g., see Faivovich, 2002). The internal, subgular vocal sac is not a synapomorphy of all Scinax, but only of the S. catharinae clade. Caramaschi (1998) added X. eugenioi, a second species for the genus. We include X. truncata in our study.

Character Sampling

Gene Selection

Because this study involves the simultaneous analysis of taxa of disparate levels of divergence, we assembled a large data set, including four mitochondrial and five nuclear genes, spanning a broad range of variation, from the fast-evolving cytochrome b (Graybeal, 1993) to the much conserved nuclear genes such as 28S (Hillis and Dixon, 1991).

Ribosomal mitochondrial genes and cytochrome b have been employed recently in several phylogenetic studies of various anuran groups at various levels of divergence (Read et al., 2001; Vences and Glaw, 2001; Cunningham, 2002; Salducci et al., 2002). Nuclear genes have been poorly explored for their use in anuran phylogenetics. The 28S ribosomal nuclear gene has been used in amphibians by Hillis et al. (1993). The protein-coding genes rhodopsin, tyrosinase, RAG-1, and RAG-2 were used to study problems at different levels by Bossuyt and Milinkovitch (2000), Biju and Bossuyt (2003), and Hoegg et al. (2004). In this study we include 12S, tRNA valine, 16S, and fragments of cytochrome b, rhodopsin, tyrosinase, 28S, RAG-1, and seventh in absentia. The last gene is used here for the first time in amphibians.

DNA Isolation and Sequencing

Whole cellular DNA was extracted from frozen and ethanol-preserved tissues (usually liver or muscle) using either phenol-chloroform extraction methods or the DNeasy (QIAGEN) isolation kit. See table 2 for a list and sources of the primers employed.

Amplification was carried out in a 25-μl-volume reaction using either puRe Taq Ready-To-Go PCR beads (Amersham Biosciences, Piscataway, NJ) or Invitrogene PCR SuperMix. For all the amplifications, the PCR program included an initial denaturing step of 30 seconds at 94°C, followed by 35 or 38 cycles of amplification (94°C for 30 seconds, 48–60°C for 60 seconds, 72°C for 60 seconds), with a final extension step at 72°C for 6 min.

Polymerase chain reaction (PCR)-amplified products were cleaned either with a QIAquick PCR purification kit (QIAGEN, Valencia, CA) or with ARRAY-IT (TeleChem International, Sunnyvale, CA) and labeled with fluorescent-dye labels terminators (ABI Prism Big Dye Terminators v. 3.0 cycle sequencing kits; Applied Biosystems, Foster City, CA). Depending on whether the cleaned product was purified with QIAquick or Array-It, the sequencing reaction was carried out in either 10 μl or 8 μl volume reaction following standard protocols. The labeled PCR products were isopropanol-precipitated following the manufacturer's protocol. The products were sequenced either with an ABI 3700 or with an ABI Prism 377 sequencer. Most samples were sequenced in both directions.

Chromatograms obtained from the automated sequencer were read and contigs made using the sequence editing software Sequencher 3.0. (Gene Codes, Ann Arbor, MI). Complete sequences were edited with BioEdit (Hall, 1999).

Morphology

Because the present study is mostly based on molecular data, the failure to include a thorough morphological data set doubtless is its weakest point. As trained morphologists, most of the authors of this paper think that a phylogenetic hypothesis that explains all the available data is the best hypothesis that we can aspire to, and that no class of data is better than any other. Apart from da Silva's unpublished dissertation, which is commented upon below, published comparative studies involving a diversity of hylid exemplars are rare. Major exceptions are the thorough osteological studies by Trueb (1970a) and those on hand muscles of Pelodryadinae (Burton, 1996), distal extensor muscles of anurans (Burton, 1998a), and foot muscles of Hylidae (Burton, 2004). Explicit character descriptions in the context of phylogenetic comparisons include those by Duellman and Trueb (1983), Campbell and Smith (1992), Duellman and Campbell (1992), Duellman and Wiens (1992), Fabrezi and Lavilla (1992), Kaplan (1994, 1999), Cocroft (1994), Burton (1996, 1998a, 2004), Haas (1996, 2003), da Silva (1997), Duellman et al. (1997), Kaplan and Ruiz-Carranza (1997), Mendelson et al. (2000), Sheil et al. (2001), Faivovich (2002), and Alcalde and Rosset (“2003” [2004]). Most of these studies were targeted in general to very specific apparent clades or to very large clades using very few terminals, which leaves particular sets of characters known for very few terminals. Unfortunately, for the inclusion of the characters employed in these studies to be informative, detailed anatomic work would be required on a very large number of terminals (besides the potentially serious need to redefine several characters), a task that we find impossible to pursue at this time. Much to our regret, we find that there are almost no published studies from which we could derive character scorings to enrich our data set without extensive work. The only data set that we thought could be included, due to its relatively dense taxon sampling, is the one resulting from the collection of observations presented by Burton (2004). Although its sampling of nonhylid taxa that match our sampled taxa is particularly sparse, we consider Burton's study to be an important addition to this analysis. Characters are listed and discussed in appendix 3.

da Silva's (1998) Dissertation

da Silva (1998) presented his Ph.D. dissertation on phylogeny of hylids with emphasis on Hylinae. Although da Silva's dissertation has not been published, some of its results and conclusions were described and commented in detail by Duellman (2001). Because the present paper deals specifically with the phylogeny of Hylinae, we cannot avoid a few comments dealing with da Silva's work. Considering the mostly coincident scope of both da Silva's dissertation and this paper, it is evident that a thorough discussion and comparison of his results with ours would almost amount to the publication of his chapter on Hylinae relationships. This is a situation with which we feel most uncomfortable, because we think that this is a responsibility that rests on Helio R. da Silva.

From a purely practical perspective, at this point the integration of da Silva's data set with ours is impracticable for two reasons: (1) The data matrix as printed in the dissertation distributed by the University of Michigan is incomplete, as it lacks the scorings for 10 characters (chars. 110–120) for all taxa. This is also the situation with the thesis that is deposited at the Department of Herpetology library of the University of Kansas, Natural History Museum (Faivovich, personal obs.). (2) A few scorings for groups that we are familiar with are not coincident with our observations on the same species, something suggestive either of polymorphism in those characters or mistaken scorings.21 If this were the case, it would not be surprising, as scoring mistakes are to be expected in such an impressive data set. The problem with them is that once detected, they have to be corrected and the analysis has to be redone. It is evident that a revision of the data set is necessary before any integration can take place.

Phylogenetic Analysis

Our optimality criterion to choose among trees is parsimony. The logical basis of parsimony as an optimality criterion has been presented by Farris (1983). However, parsimony has repeatedly been attacked from different perspectives, all of which tend to portray parsimony as inferior to such model-based approaches as maximum likelihood. Criticisms of parsimony have centered on two main topics: statistical inconsistency and the notion that parsimony is an overparameterized likelihood model. As stated by Goloboff (2003), the emphasis on statistical consistency decreased following several studies showing that: (1) maximum likelihood can be inconsistent even with minor violations of the model when they were generated with a mix of models (Chang, 1996); (2) given some evolutionary models, maximum likelihood estimators could be inconsistent (Steel et al., 1994; Farris, 1999); (3) parsimony can be consistent (Steel et al., 1993); (4) assuming likelihood as a more accurate method, inferences based on trees suboptimal under the maximum likelihood could be less reliable than inferences made on trees optimal under otherwise inferior but faster criteria (Sanderson and Kim, 2000); and (5) at least under some conditions, parsimony may be more likely than maximum likelihood to find the correct tree, given finite amounts of data (Yang, 1997; Siddall; 1998; Pol and Siddall, 2001). Tuffley and Steel (1997) demonstrated that parsimony is a maximum likelihood estimator when each site has its own branch length. Farris (1999, 2000) and Siddall and Kluge (1999) suggested that the results of Tuffley and Steel (1997) were an indication that the model implied by parsimony (“no special model of evolution” or “no common mechanism model”) was indeed more realistic. However, likelihood advocates (Steel and Penny, 2000; Lewis, 2001; Steel, 2002) countered that models that assume constant probabilities of change across all sites are to be preferred on the grounds of simplicity (i.e., as having fewer parameters to estimate). Goloboff (2003) demonstrated that parsimony could actually be derived from models that require even fewer parameters than the commonly used likelihood models.

The use of Bayesian Markov chain Monte Carlo (BMCMC) techniques has become quite popular among evolutionary biologists. However, for reasons outlined by Simmons et al. (2004), the posterior probability values of the clades cannot be interpreted as values of truth or support. Furthermore, Kolaczkowski and Thornton (2004) demonstrated, by using simulations in the presence of heterogeneous data, that parsimony performs better than both maximum likelihood and BMCMC over a wide range of conditions.

We contend that all serious criticisms of parsimony have been rebutted. We consider that while the first point mentioned (inconsistency of likelihood when the data are generated with different models) could certainly occur in any analysis, it is particularly problematic in the present one, because we are combining morphology with both mitochondrial and nuclear coding and non-coding genes. Furthermore, for a data set of this size, maximum likelihood is quite impractical to apply for computational reasons.

For the phylogenetic analyses of the DNA sequence data, we used the method of Direct Optimization (Wheeler, 1996, 1998, 2002), as implemented in the program POY (Wheeler et al., 2002), a heuristic approximation to the optimal tree alignment methods of Sankoff (1975) and Sankoff and Cedergren (1983). Sequence alignment and tree searching have traditionally been treated as two independent steps in phylogenetic analyses: sequences are first aligned, and a fixed or static multiple alignment is then treated as a standard character matrix that is the basis for tree searching in the test of character congruence. However, there may be other equally defensible multiple sequence alignments that would require fewer hypothesized transformations to explain the observed sequence variation; an explanation that requires fewer transformations is more parsimonious and is therefore objectively preferred over explanations that require a greater number of transformations (see De Laet [2005] for a much more sophisticated approach to the problems of constructing multiple alignments prior to tree searching). Direct Optimization seeks the cladogram-alignment combination (i.e., the optimal tree alignment) that minimizes the total number of hypothesized transformation events required to explain the observations. Within this framework, insertion/deletion events (indels, gaps) are historical evidence that is taken into account when hypothesizing common ancestry.

The simplest minimization of transformations is obtained when tree searches are conducted under equal weights for indels and all substitutions (1:1:1, this is the ratio of the cost of opening gap:extension gap:substitutions) (Frost et al., 2001). This weighting scheme implies that indels are as costly as the number of nucleotides they span. This is not a situation with which we are comfortable, inasmuch as a single deletion event could entail more than a single nucleotide and hence necessarily require a lower cost than if all the nucleotides it includes were lost independently of each other. However, theoretical justifications for the selection of differential costs for gap opening and gap extension are not evident.

De Laet and Smets (1998) suggested that parsimony analysis searches for the trees on which the highest number of compatible independent pairwise similarities can be accommodated; that is, they described parsimony as a two-taxon analysis. When dealing with static data sets, this approach and the minimization of transformations give the same rank of tress. However, De Laet (2005) showed that when considering parsimony as a two-taxon analysis in the presence of inapplicable character states (e.g., unequal-length sequences), the minimization of transformations (as obtained under 1:1:1) does not maximize the number of accommodated compatible independent pairwise a priori similarities. De Laet (2005) suggested that sequence homology has two components, homology of subsequences (the fragments of sequences that are comparable across a branch) and base-to-base homology within homologous subsequences. When maximization of homology is transformed into a problem of minimization of changes, the optimization of the two components that maximizes the accommodated independent pairwise similarities is obtained by summing up the cost regimes that are involved for each component. The number of subsequences is quantified by counting the number of insertion/deletion events (independent of their length, and therefore represented each as a whole by a unit opening gap). Base-to-base homology within homologous subsequences is maximized when substitutions are weighted twice as much as unit gaps (Smith et al., 1981). These result in a substitution cost of 2, a gap opening cost of 2 + 1 (the same cost of a substitution plus the cost of the first unit gap), and a gap extension cost of 1. All this development rests on the perspective of parsimony as a two-taxon analysis (De Laet and Smets, 1998). The most immediately appealing aspect of De Laet's perspective is that it offers a rationale for the use of gap-extension costs different from substitution costs, thus avoiding giving an insertion/deletion event of n nucleotides the same weight of n substitutions.

We conducted our searches using equal weights for minimizing transformations. In order to examine the effect of the gap treatment in our results, and following De Laet's development (2005), we also submitted our final tree to a round of tree-bisection and reconnection branch swapping (TBR) by using a weighting scheme of 2 for substitutions and morphological transformations, 3 for a gap opening, and 1 for a gap extension.

This study is guided by the idea that a simultaneous analysis of all available evidence maximizes explanatory power (Kluge, 1989; Nixon and Carpenter, 1996). Consequently, we analyzed all molecular and available morphological evidence simultaneously. The analysis was performed using subclusters of 60–100 processors of the American Museum of Natural History parallel computer cluster.

Heuristic algorithms applied to both tree searching and length calculation (i.e., alignment cost) were employed throughout the analysis. As with any heuristic solution, the optimal solution from these analyses under Direct Optimization represents the upper bound, and more exhaustive searching could result in an improved solution. Considering the large size of our data set, we tried two different approaches. The first strategy tries to collect many locally optimal trees from many replications to input them into a final round of tree fusing (Goloboff, 1999). For the second strategy, quick concensus estimates (Goloboff and Farris, 2001) are used as constraints for additional tree searching, following the suggestion of Pablo Goloboff (personal commun.).

For maximizing the number of trees for tree fusing, we employed two different routines:

  1. Three hundred fifty random addition sequences were done in groups of 5 or 10, followed by a round of tree fusing, sending the best tree to 10–25 parsimony ratchet cycles (Nixon, 1999a) using TBR, reweighting between 15 and 35% of the fragments, keeping one tree per cycle, and by setting the character weight multiplier between two and five in different replicates, with a final round of TBR branch swapping. Tree fusing was always done fusing sectors of at least three taxa with two successive rounds of fusing.

  2. One hundred fifty random addition sequences were built in groups of 5, 7, or 10 by submitting the best of each group to 10–25 ratchet cycles using TBR.

The 40 best trees resulting from these analyses where submitted to tree fusing in groups of five, and the resulting eight trees were subsequently fused. This final tree was submitted to 30 replicates of Ratchet using the same settings as above, with the resulting trees being submitted to a final round of TBR branch swapping.

Alternatively, we did 50 random addition sequences followed by a round of TBR and made an 85% majority rule consensus, as suggested by Goloboff and Farris (2001) to quickly estimate the groups actually present in the consensus of large data sets without having to do intensive searches. The approach of Goloboff and Farris (2001) assumes that groups that are present in all or most independent searches are more likely to be actually supported by the data. To speed up the searches for the estimation of the quick consensus, we treated the partial sequences of the RAG-1, rhodopsin, SIA, and tyrosinase genes as prealigned. Once the quick consensus was estimated, it was inputted in POY as a constraint file, with which we built 100 Wagner trees, each followed by 10 ratchet replicates. All trees resulting from these constrained searches were fused in groups of different size, and the final trees were submitted to a round of TBR. The original constraint file was not used during the fusing and final TBR steps.

While all searches were done using standard direct optimization, all were submitted to final rounds of TBR under the command “iterative pass” (Wheeler, 2003a). This routine does a three-dimensional optimization, taking into account the states of the three adjacent nodes of the internal node of interest. Because any change in the reconstructed sequence could potentially affect adjacent nodes, the procedure is done iteratively until stabilization is achieved.

The large size of the data set imposes a heavy burden in computer times to estimate support measures. Bremer supports (Bremer, 1988) were calculated using POY, without using “iterative pass”. Parsimony Jackknife values (Farris et al., 1996) were calculated using the implied alignment (Wheeler, 2003b) of the best topology. In turn, this implies that the parsimony jackknife values could be overestimated. Parsimony Jackknife was calculated in TNT (Goloboff et al., 2000); 1000 pseudoreplicates were performed. For each pseudoreplicate the best topology was searched for by using sectorial searches and tree fusing, starting with two Wagner trees generated through random addition sequences.

Final tree lengths under the 1:1:1 weighting scheme were checked with TNT. Lists of synapomorphies were generated with TNT; only unambiguous transformations common to all most parsimonious trees were considered.

For the analysis, the complete 12S-tRNA valine-16S sequence was cut into 14 fragments and the partial 28S sequence was cut into 4 fragments coincident with conserved regions (Giribet, 2001). Although this constrains homology assessment, the universe of alternative ancestral sequences that has to be explored is a more tractable problem than using long single fragments. The sequence files as they were input into POY are available from  http://research.amnh.org/users/julian. Tree editing was done using WinClada (Nixon, 1999b).

RESULTS

In total, we sequenced 256 terminals. The contiguous 12S, tRNA valine, and 16S genes were sequenced for all but seven terminals. For these terminals we were unable to amplify or sequence one or two of the overlapping PCR fragments. The partial cytochrome b fragment was sequenced for all but 12 terminals. The success with the nuclear loci varied from 232 terminals sequenced for the first exon of rhodopsin to as few as 166 sequenced for 28S. See appendix 2 for a complete list of the loci sequenced for each taxon, voucher specimens, locality data, and GenBank accessions. All sequences were produced for this project and for that of Faivovich et al. (2004) with the exception of 21 sequences taken from GenBank that were produced by Bijou and Bossuyt (2003) and by Darst and Cannatella (2004). The fact that the morphological characters are not scored for 70% of the terminals led to several ambiguous optimizations. A list of nonambiguous morphological synapomorphies is provided in appendix 3, many of which are mentioned throughout the discussion and in the section “Taxonomic Conclusions: A New Taxonomy of Hylinae and Phyllomedusinae”.

The phylogenetic analysis resulted in four most parsimonious trees of 65,717 steps. One of these trees resulted from one of the rounds of tree fusing of the trees resulting from the constrained search, and the other three trees were obtained after a round of TBR swapping of the first one. Parsimony Jackknife and Bremer support values are generally high. Most of the 272 nodes of the strict consensus (figs. 1–5) are well supported, with 226 nodes having a Bremer support of ≥10 and 162 nodes with a Bremer support of ≥20; additionally, 255 nodes have a jackknife value of ≥75% and 245 nodes have a jackknife value of ≥90%.

All conflict among the trees is restricted to two points: (1) the relationships among Hyla circumdata, H. hylax, and the undescribed species Hyla sp. 4 (fig. 3); and (2) the relationships of H. femoralis with the H. versicolor and H. eximia groups (fig. 5).

When the best trees were submitted to a round of TBR using a weighting scheme of 3:1:2 as suggested by De Laet (in press) and mentioned earlier, the resulting tree differs from the original ones only in that (1) the clade composed of Cryptobatrachus and Stefania moves to be the sister group of Flectonotus and Gastrotheca (fig. 2), and (2) the clade composed of Lysapsus, Pseudis, and Scarthyla moves from the sister taxon of Scinax to the sister taxon of the 30-chromosome Hyla groups, Sphaenorhynchus, and Xenohyla (fig. 4).

DISCUSSION

Major Patterns of Relationships of Hylidae and Outgroups

As in previous analyses (Ruvinsky and Maxson, 1996; Haas, 2003; Darst and Cannatella, 2004), our results do not recover Hylidae as a monophyletic taxon (figs. 1, 2). Hemiphractinae appears as only distantly related to the Hylinae, Pelodryadinae, and Phyllomedusinae, each of which is monophyletic. For this reason, we exclude Hemiphractinae from Hylidae, being thereby restricted to Hylinae, Pelodryadinae, and Phyllomedusinae. In the same way, Centrolenidae, for a long time suspected to be related with hylids, appears as a distantly related clade, as suggested by previous studies (Haas, 2003; Darst and Cannatella, 2004).

Hylidae, as understood here, excludes the Hemiphractinae. Otherwise, the major clades within the Hylidae are coincident with the remaining subfamilies currently recognized. The Pelodryadinae is the sister taxon of Phyllomedusinae, corroborating the results of Darst and Cannatella (2004); in turn, Pelodryadinae + Phyllomedusinae is the sister taxon of Hylinae (figs. 1, 2).

Ranoids appear as monophyletic, with the two microhylid exemplars being the sister taxon of the Astylosternidae + remaining ranoids (fig. 2). Ranidae forms a paraphyletic melange, with the exemplars of Hemisotidae, Mantellidae, and Rhacophoridae being nested among the few ranid exemplars. Ranoids are the sister taxon of all remaining terminals (fig. 2).

Within hyloids, as expected, Leptodactylidae is rampantly paraphyletic, with all other included families nested within it (figs. 1, 2). Ceratophryinae, Eleutherodactylinae, Leptodactylinae, and Telmatobiinae are not monophyletic (fig. 2).

At the base of hyloids, the two exemplars of Eleutherodactylus are the sister taxon of a clade composed of Hemiphractus helioi, Brachycephalus ephippium, and Phrynopus sp. This situation renders Eleutherodactylinae and Hemiphractinae nonmonophyletic (fig. 2). The nonmonophyly of Hemiphractinae is further given by the fact that in the 1:1:1 analysis, Stefania + Cryptobatrachus and Gastrotheca + Flectonotus are not monophyletic but occur as a grade leading to the other hyloids (fig. 2); however, the group is monophyletic in the 3:1:2 analysis. Moving upward in the tree finds two large clades: one composed of Hylidae in the sense used here (i.e., excluding Hemiphractinae), and the other composed of the remaining hyloid families and subfamilies of Leptodactylidae. Leptodactylinae as defined by Laurent (1986) is not monophyletic in that Limnomedusa is only distantly related to the remaining “Leptodactylinae”, being the sister taxon of Odontophrynus. Note that this arrangement is congruent with Leptodactylinae as defined by Lynch (1971). Centrolenidae obtains as monophyletic and as the sister taxon of Allophrynidae. The only cycloramphine exemplar, Crossodactylus schmidti, is the sister taxon of the dendrobatid exemplars.

Telmatobiinae is not monophyletic for having one of the Ceratophryinae exemplars, Ceratophrys cranwelli, nested within it. Furthermore, the other two Telmatobiinae exemplars, Alsodes gargola and Euspsophus calcaratus, form a clade with the Leptodactylinae exemplar Limnomedusa macroglossa and the other Ceratophryinae exemplar Odontophrynus americanus. This clade is also the sister taxon of all Bufonidae exemplars (fig. 2).

In general, most results concerning the relationships among outgroup taxa should be considered cautiously, because the taxon sampling of this analysis was not designed to address those specific questions. Some results are nonetheless expected or at least suggestive. In the former group we include, for example, the monophyly of Bufonidae, Dendrobatidae, Centrolenidae, and Ranoidea. The relationship between Crossodactylus and dendrobatids is consistent with the results of Haas (2003).

The fact that hemiphractines are not related to the Hylidae, and are likely nonmonophyletic, requires a change in how study of this group is approached. For instance, relationships of Hemiphractinae as recovered here are quite different from the results of the cladistic analysis based on morphology and life-history data presented by Mendelson et al. (2000). These authors found Hemiphractus to be nested within Gastrotheca, a result that Duellman (2001) considered implausible. Although we did not incorporate their data set into our analysis, the fact that they employed only hylid outgroups could have affected their results. With the important difference that we do not recover a monophyletic Hemipractinae, our results corroborate the sister taxon relationship between the northern Andean Cryptobatrachus and the Guayanan Stefania, as well as the sister group relationship between Flectonotus and Gastrotheca, as suggested by Duellman and Hoogmoed (1984) and Wassersug and Duellman (1984). Regarding the monophyly and actual position of Hemiphractinae within Neobatrachia, our results are inconclusive (similar to those of Darst and Cannatella, 2004) most likely because of a lack of the appropriate taxon sampling to address the problem. Their positions in the tree suggest that a much denser taxon sampling of “Leptodactylidae” and perhaps Eleutherodactylinae will be necessary to better understand their relationships.

Pelodryadinae and Phyllomedusinae

Our results of a monophyletic Pelodryadinae + Phyllomedusinae corroborate early suggestions by Trewavas (1933), Duellman (1970), Bagnara and Ferris (1975), and more recent results by Darst and Cannatella (2004) and Hoegg et al. (2004). The monophyly of Pelodryadinae and Phyllomedusinae was not recovered in the analyses by Duellman (2001), Burton (2004), and Haas (2003). Discrepancies between the analyses of Burton (2004) and Haas (2003) and our's may be the result of different taxon sampling and assumptions. Duellman (2001) assumed that Hylidae, in the classical sense (including Hemiphractinae), was monophyletic, and Burton (2004) assumed that Hylidae, Centrolenidae, and Allophrynidae formed a monophyletic group. Our analysis did not include the morphological characters employed by Haas (2003) in his analysis, nor does our pelodryadine taxon sampling match his. For this reason, our results are not directly comparable to his, and we do not consider the monophyly of the Pelodryadinae a settled issue.

In our analysis, the presence of a tendon of the m. flexor ossis metatarsi II arising only from distal tarsal 2–3 is a synapomorphy of Pelodryadinae plus Phyllomedusinae. Furthermore, the presence of the pigment pterorhodin (Bagnara and Ferris, 1975) may be a synapomorphy of this clade, although the distribution of this character state requires further elucidation. Both Pelodryadinae and Phyllomedusinae share the presence of supplementary elements of the m. intermandibularis. These elements are apical in Pelodryadinae and posterolateral in Phyllomedusinae (Tyler, 1971). Both character states have previously been considered nonhomologs (Tyler and Davies, 1978a) that separately support the monophyly of each of these groups (Duellman, 2001). In the context of our analysis, however, the sole presence of supplementary elements is more parsimoniously interpreted as a putative synapomorphy of this clade, while it is ambiguous which of the positions of the elements (apical or posterolateral) is the plesiomorphic state. Note that this ambiguity is a potential challenge to the only known morphological synapomorphy of Pelodryadinae. Future anatomical work will corroborate whether these two morphologies could be considered as states of the same transformation series, as is tentatively being done here.

Pelodryadinae

As stated previously, our analysis does not include enough of a comprehensive taxon sampling of Pelodryadinae to address its internal relationships in a meaningful way. Nonetheless, our results corroborate the long-held idea (see previous discussions) that Cyclorana and Nyctimystes are nested within Litoria. For the reasons detailed earlier, our analysis is not an overly strong test of the positions of the former two genera within Litoria. Nevertheless, the single exemplar of Cyclorana is the sister taxon of L. aurea, an exemplar of the L. aurea group with which Cyclorana is supposed to be related based on various sources of evidence (King et al., 1979; Tyler, 1979; Tyler et al., 1981). Nyctimystes is the sister taxon of L. infrafrenata, one of the groups of Litoria that Tyler and Davies (1979) considered as possibly related to Nyctimystes based on morphological similarities of its skull with that of N. zweifeli. The other groups they considered are mostly the montane species of Litoria; from these we included a single exemplar, L. arfakiana, that is quite distant from Nyctimystes, being the sister taxon of L. meiriana (with which, incidentally, it also shares the presence of a flange in the medial surface of metacarpal III; Tyler and Davies, 1978b). In our analysis, the fibrous origin of the m. extensor brevis superficialis digiti III on the distal end of the fibulare is a synapomorphy of Pelodryadinae; we are skeptical, however, that this optimization will hold with better sampled outgroups for muscular characters, because Burton (2004) found the same character state in several leptodactylids, none of which is included in our outgroup sample.

Phyllomedusinae

Several authors (Funkhouser, 1957; Duellman, 1970; Donnelly et al., 1987; Hoogmoed and Cadle, 1991) noticed the distinctiveness of Agalychnis calcarifer and its presumed sister taxon, A. craspedopus, from the other species of Agalychnis. Corroborating the results of Duellman (2001), we found no evidence for the monophyly of Agalychnis. Our results indicate that A. calcarifer is the sister group of the remaining Phyllomedusinae, and it has no close relationship with the other exemplars of Agalychnis.

Phyllomedusa lemur, the only exemplar of the P. buckleyi group available for this analysis, is recovered, although with low Bremer support (3), as the sister group of Hylomantis, and it is only distantly related with the other exemplars of Phyllomedusa. This situation corroborates previous suggestions (Funkhouser, 1957; Cannatella, 1980; Jungfer and Weygoldt, 1994) that the P. buckleyi group should not be included in Phyllomedusa. Cruz (“1988” [1989]) suggested, on the basis of iris coloration, skin texture, poor development of webbing, and slender body, that Hylomantis is related to two species of the P. bukleyi group, P. buckleyi and P. psilopygion. On the basis of the same character states, Cruz (1990) associated the P. buckleyi group with both Hylomantis and Phasmahyla. Our results support these ideas only in part, because while our only exemplars of Hylomantis and the P. buckleyi group are each monophyletic, Phasmahyla is more closely related to Phyllomedusa (excluding the P. buckleyi group).

We had no clear idea regarding the position of Hylomantis and Phasmahyla. Morphologically, the evidence is conflicting in that each one shares at least one different possible synapomorphy with the restricted Phyllomedusa (Phyllomedusa excluding the P. buckleyi group). Phasmahyla has the same type of nest where the eggs are wrapped in a leaf; nests are unknown in Hylomantis, but species of this genus share with Phyllomedusa (excluding the P. buckleyi group) the presence of the slip of the m. depressor mandibulae that originates from the dorsal fascia at the level of the m. dorsalis scapulae (Cruz, 1990; Duellman et al., 1988b), a character state that is absent in all other Phyllomedusinae. Our analysis recovers Phasmahyla as the sister group of the restricted Phyllomedusa, suggesting that the eggs wrapped in a leaf are a synapomorphy of this clade.

Major Patterns of Relationships Within Hylinae

For purposes of discussion, we consider Hylinae to be composed of four major clades (fig. 1), called here: (1) the South American clade I; (2) South American clade II (SA-II); (3) Middle American/Holarctic clade; and (4) South American/West Indian Casque-headed Frogs. These major sections and their subclades will be discussed in this order.

South American Clade I

This clade is composed of all Gladiator Frogs, the Andean stream-breeding Hyla, the genus Aplastodiscus, and a Tepuian clade of Hyla. It contains five major clades (fig. 3). The first of these is called the Tepuian clade, and is composed solely of two exemplars of the H. aromatica and H. geographica groups. The second clade is composed of all Andean stream-breeding Hyla. The third is composed of all the exemplars of the H. circumdata, H. martinsi, and H. pseudopseudis groups, from southeastern Brazil, and we are calling it informally the Atlantic/Cerrado clade. The fourth is composed of the southeastern Brazilian H. albosignata and H. albofrenata complexes of the larger, nonmonophyletic H. albomarginata group plus the two species of Aplastodiscus, and we are calling it informally the Green clade. The fifth clade is composed of all the remaining species groups (H. geographica, H. pulchella, H. boans, H. granosa, H. punctata, H. albomarginata complex of the H. albomarginata group) and unassigned species associated in the past with the Gladiator Frogs, and we are calling it informally the TGF clade (for True Gladiator Frogs.)

Six currently recognized species groups within the South American clade I are not monophyletic. The Hyla albomarginata group is not monophyletic because its three “complexes” defined by Cruz and Peixoto (“1985” [1987]) are spread throughout the Green clade and the TGF clade. The H. albomarginata complex is not monophyletic, with its species being related with different groups in the TGF clade (see below). The H. albosignata complex is monophyletic, as is the H. albofrenata complex. These two complexes, however, do not form a monophyletic group, because Aplastodiscus is the sister group to the H. albosignata complex, and this clade is sister to the H. albofrenata complex. Within the TGF Clade, the only group that is not represented by a single exemplar (the Hyla punctata group) that results as monophyletic is the H. pulchella group. The H. albomarginata complex, H. albopunctata, H. boans, H. geographica, and H. granosa groups are nonmonophyletic.

Hyla pellucens and H. rufitela are the sister group of the clade composed of H. heilprini and the paraphyletic H. albopunctata group (see below); H. albomarginata is the sister taxon of a fragment of the H. boans group (see below). The H. albopunctata group is paraphyletic inasmuch as H. fasciata plus H. calcarata is nested within it. The H. boans group is polyphyletic because the mostly southeastern Brazil/northeastern Argentina exemplars (H. faber, H. lundii, H. pardalis, H. crepitans, and H. albomarginata) together with H. albomarginata are the sister taxon of the H. pulchella group and are only distantly related to H. boans. Hyla boans is the sister taxon of H. geographica plus H. semilineata. The H. geographica group is rampantly polyphyletic, with its exemplars partitioned into five different clades within the South American clade I: (1) H. kanaima is the sister taxon of H. inparquesi, the single exemplar of the H. aromatica group; (2) H. roraima and H. microderma form a monophyletic group with four Guayanese and one Amazonian species; (3) Hyla picturata is related to the exemplars of the H. punctata and H. granosa groups; (4) Hyla semilineata + H. geographica are related to H. boans; and (5) H. fasciata + H. calcarata are nested within the H. albopunctata group as detailed above. The H. granosa group is paraphyletic by having H. picturata and H. punctata nested within it.

Andean Stream-Breeding Hyla and the Tepuian Clade

The monophyly of the Andean stream-breeding Hyla is congruent with suggestions presented by Duellman et al. (1997) and Mijares-Urrutia (1997), who noticed similarities in larval morphology of the H. bogotensis and H. larinopygion groups. Duellman et al. (1997) presented a phylogenetic analysis restricted to wholly or partially Andean species groups of Hyla. In their most parsimonious tree, the H. armata, H. bogotensis, and H. larinopygion groups formed a monophyletic group supported by three transformations in tadpole morphology: the enlarged, ventrally oriented oral disc; the complete marginal papillae; and a labial tooth row formula 4/6 or higher.

Duellman et al. (1997) suggested a close relationship between the H. armata and H. larinopygion groups based on the presence in males of a greatly enlarged prepollex lacking a projecting spine. While our results are congruent with this, note that males of the H. bogotensis group also have a prepollex with the same external morphology as those of the H. armata and H. larinopygion groups. Kizirian et al. (2003) suggested that the H. armata group was nested in the H. larinopygion group. Our results do not support this suggestion. However, this could be a consequence of the few exemplars of the H. larinopygion group available for our study.

Kizirian et al. (2003) had doubts about the placement of Hyla tapichalaca. Faivovich et al. (2004) showed that this species is related to the H. armataH. larinopygion groups (although they only included H. armata in their analysis). Our results go a step further, indicating a closer relationship with the H. larinopygion group.

Hyla inparquesi and H. kanaima forming the sister taxon of all the remaining South American clade I is an unexpected result. On the basis of morphology, we expected our only exemplar of the H. aromatica group, H. inparquesi22, to be related to the Andean stream-breeding clade of Hyla, because both share the character states that Duellman et al. (1997) suggested as synapomorphies in support of the monophyly of the Andean stream-breeding Hyla: (1) known larvae with ventral, enlarged oral discs; (2) complete marginal papillae; and (3) with a minimum labial tooth row formula of 4/6. Furthermore, adults of the H. aromatica group share a greatly enlarged prepollex without a projecting spine in males that, as we mentioned above, is present in most species of Andean stream-breeding Hyla (the only known exception being H. tapichalaca; Kizirian et al., 2003).

This sister-group relationship between the Tepuian clade and all the remaining groups of the South American clade I has further implications. Based on the available material, Duellman et al. (1997) considered the prepollex greatly enlarged without a projecting spine to be an intermediate state in an ordered transformation series from prepollex not greatly enlarged to prepollex greatly enlarged with a projecting spine. Our topology implies that the greatly enlarged prepollex without a projecting spine is a synapomorphy of the whole South American clade I (with subsequent transformations, including the development of a projecting spine). However, H. kanaima does not have an enlarged prepollex as prominent as that found in the H. armata, H. aromatica, H. bogotensis, and H. larinopygion groups. In order to clarify this situation, it would be necessary to (1) define the prepollex character states osteologically, and (2) include a denser sampling of the H. aromatica group, to better understand its relationship with H. kanaima. (Perhaps the character state of H. kanaima could be interpreted as a reversal.)

Our topology implies an interesting scenario regarding the evolution of larval morphology in the South American clade I; that is, that larval morphology of the Atlantic/ Cerrado, Green, and TGF clades evolved from an ancestor with the highly modified morphology typical of stream larvae (including large numbers of labial tooth rows, a large oral disc with complete marginal papillae, and relatively low fins) that during evolution, underwent a transformation of these character states (specifically, reduction in labial tooth row formulae, a reduced oral disc, and formation of an anterior gap in the marginal papillae). These transformations were coincident with distributional shifts from high-elevation mountain streams (as in the Tepuian and Andean stream-breeding clades) toward lower elevation forest mountain streams (the cases of the Atlantic/Cerrado clade, the Green clade, and, and some taxa of the TGF clade), and Amazonian lowlands and the Cerrado-Chaco (several taxa of the TGF clade).

Gladiator Frogs

Duellman et al. (1997) suggested the existence of a clade composed of the Hyla albomarginata, H. albopunctata, H. boans, H. circumdata, H. geographica, and H. pulchella groups. The synapomorphy supporting this clade is, according to these authors, the presence of “an enlarged prepollical spine lacking a quadrangular base”. Faivovich et al. (2004), based on the analysis of mitochondrial DNA sequences and on the observation (Garcia and Faivovich, personal obs.) that H. punctata and the H. polytaenia group show the same morphology of the prepollical spine, argued that these additional groups also belong to that clade (these authors further included the H. polytaenia group within the H. pulchella group), which was called earlier in this paper “Gladiator Frogs”. Our results indicate that a clade with the composition suggested by Duellman et al. (1997) and Faivovich et al. (2004) is paraphyletic because Aplastodiscus is nested within it; furthermore, H. kanaima of the H. geographica group is only distantly related to this clade.

Atlantic/Cerrado Clade

Our results corroborate the long-suspected association of the Hyla circumdata group with the groups of H. pseudopseudis and H. martinsi (Bokermann, 1964a; Cardoso, 1983; Caramaschi and Feio, 1990; Pombal and Caramaschi, 1995), even though the monophyly of these two groups could not be tested by our analysis. We also consider as corroborated the suspected relationship of the Hyla circumdata and H. pseudopseudis groups with H. alvarengai (Bokermann, 1964a; Duellman et al., 1997), because Hyla sp. 9 (aff. H. alvarengai) is nested in this clade.

Unfortunately, due to the unavailability of samples, we could not test the relationships of the Hyla claresignata group with this clade. Considering the phylogenetic context of the Atlantic/Cerrado clade within the South American clade I, we must revisit the apparent synapomorphies of the H. claresignata group mentioned earlier (oral disc completely surrounded by marginal papillae, and 7/12–8/ 13 labial tooth rows). The presence and distribution of these character states in the Tepuian and Andean stream-breeding Hyla clades is suggestive, not of a closer relationship of the H. claresignata group with any of them (these character states are plesiomorphies in this context), but of the nature of its relationship with the Atlantic/Cerrado clade. Perhaps the H. claresignata group is not a member of the Atlantic/Cerrado clade, but is a basal group related either to the Tepuian or Andean stream-breeding clade, or perhaps it is even the sister taxon of the clade composed of the Atlantic/Cerrado, Green, and TGF clades. At this point we are not aware of evidence favoring any of these alternatives.

Green Clade

Lutz (1950) was the first to suggest that Aplastodiscus was related to species latter included in the Hyla albosignata complex, as defined by Cruz and Peixoto (“1985” [1987]). This was supported by Garcia et al. (2001a) based on the presence of enlarged internal metacarpal and metatarsal tubercles and unpigmented eggs. More recently, Haddad et al. (2005) described the reproductive mode of Aplastodiscus perviridis, which includes egg deposition in a subterranean nest excavated by the male, where exotrophic larvae spend the early stages of development until flooding releases them to a nearby water body. This mode is the same as that observed in one species of the H. albosignata complex, H. leucopygia (Haddad and Sawaya, 2000), and for the undescribed species of the H. albofrenata complex included in our analysis, Hyla sp.1 (aff. H. ehrhardti) (Hartmann et al., 2004); this mode is further suspected to occur in all species of both the H. albosignata and H. albofrenata complexes (Haddad and Sawaya, 2000, Hartmann et al., 2004). Species included in the H. albomarginata complex instead lay their eggs on the water film surface (Duellman, 1970). Based on the shared reproductive mode, Haddad et al. (2005) suggested that Aplastodiscus could be related to the H. albofrenata and H. albosignata complexes. Our results support the monophyly of both the H. albofrenata and H. albosignata complexes and their close relationship with Aplastodiscus, that is the sister taxon of the H. albosignata complex.

While we are not aware of nonmolecular synapomorphies for several nodes supported by molecular evidence, the few osteological data available for the South American clade I indicate that the Green clade and the TGF clade share the presence of transverse processes of the sacral diapophyses notably expanded distally, while species of the Atlantic/ Cerrado clade (Bokermann, 1964a; Garcia 2003) and H. tapichalaca (Kizirian et al., 2003), the only Andean stream-breeding Hyla with any described postcranial osteology, have the transverse processes poorly expanded or not expanded at all. However, the distribution of this character state is in conflict with the presence of a prepollical spine in the Atlantic/Cerrado clade and the TGF clade, which is absent in the Green clade, where there is a fairly enlarged prepollex but no spine. A detailed study of prepollex morphology in these taxa would help to better define the relevant transformation series and it transformation sequences in the tree.

True Gladiator Frog Clade

The results imply the nonmonophyly of several species groups within this clade, as described earlier. This situation is not unexpected considering the paucity of evidence of monophyly previously available for most of them.

The monophyly of the group composed of the two unassigned species from the Guayana Highlands, Hyla benitezi, H. lemai, Hyla sp. 2, two members of the H. geographica group (H. microderma and H. roraima), and Hyla sp. 8 is further supported by the presence of a mental gland in males (Faivovich et al., in prep.)

In the DNA-based phylogenetic analysis of the Hyla pulchella group performed by Faivovich et al. (2004), H. punctata is the sister species of H. granosa, with this overall clade forming the sister taxon of a clade composed of the H. albopunctata, H. geographica, H. albomarginata, H. boans, and H. pulchella groups. In our analysis, H. punctata, our only exemplar of the group, is the sister taxon of H. granosa, and this taxon is at the apex of a pectinated series that includes H. sibleszi and, curiously, H. picturata. Prior to the analysis, we had no idea as to with which species group H. picturata would be related, but certainly we did not expect this colorful frog to be nested within a group of green species.

Our results lend only partial support for a relationship between Hyla heilprini and the H. albomarginata group, as tentatively suggested by Duellman (1974) and Trueb and Tyler (1974) based on overall pigmentation and the white peritoneum, because H. heilprini is the sister taxon of the H. albopunctata group (including a fragment of the H. geographica group), with H. heilprini plus this unit being the sister taxon of a fragment of the H. albomarginata group. The nonmonophyly of the H. albopunctata group corroborates comments advanced by de Sá (1995, 1996) as to the lack of evidence for its monophyly.

Hyla crepitans, H. faber, H. lundii, and H. pardalis form, together with H. albomarginata, a monophyletic group only distantly related to H. boans, the species that gives the name to the former group. Within this clade, the nest builders23 H. faber, H. lundii, and H. pardalis, are monophyletic.

The polyphyly of the Hyla boans group has implications for the evolution of reproductive modes, in that it implies independent origins of nest-building behavior by males. Although theoretically possible, certainly no author had ever suggested that such a characteristic behavior as the nest building could be a homoplastic feature.24 However, the molecular evidence points that way, and further evidence indicates that this or a similar reproductive mode also occurs in at least some species of the H. circumdata group (Pombal and Haddad, 1993, Pombal and Gordo, 2004), implying then at least three independent occurrences within the South American clade I. In a wider context, nest building was reported as well in Pelodryadinae in males of Litoria jungguy (Richards, 1993; using the name L. lesueuri, see Donnellan and Mahony [2004]), thus implying a fourth instance of homoplasy within Hylidae.

Our topology for the Hyla pulchella group is identical to that of Faivovich et al. (2004), including the exemplars of the former H. polytaenia group nested within it. Following Faivovich et al. (2004), we continue to recognize a H. polytaenia clade within the H. pulchella group. These authors stated that the lack of any pattern on the hidden surfaces of thighs was one of two possible morphological synapomorphies of this clade (with the other being the mostly striped dorsal pattern). This observation is mistaken, because the same character state occurs in H. ericae (Caramaschi and Cruz, 2000), H. joaquini, H. marginata, H. melanopleura, H. palaestes, and H. semiguttata (Duellman et al., 1997; Garcia et al., 2001b), suggesting that it actually may be a synapomorphy of a more inclusive clade whose contents are still undefined.

South American II Clade

The South American II clade (fig. 4) is composed of the 30-chromosome Hyla, the Hyla uruguaya group, Lysapsus, Pseudis, Scarthyla, Scinax, Sphaenorhynchus, and Xenohyla. It contains two main clades: one composed of Lysapsus, Pseudis, Scarthyla, and Scinax (including the H. uruguaya group), and the other composed of Sphaenorhynchus, Xenohyla, and all the exemplars of 30-chromosome Hyla species groups.

Within this clade, Scinax and the Hyla microcephala group are not monophyletic. Scinax has H. uruguaya, an exemplar of the H. uruguaya group, nested within it. The H. microcephala group is paraphyletic with respect to the available exemplars of the H. decipiens and H. rubicundula groups.

Relationships of Scinax

Several hypotheses have been advanced on the relationships of Scinax. Aparasphenodon was considered by Trueb (1970a) to be closely related to Corythomantis and, in turn, she considered these two genera to be nested within the (then) Hyla rubra group (currently the genus Scinax), based on overall similarities in cranial morphology. Scarthyla was considered to be related to Scinax by Duellman and de Sá (1988). Duellman and Wiens (1992) extended this to suggest that Scarthyla is the sister taxon of Scinax, which together form the sister taxon of Sphaenorhynchus. According to Duellman and Wiens (1992), character states supporting the monophyly of these three genera are narrow sacral diapophyses, anteriorly inclined alary processes of the premaxillae, and tadpoles with large, laterally placed eyes. Duellman and Wiens (1992) suggested that possible morphological synapomorphies of Scinax and Scarthyla are reduced webbing on the hand and the presence of an anterior process of the hyale. Tepuihyla was suggested to be closely related with Scinax, this being supported by the absence or extreme reduction of webbing between toes I and II, adhesive discs wider than long, and the presence of double-tailed sperm (Ayarzagüena et al., “1992” [1993b]). This association was questioned by Duellman and Yoshpa (1996) on the grounds that the absence or extreme reduction of webbing between toes I and II was homoplastic among hylids (although Duellman and Wiens [1992] suggested this same character state to be a synapomorphy of Scinax). These authors suggested that the only evidence uniting Tepuihyla with Scinax could be the double-tailed sperm reported by Ayarzagüena et al. (“1992” [1993b]) for Tepuihyla and by Fouquette and Delahoussaye (1977) for Scinax.25

Mijares-Urrutia et al. (1999) again suggested a close relationship of Tepuihyla with Scinax, but also with Scarthyla and Sphaenorhynchus– Scinax relatives, as suggested by Duellman and Wiens (1992). Mijares-Urrutia et al. (1999) also noted that Tepuihyla has rounded sacral diapophyses as found in these three genera (Duellman and Wiens, 1992).

Our results do not support a relationship of Scinax with Aparasphenodon, Corythomantis, or Tepuihyla because these three genera are nested within the South American/ West Indies Casque-headed Frog clade. Furthermore, Scinax is not the sister group of Scarthyla but of a clade composed of Scarthyla plus Lysapsus and Pseudis (in the 1:1: 1 analysis) or of all the remaining genera included in the South American II clade (in the 3:1:2 analysis).

Scinax is also paraphyletic with respect to the Hyla uruguaya group, for which Bokermann and Sazima (1973a) and Langone (1990) could not suggest affinities with any other hylids. Most recently, Kolenc et al. (“2003” [2004]) observed in the larvae of H. uruguaya and H. pinima the morphological synapomorphies of the larvae of the Scinax ruber clade that were reported by Faivovich (2002), and they suggested a possible relationship between the H. uruguaya group and the Scinax ruber clade. Adults of the H. uruguaya group are quite characteristic morphologically, and perhaps as a consequence this group was never associated with any species of Scinax prior to Kolenc et al. (“2003” [2004]).

Our results reveal that none of the outgroups employed by Faivovich (2002) in the phylogenetic analysis of Scinax is particularly close to Scinax; instead, all other components of the South American II clade are much more suitable to establish character-state polarities in this genus. Consequently, exemplars of these closer neighbors of Scinax need to be added, and the synapomorphies of Scinax resulting from that analysis need to be reevaluated.

Lysapsus, Pseudis, and Scarthyla

The sister-group relationship between Scarthyla goinorum and “pseudids” (Lysapsus + Pseudis), and this group being nested within Hylinae, corroborates recent findings by Darst and Cannatella (2004) and Haas (2003). Burton (2004) reported a likely synapomorphy for the Scarthyla plus the “pseudid” clade that is corroborated in the present analysis; that is, the m. transversus metatarsus II oblique, with a narrow, proximal connection to metatarsus II, and a broad, distal connection to metatarsus III. Another character state described by Burton (2004), the undivided tendon of the m. flexor digitorum brevis superficialis, optimizes in this analysis as a synapomorphy of this clade plus Scinax.

Besides the molecular data, the monophyly of Lysapsus plus Pseudis is further supported by the tendo superficialis pro digiti III arising from the m. flexor digitorum brevis superficialis, with no contribution from the aponeurosis plantaris; the origin of m. flexor ossis metatarsi IV and the joint tendon of origin of mm. flexores ossum metatarsorum II and III crossing each other; the m. flexor ossis metatarsi IV very short, inserting on the proximal two-thirds of metatarsal IV or less; absence of a tendon from the m. flexor digitorum brevis superficialis to the medial slip of the medial m. lumbricalis brevis digiti V; and m. transversus metatarsus III oblique, with a narrow, proximal connection onto metatarsal III, and a broad, distal connection to metatarsal IV. Another likely morphological synapomorphy is the elongated intercalary elements.

Vera Candioti (2004) noticed that Lysapsus limellum and most of the 30-chromosome Hyla (H. nana and H. microcephala) studied by her and Haas (2003) share two of the synapomorphies that Haas (2003) reported for Pseudis paradoxa and P. minuta: insertion of the m. levator mandibulae lateralis in the nasal sac, and a distinct gap in the m. subarcualis rectus II–IV. Haas (2003) observed different character states for H. ebraccata (the m. levator mandibulae lateralis inserts in tissue close to posterodorsal process of suprarostral cartilage or adrostral tissue; continuous m. subarcualis rectus II–IV), suggesting the need for additional studies on its taxonomic distribution within the 30-chromosome Hyla and in the other genera of the South American II clade.

Sphaenorhynchus, Xenohyla and the 30-Chromosome Hyla

Izecksohn (1959, 1996) suggested possible relationships of Xenohyla with Sphaenorhynchus and Scinax (Izecksohn, 1996). These ideas are partially corroborated by our 1:1:1 results, with the exception that they also suggest that Xenohyla is the sister group of the 30-chromosome Hyla. The karyotype is still unknown in Xenohyla, and this poses an obstacle to our understanding of the limits of the 30-chromosome Hyla. Interestingly, while both Sphaenorhynchus and Xenohyla do have a quadratojugal, in both cases it does not articulate with the maxilla (Duellman and Wiens, 1992; Izecksohn, 1996), which could be seen as an intermediate step before the extreme reductions of the quadratojugal seen in the 30-chromosome Hyla (Duellman and Trueb, 1983). Tadpoles of Xenohyla and several species of 30-chromosome Hyla share the presence of the tail tip extended into a flagellum, as well as the presence of high caudal fins (e.g., see Bokermann, 1963; Kenny, 1969; Gomes and Peixoto, 1991a, 1991b; Izecksohn, 1996; Peixoto and Gomes, 1999).

Phylogenetic hypotheses of the 30-chromosome Hyla species groups using morphological characters were presented by Duellman and Trueb (1983), Duellman et al. (1997), Kaplan (1991, 1994), and Kaplan and Ruíz (1997); none of these tested the monophyly of the contained species groups. A summary of their proposals and the supporting evidence are depicted in figure 6.

Chek et al. (2001) presented a phylogenetic analysis using partial 16S and cytochrome b sequences of the Hyla leucophyllata group, including exemplars of other 30-chromosome Hyla species groups. Because they did not include non-30-chromosome hylids, they did not test the monophyly of this clade.

The distribution of certain characters in several species associated with the currently recognized species groups suggests problems in our phylogenetic understanding of these frogs. The monophyly of a group composed of the Hyla leucophyllata, H. marmorata, H. microcephala, and H. parviceps groups is currently supported by the absence of labial tooth rows in their larvae (Duellman and Trueb, 1983). However, within the H. parviceps group, H. microps (Santos et al., 1998) and H. giesleri (Bokermann, 1963; Santos et al., 1998) have at least one labial tooth row. Similarly, Gomes and Peixoto (1991a) and Peixoto and Gomes (1999) noticed in the H. marmorata group the presence of one labial tooth row in the larvae of H. nahdereri, H. senicula, and H. soaresi. Based on these facts and similarities in tail depth, tail color, general body shape, and predatory habits, they suggested that the H. marmorata group could instead be more closely related to H. minuta than to the groups suggested by Duellman and Trueb (1983). Gomes and Peixoto (1991b) pointed out the presence of a labial tooth row in the larva of H. elegans. Wild (1992) further noted the absence of marginal papillae (an apparent synapomorphy of the H. microcephala group) in the larva of H. allenorum (a species of the H. parviceps group).

The reproductive modes of the different species are also informative. According to Duellman and Crump (1974), Hyla parviceps deposits its eggs directly in the water, as does H. microps (Bokermann, 1963), whereas H. bokermanni and H. brevifrons oviposit on leaves overhanging ponds; upon hatching, the tadpoles drop into the water where they complete development. Hyla ruschii oviposits on leaves overhanging streams (Weygoldt and Peixoto, 1987). The oviposition on leaves occurs in most species of the H. leucophyllata group, whereas both reproductive modes occur in the H. microcephala group.

Our results recover the 30-chromosome Hyla species as monophyletic; however, our topology differs from previous hypotheses. In our topology, the root is placed between the H. marmorata group and the other exemplars, instead of between the H. labialis group and the other exemplars, as was assumed in previous analyses (Duellman and Trueb, 1983; Kaplan, 1991, 1994, 1999; Chek et al., 2001).

Topological differences from previous hypotheses are not due merely to a re-rooting of the previously accepted tree; the relationships obtained by our analysis are quite different from previous proposals. Our analysis does not recover as monophyletic the exemplars of the three species groups once thought to be monophyletic on the basis of lacking labial tooth rows, that is, the Hyla leucophyllata, H. microcephala, and H. parviceps groups. Instead, the H. microcephala group (including the taxa imbedded within it) is the sister taxon of a clade composed of H. anceps and the exemplars of the H. leucophyllata group. The exemplars of the H. parviceps group are the sister taxon of a clade composed of the exemplars of the H. columbiana and H. labialis groups. This shows that the scenario of labial tooth row evolution is more complex than previously thought, because it implies several transformations in both directions between presence and absence of labial teeth within the clade.

Observations by Wassersug (1980), Spirandeli Cruz (1991), and Kaplan and Ruiz-Carranza (1997) on the internal oral features of larvae of representatives of the Hyla leucophyllata (H. ebraccata and H. sarayacuensis), H. microcephala (H. microcephala, H. nana, H. phlebodes, and H. sanborni), and H. garagoensis (H. padreluna and H. virolinensis) groups revealed a reduction of internal oral structures (including reduction of most internal papillation, reduction of branchial baskets, reduction or absence of secretory ridges and secretory pits) that is most extreme in the representatives of the H. microcephala group. Hyla minuta does not show the reductions seen in these species groups (Spirandeli Cruz, 1991). This species also shares with representatives of the H. leucophyllata group described by Wassersug (1980) a reduction in the density of the filter mesh of the branchial baskets in comparison with other hylid tadpoles. It is clear that the study of internal oral features will provide several additional characters relevant for the study of the 30-chromosome species of Hyla.

The exemplars of the Hyla parviceps group obtain as monophyletic. However, we included only 3 of the 15 species currently included in this problematic group. We are not confident that the monophyly of the H. parviceps group will be maintained as more taxa are added. Regarding the exemplars of the H. leucophyllata group, their relationships are equivalent to those obtained by Chek et al. (2001).

The sister-group relationship of Hyla anceps and the H. leucophyllata group corroborates early suggestions by Lutz (1948, 1973) that these could be related on the basis of sharing a large axilar membrane and flash coloration.

While we could not test the monophyly of the Hyla minima and H. minuta groups, our exemplars of these groups are sister taxa, and they are only distantly related with the exemplars of the H. parviceps group. This position does not support Duellman's (2001) tentative suggestion that the species of the H. minima group should be included in the H. parviceps group.

The paraphyly of the Hyla microcephala group with respect to the H. rubicundula group is an expected result, as historically its species were associated with H. nana and H. sanborni (Lutz, 1973). Nevertheless, the association of the H. microcephala and H. rubicundula groups were reinforced by Pugliese et al. (2001), who described the larva of H. rubicundula and noted similarities (like the lack of marginal papillae) with the larvae of members of the H. microcephala group. In particular, these authors noticed similarities with H. nana and H. sanborni, with the latter being the sister taxon of H. rubicundula in our analysis.

Carvalho e Silva et al. (2003) segregated the Hyla decipiens group from the H. microcephala group on the basis that the larvae of these species lack the possible morphological synapomorphies currently diagnostic of the H. microcephala group (body of tadpole depressed, labial papillae absent in tadpoles) and the putative clade composed of the H. leucophyllata, H. microcephala, and H. parviceps groups (absence of labial tooth rows). However, as mentioned above, our results imply a complex scenario for labial tooth row transformations and place the H. decipiens group within the H. microcephala group. The available taxon sampling did not allow testing the monophyly of the H. decipiens group. The fact that its known species share the oviposition on leaves above the water, and the reversals in larval morphology that led Carvalho e Silva et al. (2003) to consider them unrelated to the H. microcephala group, probably indicates that, even if nested inside this group, the species assigned to the H. decipiens group could be a monophyletic unit.

The relationships of the Hyla garagoensis group, from which no exemplar was available for this study, were discussed by Kaplan and Ruiz-Carranza (1997). Based on the absence of labial tooth rows, they placed the H. garagoensis group in a polytomy together with the H. marmorata group and the clade composed of the H. microcephala, H. parviceps, and H. leucophyllata groups. Duellman et al. (1997) presented a cladogram for most of the 30-chromosome Hyla groups, where the H. garagoensis and H. marmorata groups appear together as a clade supported by the presence of one ventral row of small marginal papillae in larvae. This character state needs further assessment, as indicated by Gomes and Peixoto (1991a) and Peixoto and Gomes (1999), because tadpoles of the H. marmorata group have either one (H. nahdereri) or two rows of marginal papillae (H. senicula; H. soaresi); the tadpoles of H. padreluna, a species of the H. garagoensis group, also has a double row (Kaplan and Ruiz-Carranza, 1997). Considering this and earlier comments, we do not see evidence that associates the H. garagoensis group with the H. marmorata group more than with any other group within the 30-chromosome Hyla clade.

Middle American/Holarctic Clade

This clade is composed of most of the Middle American/Holarctic genera and species groups of treefrogs (fig. 5). For the purposes of discussion, we divide it into four large clades. The first of these includes Acris and Pseudacris. The second includes Plectrohyla, the Hyla bistincta group, the H. sumichrasti group, and various elements of the H. miotympanum group. The third clade includes Duellmanohyla, Ptychohyla, H. miliaria, H. bromeliacia (the sole exemplar of the H. bromeliacia group), and one element of the H. miotympanum group. The fourth clade includes Smilisca, Triprion, Anotheca, and the exemplars of the H. arborea, H. cinerea, H. eximia, H. godmani, H. mixomaculata, H. pictipes, H. pseudopuma, H. taeniopus, and H. versicolor groups.

Within the Middle American/Holarctic clade, the genera Ptychohyla and Smilisca, as well as the Hyla arborea, H. cinerea, H. eximia, H. miotympanum, H. tuberculosa, and H. versicolor groups, are not monophyletic. The H. miotympanum group is polyphyletic; its exemplars split among three different clades: H. miotympanum is the sister taxon of one of the exemplars of the H. tuberculosa group, H. miliaria; H. arborescandens and H. cyclada are related to an undescribed species close to H. thorectes, and together are related to exemplars of the H. bistincta group; H. melanomma and H. perkinsi are at the base of the H. sumichrasti group. Smilisca is not monophyletic, having Pternohyla fodiens nested within it. Ptychohyla is paraphyletic with respect to H. dendrophasma (H. tuberculosa group). The H. arborea group is polyphyletic, with H. japonica nested within the H. eximia group. The H. cinerea group is not monophyletic, with H. femoralis being more closely related to members of the H. eximia and H. versicolor groups than to H. cinerea, H. gratiosa, and H. squirella. The H. versicolor group is not monophyletic because H. andersonii is more closely related to the H. eximia group.

The monophyly of all genera and species groups of Hyla contained in this clade was maintained by Duellman (1970, 2001) based mostly on biogeographic grounds, because morphological evidence of monophyly was lacking. Duellman (2001) further presented a diagram depicting “suggested possible evolutionary relationships” among Middle and North American Hylinae, using as terminals the species groups of Hyla and the different genera (redrawn here as fig. 7). Duellman (2001) envisioned a North American basal lineage being the sister taxon of what he called the Middle American basal lineage. This Middle American basal lineage is further divided into a lower Central American lineage (itself divided into an isthmian highland lineage and a lowland lineage) and the Mexican-Nuclear Central American lineage (in turn divided into a Mexican-Nuclear Central American highland lineage and a lowlands lineage).

While the basal position of Acris and Pseudacris in this clade is consistent with Duellman's (2001) intuitive suggestion of a North American basal lineage, it differs in that the Holarctic species groups of Hyla are only distantly related to them.

We found no evidence supporting Duellman's (2001) exclusively Mexican-Nuclear Central American lineage, nor of his Mexican-Nuclear Central American highland lineage. The latter has nested within it the Mexican-Nuclear Central American lowland clade (the H. godmani group), a clade reminiscent of Duellman's (2001) isthmian highland-lowlands lineage, and also all species groups of Holarctic Hyla. Biogeographic implications of the discordant nature of our results with Duellman's suggested relationships between Middle- and North American Hylinae will be dealt with from a biogeographic perspective later in this paper.

The nonmonophyly of North American Hylinae does not agree with previous analyses (Hedges, 1986; Cocroft, 1994; da Silva, 1997; Moriarty and Cannatella, 2004) because those analyses assumed implicitly that Acris, the Holarctic Hyla, and Pseudacris are monophyletic. In spite of this, the internal relationships of Pseudacris recovered in this analysis are consistent to those obtained by Moriarty and Cannatella (2004).

Previous analyses either did not find evidence that Acris was particularly close to any group of North American hylids (Cocroft, 1994), or else suggested relationships with different species groups of North American Hyla (Hedges, 1986; da Silva, 1997). Two morphological synapomorphies of this Acris + Pseudacris clade could be the spherical or ovoid testes (as opposed to elongate testes) and the presence of dark pigmentation in the peritoneum surrounding the testes (Ralin, 1970, as cited by Hedges, 1986).

The nonmonophyly of the Hyla miotympanum group is not surprising because, as discussed in the section on taxon sampling, it did not appear that any of its putative synapomorphies could withstand a test with a broader taxon sampling. Duellman (2001) merged the formerly recognized H. pinorum group (Duellman, 1970) with the H. miotympanum group. With the notable exception of H. miotympanum, our results are close to recovering both groups as originally envisioned by Duellman (1970), because H. cyclada and H. arborescandens (H. miotympanum group) are recovered as monophyletic, and the former H. pinorum group is recovered as a paraphyletic assemblage that includes the H. sumichrasti group nested within it. In his analysis (Duellman, 2001: 912), the two species included by Duellman (1970) in the H. pinorum group (H. melanomma and H. pinorum), plus the two species that were later associated with this group (H. perkinsi and H. juanitae), form a monophyletic group supported by a single synapomorphy, the presence of an extensive (equal to or more than one-half length of upper arm) axillary membrane.

The dubious monophyly of the 17 species assigned to the Hyla bistincta group was not seriously tested in our analysis, because only 2 species were available. These two exemplars form the sister group of two taxa previously associated with the H. miotympanum group, and together they form the sister taxon of Plectrohyla. According to Duellman and Campbell (1992), character states supporting a monophyletic H. bistincta group plus Plectrohyla are: medial ramus of pterygoid long, in contact with otic capsule; dorsal skin thick (but see Mendelson and Toal [1995] and Duellman [2001] for discussions of this character); complete marginal papillae of the oral disc; and presence of at least one row of submarginal papillae (called by these authors accessory labial papillae) on the posterior labium (but see Wilson et al. [1994a] for discussion of this character state). From these, the complete marginal papillae of the oral disc occur in all known larvae of the clade containing Plectrohyla, and the H. bistincta, H. sumichrasti, and fragments of the H. miotympanum group, as well as in larvae of several other nearby clades (Ptychohyla, Duellmanohyla, the H. mixomaculata, and H. taeniopus groups; see Duellman 1970, 2001). Furthermore, the row of submarginal papillae in the larval oral disc presents a fair amount of variation in the extent and distribution of the papillae, within which could probably be subsumed the morphology seen in all known larvae of the clade mentioned above (see illustrations of all these oral discs in Duellman, 1970, 2001). Besides discussions provided by Mendelson and Toal (1995) and Duellman (2001) regarding the definition of the character state “thick skin”, it does not occur in the following species currently assigned to the H. bistincta group: H. calvicollina, H. charadricola, H. chryses, H. labedactyla, and H. sabrina (Duellman, 2001). Considering that 15 of 17 species currently included in the H. bistincta group and 15 of 18 included in Plectrohyla could not be included in the analysis, we do not consider our results a strong test of their intrarelationships, particularly when several species of the H. bistincta group that present suspicious character state combinations, like the ones mentioned above, were not available. Our results relating H. arborescandens with species of the H. bistincta group corroborate earlier suggestions by Caldwell (1974) that relate this species to species currently placed in the H. bistincta group (H. mykter, H. robertsorum, and H. siopela.). Mendelson and Toal (1996) also suggested affinities of H. arborescandens and H. hazalae with the H. bistincta group on the basis of unpublished osteological data.

Duellman (2001) noted the lack of evidence for the monophyly of the Hyla tuberculosa group. Although poor, our taxon sampling does not recover it as monophyletic, because H. dendrophasma is nested within Ptychohyla, and H. miliaria is the sister taxon of H. miotympanum. Duellman (2001) also referred to the possibility advanced by da Silva (1997) of a relationship of the H. tuberculosa group with the Gladiator Frogs; at this point the evidence presented herein does not support this idea, but in case a denser sampling of the group still corroborates its polyphyly, we would not be surprised if some of its elements (particularly H. tuberculosa26) are shown to be related with the TGF clade.

Hyla miotympanum has repeatedly been considered a generalized Middle American hyline (Duellman, 1963, 1970; Campbell and Smith, 1992; Duellman, 2001), largely because the larva of H. miotympanum exhibits a labial tooth row formula of 2/3 and a relatively small oral disc with an anterior gap in the marginal papillae. We are not aware of any morphological synapomorphy supporting its relationship with H. miliaria, although our molecular data firmly place it there. According with our results, there is a morphological synapomorphy supporting the monophyly of the clade composed of these two species plus Duellmanohyla, the H. bromeliacia group, and Ptychohyla (including H. dendrophasma): the tendo superficialis hallucis that tapers from an expanded corner of the aponeurosis plantaris, with fibers of the m. transversus plantae distalis originating on distal tarsal 2–3 that insert on the lateral side of the tendon.

Considering its overall external appearance, we are surprised by the position of the poorly known Hyla dendrophasma. This species was originally considered to be a member of the H. tuberculosa group (Campbell et al., 2000) based on its large snout–vent length and extensive hand webbing, although with the caveat that it lacks dermal fringes, the only character state shared by all other species placed in the H. tuberculosa group. DNA was isolated and sequenced twice from tissues of the female holotype, the only known specimen (Campbell et al., 2000). Inasmuch as most previous notions of relationships among species of Ptychohyla derive from adult male morphology and tadpoles, the discovery of at least one male specimen of H. dendrophasma could hopefully allow us to better understand its relationships within Ptychohyla.

Campbell and Smith (1992) and Duellman (2001) suggested five morphological synapomorphies for Ptychohyla. One of these is apparently unique to Ptychohyla (pars palatina of the premaxilla with well-developed lingual flange), while the other four show a more extensive taxonomic distribution. (1) The cluster of ventrolateral mucous glands in breeding males is present in Duellmanohyla chamulae, D. ignicolor, and D. schmidtorum (Campbell and Smith, 1992; see also Thomas et al., 1993). (2) The presence in the ventrolateral edge of forearm of tubercles coalesced into a ridge (as opposed to the absence of tubercles) was reported for D. lythrodes, D. salvavida, D. schmidtorum, and D. soralia (Duellman, 1970; 2001); H. bromeliacia has an indistinct row of tubercles that do not coalesce into a ridge (absent in H. dendroscarta) (Duellman, 1970). (3) The double row of marginal papillae is present as well in larvae of the H. bromeliacia group (Duellman, 1970). Finally, (4) larvae of the H. bromeliacia group have a labial tooth row formula of 2/4 or 2/5, and all known larvae of Duellmanohyla have a labial tooth row formula of 3/3; the minimum known for a species of Ptychohyla is 3/5 (P. legleri and P. salvadorensis) (Duellman, 1970, 2001; Campbell and Smith, 1992).

The monophyly of the group composed of Ptychohyla euthysanota, P. hypomykter, P. leonhardschultzei, and P. zophodes is congruent with the results of Duellman (2001), who supported the monophyly of these taxa based on the presence of a thick, rounded tarsal fold. These species further share the presence of hypertrophied ventrolateral glands in breeding males with two species we could not include in our analysis: P. macrotympanum and P. panchoi. Furthermore, all these species also share with P. spinipollex the presence of the nuptial excrescences composed of enlarged individual spines. The states of these characters are unknown in Ptychohyla sp. and Hyla dendrophasma because the only available specimens are females. The nonmonophyly of P. hypomykter plus P. spinipollex is most surprising, considering that both were considered to be a single species (Wilson and McCranie, 1989; see also McCranie and Wilson, 1993).

Although we included several species of Ptychohyla in our analysis, we do not think that we have apprehended a good representation of the morphological diversity of the group, and the absence of species like P. erythromma, P. legleri, and P. sanctaecrucis certainly weakens the test of monophyly of Ptychohyla. This is more so considering the fact that several of the putative morphological synapomorphies of Ptychohyla are actually shared with some species of its sister taxon, as discussed earlier, and that the monophyly of our exemplars of Ptychohyla is weakly supported. The low Bremer support (3) for Ptychohyla also suggests that the evidence for its monophyly deserves further attention.

The Hyla bromeliacia group was tentatively associated with the polyphyletic H. miotympanum group by Duellman (2001: 779). Other than this, we are not aware of it being associated with any other group. Besides the molecular evidence, we are aware of at least one likely morphological synapomorphy supporting the monophyly of Duellmanohyla plus the Hyla bromeliacia group: the presence of pointed serrations of the larval jaw sheaths (Campbell and Smith, 1992; Duellman, 1970; 2001). These are apparently longer in some species of Duellmanohyla than in the H. bromeliacia group, but both seem to be notably more pointed than in Ptychohyla (see descriptions and illustrations in Duellman, 1970).

We share with Duellman (2001) and Mendelson and Campbell (1999) doubts regarding the monophyly of the Hyla taeniopus group. Nevertheless, our two exemplars are recovered as monophyletic in the analysis. Duellman (2001) examined the possibility of a relationship between this group and the H. bistincta group, based on the fact that both have large stream-adapted tadpoles with small, ventral oral discs with complete marginal papillae and bear a labial tooth row formula of 2/3 (but noting that the tooth-row formula is slightly higher for H. nephila and H. trux). Our results suggest instead that the H. taeniopus group is the sister taxon of a clade composed of H. mixe (the only available exemplar of the H. mixomaculata group) plus the clade composed of the Holarctic Hyla groups, the H. godmani, H. pictipes, and H. pseudopuma groups, and Anotheca, Smilisca (including Pternohyla), and Triprion. Furthermore, in the context of our results, the ventral oral disc with complete marginal papillae seems to be a synapomorphy of the whole Middle American clade, with subsequent transformations in the clade just mentioned and in other points of the tree.

We were unable to test the monophyly of the Hyla mixomaculata group, because H. mixe was the only taxon available. Regardless, and until a rigorous test is possible, the monophyly of this group could be reasonably assumed based on the presence of the enlarged oral disc with 7/10 or 11 labial tooth rows. At this point it should be stressed that the sequenced sample comes from a tadpole that was assigned to the H. mixomaculata group based that on that characteristic, and that it was tentatively assigned to H. mixe for being the only species of the group known from the region where the larva was collected; thus, considering the uncertainty in its determination, its position in the tree should be viewed cautiously.

Duellman (2001) included the species of the former Hyla picta group in the H. godmani group. Unfortunately, the two exemplars available to us for this analysis are only the two members of the former H. picta group and none of the restricted H. godmani group; therefore, this is not a satisfactory test of the monophyly of the H. godmani group (sensu lato).

The Lower Central American Lineage

The monophyly of the included exemplars of the Hyla pictipes and H. pseudopuma groups, and its relationship with a clade composed of Anotheca, Smilisca, Triprion, and Pternohyla, is quite consistent (in the sense that it contains almost the same groups) with Duellman's intuitive proposal of a lower Central American clade that contains an Isthmian Highland lineage and a Lowland lineage (fig. 7), with the only exception being that he tentatively considered the H. miliaria group related to Anotheca.

The monophyly of a group composed of Hyla pseudopuma and H. rivularis, the only two exemplars available from the H. pseudopuma and H. pictipes groups, could be suggestive of a lineage of highland isthmian Hylinae as suggested by Duellman (2001). Although the monophyly of each of these two groups has not been tested here, the position of the undescribed Mexican species Hyla sp. 5 (aff. H. thorectes) deserves some comments.

Duellman (1970) recognized the Hyla hazelae group in which he included the nominal species and H. thorectes. Reasons for recognizing this group were “the combination of large hands with vestigial webbing, half webbed feet … and presence of a tympanum are external features which separate these species from other small stream-breeding Mexican Hyla. Furthermore both species have small, relatively narrow tongues and large tubercles below the anal opening. The nature of the nasals and sphenethmoid are unique among northern Middle American hylids” (Duellman, 1970: 384). It is unclear if any of these character states could have been considered as evidence of monophyly of the group. It is also unclear on what basis Duellman (2001) dismantled the group and placed H. hazelae in the H. miotympanum group, while H. thorectes was transferred to the H. pictipes group. Wilson et al. (1994b) suggested that H. thorectes could be related to H. insolita and H. calypsa (under the name H. lancasteri; see Lips, 1996) because they share oviposition on leaves overhanging streams and have dark ventral pigmentation; these character states were not included in Duellman's (2001) analysis of the group. Although we do not have data to take a position regarding these actions, the fact that Hyla sp. 5 (aff. H. thorectes) is unrelated to H. rivularis is here taken as evidence that H. thorectes should not be included in the H. pictipes group. Furthermore, all character states advanced by Duellman (2001) as shared by H. thorectes and the species of the H. pictipes group are also shared by H. thorectes and the taxa to which Hyla sp. 5 (aff. H. thorectes) appears to be closely related in our analysis. Because we were unable to include H. calypsa, H. insolita, or H. lancasteri, we do not have elements to test the hypothesis of Wilson et al. (1994b) regarding the close relationship of H. calypsa, H. insolita, and H. thorectes.

In order to better understand the relationships of the Hyla pictipes group, it would be important to add to this analysis exemplars of the former H. zeteki and H. lancasteri groups, because together with the original H. pictipes and H. rivularis groups (as defined by Duellman, 1970) they represent the three main morphological extremes of the group. The fact that so few exemplars of these two groups were available is one of the weaker points of our analysis.

The paraphyly of Smilisca is partly consistent with Duellman's (2001) phylogenetic analysis of this genus in that Pternohyla is nested within it. However, we did not recover Triprion nested within Smilisca, as did Duellman (2001).

A possible relationship between Triprion and Anotheca was first advanced by Lutz (1968) because she considered them to be the extreme of one specialization consisting “in excessive ossification of the head, accompanied at some stages by extra dentition” (Lutz, 1968: 10). Within this same line, she included all casque-headed frogs, including together South American and Middle American forms. Duellman and Trueb (1976) suggested a possible link of Anotheca with Nyctimantis (discussed below). Duellman (2001: 332) proposed a tentative relation of Anotheca with the Hyla miliaria group based on the oophagous tadpoles that develop in bromeliads or tree-holes (known for the only species of the H. tuberculosa group with a known tadpole, H. salvaje; see Wilson et al., 1985). While our results support a sister-group relationship of Anotheca and Triprion as suggested by Lutz (1968), it occurs within the Holarctic/Middle American clade and not within a group composed of all casque-headed frogs as she suggested. In the context of this analysis, both Triprion and Anotheca share the posterior expansion of the frontoparietals that cover almost all the otoccipital dorsally (see figures in Duellman, 1970).

Our analysis indicates that the insertion of m. extensor digitorum comunis longus on metatarsal II is a synapomorphy of a group composed of Anotheca, Triprion, and the paraphyletic Smilisca. Furthermore, Smilisca (including Pternohyla) and Triprion share the type I septomaxillary (see Trueb, 1970a) and bifurcated cavum principale of the olfactory capsule (Trueb, 1970a); the distribution of these character states should be studied in Anotheca and nearby groups to determine the level of inclusiveness of these possible synapomorphies.

Real Hyla

Our results concerning the relationships between the North American and Eurasiatic species groups of Hyla differ from previous analyses, in part likely because of the previous assumption of monophyly of North American/Holarctic Hylinae. In the first place, the molecular evidence supports a clade containing all North American and Eurasiatic species groups of Hyla, a result that differs from previous analyses where relationships either were unresolved (Cocroft, 1994) or were paraphyletic with respect to Acris and/or Pseudacris (Hedges, 1986; da Silva, 1997). The polyphyly of the Hyla arborea group and the paraphyly of the H. eximia group corroborate previous ideas by Anderson (1991) and Borkin (1999) regarding their nonmonophyly and the closer relationship of H. japonica with the H. eximia and H. versicolor groups. A likely synapomorphy of the H. eximia and H. versicolor groups, including H. japonica and H. andersonii, is the nucleolar organizer region (NOR) present in chromosome 6 instead of chromosome 10 (Anderson, 1991).

South American/West Indian Casque-Headed Frogs

This clade (fig. 5) is composed of Phyllodytes, Phrynohyas, Nyctimantis, and all South American/West Indian casque-headed frogs: Argenteohyla, Aparasphenodon, Corythomantis, Osteopilus, Osteocephalus, Trachycephalus, and Tepuihyla. It is divided basally in a group composed of the two exemplars of Phyllodytes, and another group composed of all casque-headed frog genera, including Phrynohyas and Nyctimantis.

Within this clade, other than those genera that are not monotypic (Argenteohyla, Nyctimantis, Corythomantis) or represented in this analysis by a single species (Aparasphenodon, Tepuihyla), Phyllodytes and Osteopilus are monophyletic, and Osteocephalus, Phrynohyas, and Trachycephalus are not monophyletic. Osteocephalus is not monophyletic because O. langsdorffii (the only species of the genus distributed in the Atlantic forest) is not related to the remaining exemplars of Osteocephalus, which form a monophyletic group that is the sister taxon of Tepuihyla. Phrynohyas is not monophyletic, having Trachycephalus nigromaculatus nested within it, and Trachycephalus is not monophyletic, with T. jordani forming the sister taxon of “Phrynohyas” + T. nigromaculatus.

With respect to Phyllodytes, we were unable to find any published hypothesis regarding its relationships, and considering the scant information available on its morphology, we had no previous clue as to other groups of Hylidae with which it might be related. The only morphological character state of which we are aware that Phyllodytes shares with several members of the South American/West Indian Casque-headed Frogs clade is the presence of at least four posterior labial tooth rows in the tadpole oral disc (see below, “Taxonomic Conclusions: A New Taxonomy of Hylinae and Phyllomedusinae”, for further details).

The polyphyly of Osteocephalus was not unexpected considering the lack of any evidence of its monophyly. This polyphyly results because of the position of O. langsdorffii. This is the only species of the genus present in the Atlantic forest and a species that had been particularly poorly discussed in the context of the systematics of Osteocephalus (Duellman, 1974). While we do not test the monophyly of the bromeliad-breeding/single vocal sac species (here represented by a O. oophagus), our results show that our exemplars with lateral vocal sacs are paraphyletic with respect to O. oophagus.

The monophyly of Tepuihyla was not tested in this analysis. Its sister-group relationship with Osteocephalus (excluding O. langsdorffii) is supported by our data, instead of with Scinax as first suggested (Ayarzagüena et al., “1992” [1993b]; see earlier discussion on the relationships of Scinax). This situation requires changes in the original interpretation of two character states that provided evidence of the relationships of Tepuihyla with other hylids. The presence of spicules in the dorsum of males is more parsimoniously interpreted as a putative synapomorphy of Tepuihyla plus Osteocephalus, instead of a homoplasy, as advanced by Ayarzagüena et al. (“1992” [1993b]). Similarly, the reduction of webbing between toes I and II is more parsimoniously interpreted as a putative synapomorphy of Tepuihyla (homoplastic with Scinax) instead of a synapomorphy of Scinax + Tepuihyla.

The monophyly of the four exemplars of Osteopilus corroborates in a much broader taxonomic context the results of Maxson (1992), Hedges (1996), and Hass et al. (2001), based on albumin immunological distances and still unpublished sequence data regarding its monophyly, and the recent taxonomic changes summarized by Powell and Henderson (2003b). Unfortunately, we could not include in our analysis O. marianae, O. pulchrilineatus, and O. wilderi, and we are not aware of any possible morphological synapomorphy supporting their monophyly. In the absence of other evidence, it could be suggested that the oviposition and development in bromeliads, which occurs in these three species and O. brunneus, is a possible synapomorphy uniting these with O. crucialis, which apparently also has this reproductive mode (Hedges, 1987).

The presence of paired lateral vocal sacs and biogeographic considerations led Trueb (1970b) and Trueb and Duellman (1971) to suggest the collective monophyly of Argenteohyla, Osteocephalus, Phrynohyas, and Trachycephalus (at that time the species of Osteocephalus having a single subgular vocal sac were still unknown). Furthermore, these authors considered Trachycephalus and Phrynohyas to be a monophyletic group on the basis of sharing vocal sacs that are more lateral and protrude posteriorly to the angles of the jaws when inflated. Trueb and Tyler (1974) suggested that the West Indian Osteopilus and the former Calyptahyla crucialis (now Osteopilus crucialis) were also related to this clade, although they exhibit a single, subgular vocal sac. Our results corroborate the monophyly of Phrynohyas plus Trachycephalus (see below), but they also suggest a more complex situation where the casque-headed frogs with double vocal sacs are paraphyletic, with all of the genera of casque-headed frogs that have a single subgular sac being nested within them.

Duellman and Trueb (1976) suggested that Nyctimantis was related to Anotheca, because both share the medial ramus of the pterygoid being juxtaposed squarely against the anterolateral corner of the ventral ledge of the otic capsule. Also, frogs of both genera are known (Anotheca; Taylor, 1954; Jungfer, 1996) or suspected (Nyctimantis; Duellman and Trueb, 1976) to deposit their eggs in water-filled tree cavities. Our results suggest a radically different picture, with Nyctimantis nested within the South American/West Indian Casque-headed Frogs, while Anotheca is nested within the Middle American/Holarctic clade, being the sister taxon of Triprion.

The topology has some discrepancies with previous suggestions as to the relationships of Argenteohyla, Aparasphenodon, and Corythomantis (for the latter two genera, see also comments for Scinax above). Argenteohyla siemersi was segregated by Trueb (1970b) from Trachycephalus, where it had been placed by Klappenbach (1961), because it lacks the diagnostic character states of Trachycephalus established by Trueb (1970a). Further, she suggested that Argenteohyla is a close ally of Osteocephalus based on the presence of paired lateral vocal sacs. Although Trueb (1970a) suggested that Aparasphenodon and Corythomantis are sister taxa, our evidence suggests that both Argenteohyla and Nyctimantis are closer to Aparasphenodon than to Corythomantis.

Most species in the South American/West Indies Casque-headed Frog clade frequently live in or seek refuge in bromeliads or treeholes. This has been reported for Aparasphenodon (Paolillo and Cerda, 1981; Teixeira et al., 2002), Argenteohyla (Barrio and Lutz, 1966; Cespedez, 2000), Corythomantis (Jared et al., 1999), Nyctimantis (Duellman and Trueb, 1976), Osteocephalus langsdorffii (Haddad, personal obs.), Phrynohyas (Goeldi, 1907; Prado et al., 2003), Tepuihyla (Ayarzagüena et al., “1992” [1993b]), and Trachycephalus (Lutz, 1954; Bokermann, 1966c). Furthermore, all species of Phyllodytes (Peixoto et al., 2003), some species of Osteocephalus (Jungfer and Schiesari, 1995; Jungfer and Weygoldt, 1999; Jungfer and Lehr, 2001), some species of Osteopilus (Hedges, 1987; Lannoo et al., 1987), and at least two species of Phrynohyas (Goeldi, 1907; Lescure and Marty, 2000) even lay their eggs in phytotelmata or treeholes where their exotrophic larvae develop. While bromeliads and treeholes are used as refuges or for reproduction in other groups of hylids (e.g., Anotheca spinosa, the Hyla bromeliacia group, the two bromeliad breeding frogs of the H. pictipes group, H. astartea, the H. tuberculosa group, Scinax alter, the Scinax perpusillus group of the S. catharinae clade), the South American/West Indian Casque-headed Frog clade seems to be the largest clade of hylids that consistently makes use of bromeliads or treeholes.

The phylogenetic structure of the South American/West Indies Casque-headed Frog clade implies a minimum of one instance of reversal from presence of heavily exostosed and co-ossified skulls to normal looking, albeit heavily built skulls (the case of Phrynohyas), and at least a possible second and third instance involving reversals from exostosed skulls (the cases of Tepuihyla and Osteopilus vastus).

From a morphological perspective, the paraphyly of Phrynohyas with respect to Trachycephalus nigromaculatus and the concomitant nonmonophyly of Trachycephalus are most interesting and surprising. Herpetologists have been noticing for years that T. nigromaculatus and Phrynohyas mesophaea produce hybrids throughout their overlapping ranges of distribution (Haddad, personal obs.; Pombal, personal commun.; Ramos and Gasparini, 2004). The possibility of hybridization leads us to think that perhaps the introgression of P. mesophaea mitochondria could actually be the reason for the recovered paraphyly of Trachycephalus. However, phylogenetic analyses using either tyrosinase or rhodopsin alone (the only two nuclear genes that were successfully sequenced in T. nigromaculatus) still recover a paraphyletic Trachycephalus (results not shown).

TAXONOMIC CONCLUSIONS: A NEW TAXONOMY OF HYLINAE AND PHYLLOMEDUSINAE

Below we present a new taxonomic arrangement of Hylinae and Phyllomedusinae, based on the results discussed above. While our data clearly point to the fact that we could hardly have a more paraphyletic and uninformative hylid taxonomy as the current one, we foresee some resistance to this new monophyletic taxonomy, due mostly to the lack of morphological evidence in the analysis with consequent few morphological synapomorphies in the diagnoses (for many of the groups, only the molecular evidence presented here provides the evidence of monophyly) or to insufficient numbers of exemplars. The lack of a complete, well-researched nonmolecular data set is admittedly a weakness of this project. However, our study represents the largest amount of evidence for the largest number of terminals ever put together and analyzed in a consistent way to address the phylogenetic relationships of hylids. Until a nonmolecular data set is assembled, we are left only with the evidence provided by our analysis. The alternatives are evident: either we ignore the present results and stick to the traditional, grossly uninformative taxonomy, or we dare to present a new monophyletic taxonomy based on the evidence provided here. The latter option is far closer to the goals of phylogenetic systematics than is the former one. The new taxonomy is the result of our attempt to reconcile the need to recognize only monophyletic groups and to minimize changes to the existing taxonomy while keeping it informative (e.g.: we could have included all currently recognized genera of Hylinae in the synonymy of Hyla; this would have resulted in a perfectly monophyletic though utterly uninformative taxonomy).

We have not tested the monophyly of several genera and species groups either because we could not sample them at all (e. g. the cases of Phrynomedusa and the Hyla claresignata and H. garagoensis group) or because of insufficiency in sampling (e.g., the H. pictipes, H. pseudopuma, and H. mixomaculata groups). There are, as well, seven species that we could not associate with any group (see “Incertae Sedis and Nomina Dubia” below and appendix 4). Nevertheless, we are being bold in the recognition of groups. We recognize all groups that were previously recognized but for which we have not sampled sufficiently to test their monophyly (i.e., only one species was available). These are noted in text. We also assume that the transformation series supporting the monophyly of the exemplars of any given clade are correctly extrapolated as being evidence of the monophyly of the whole group. In the worse case scenario, we will be shown to be wrong; in the best case our hypotheses will withstand further testing. By default, we hope our arrangement will stimulate further research.

The former subfamily Hemiphractinae is now tentatively considered to be part of the paraphyletic Leptodactylidae, pending further research on this vast nonmonophyletic conglomerate of hyloids.

The total number of DNA transformations supporting the monophyly of each relevant clade is informed in the respective diagnoses, with the exception of species groups whose monophyly has not been tested in our analysis. See figure 8 for a summary of the new taxonomy and figures 9–12 for the strict consensus of our analysis updated with the new taxonomy. See appendix 5 for details regarding the number of transitions, transversions, and inferred insertion/deletion events as well as the specific positions involved for each gene.

HYLINAE RAFINESQUE, 1815

Synonyms:

See sections for tribes.

Diagnosis:

This subfamily is diagnosed by 32 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these transformations. Possible morphological synapomorphies are the tendo superficialis digiti V (manus) with an additional tendon that arises ventrally from m. palmaris longus (da Silva, 1998, as cited by Duellman, 2001).

Comments:

The 2n = 24 chromosomes may be another putative synapomorphy of this clade, but it will be necessary to better understand its distribution in the more basal members of the different tribes.

Cophomantini Hoffmann, 1878

  • Cophomantina Hoffmann, 1878. Type genus: Cophomantis Peters, 1870.

  • Diagnosis:

    This tribe is diagnosed by 65 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these transformations. Possible morphological synapomorphies include a ventral oral disc, and complete marginal papillae in larvae, (these character states subsequently transform in more inclusive groups within this clade).

    Comments:

    This tribe includes the genera Aplastodiscus, Bokermannohyla new genus, Hyloscirtus, Hypsiboas, and Myersiohyla new genus.

    The increase in the number of labial tooth rows is likely another synapomorphy of Cophomantini, because all known larvae of Hyloscirtus and Myersiohyla new genus colectively have a minimum of 6/7 labial tooth rows. However, at this time the minimum number of labial tooth rows that is synapomorphic for Cophomantini is ambiguous, because the tadpole is still unknown in H. kanaima.

    An enlarged prepollex is present in all species of Hyloscirtus and in most species of Myersiohyla, new genus. (Unlike species of the H. aromatica group, in H. kanaima, the prepollex is not enlarged.) This characteristic could be a synapomorphy of Cophomantini as an intermediate state leading to the enlarged prepollex with a projecting spine, as proposed by Duellman et al. (1997). In order to understand whether this character state is a synapomorphy of Cophomantini, further research is required, including (1) more osteological work to define the character states involved, and (2) additional studies on the phylogenetic relationships within Myersiohyla, new genus, to understand whether the character state present in the former H. kanaima could be interpreted as a reversal.

    Burton (2004) suggested that the tendo superficialis hallucis tapering from an expanded corner of the aponeurosis plantaris, with fibers of the m. transversus plantae distalis originating on distal tarsal 2–3 inserting on the lateral side of the tendon, provides evidence of monophyly of a group composed of the H. albomarginata, H. albopunctata, H. boans, H. geographica, and H. pulchella groups. The lack of information on the taxonomic distribution of this character state within several terminals of Cophomantini renders its optimization ambiguous in all our most parsimonious trees. While it is clear that it is a synapomorphy of some component of Cophomantini, at this point we do not know its level of inclusiveness. (Burton points out its presence in H. phyllognatha, the only member of the H. bogotensis group that he studied.) The same point holds for the presence of an accessory tendon of the m. lumbricalis longus digiti III, which Burton (2004) considered characteristic of those same species groups.

    There are other character states that were observed in exemplars of this tribe whose taxonomic distribution needs to be assessed in its most basal taxa in order to know with more precision the limits of the clade or clades they diagnose. One of these is the point of insertion of the tendon of the m. extensor brevis medius digiti IV that Faivovich (2002) found to insert in the medial proximal margin of phalanx 2 in the exemplars of this tribe that he studied (Aplastodiscus perviridis, Hyla albopunctata, H. faber, and H. raniceps). In other hyloids this tendon is known to insert in the anterior medial margin of metacarpal IV (Burton, 1996, 1998b; Faivovich, 2002). Subsequently, this character state was observed in other species available for studies (H. albomarginata, H. andina, H. circumdata, H. clepsydra, H. geographica, H. granosa, H. multifasciata, and H. polytaenia; Faivovich, personal obs.).

    There are at least two other character states whose taxonomic distribution within this tribe deserve further scrutiny. The first of these is the presence in the dorsal surface of the larval oral cavity of an anteromedial loop of the prenarial wall into the prenarial arena. Wassersug (1980) described and reported it in Hyla rufitela, Spirandeli Cruz (1991) in Aplastodiscus perviridis, H. faber, H. lundii, and H. prasina, and D'Heursel and de Sá (1999) in H. geographica and H. semilineata. The second character state is the presence of one (most frequently) or more (a few species of the H. bogotensis group; Mijares-Urrutia, 1992b) fleshy projections of variable shape (triangular, round, or elliptic; sometimes called papillae) in the inner margin of the nostrils of the larvae, which various authors (Kenny, 1969; Peixoto, 1981; Peixoto and Cruz, 1983; Lavilla, 1984; Mijares-Urrutia, 1992b; Wild, 1992; Ayarzagüena and Señaris, “1993” [1994]; de Sá, 1995, 1996; Duellman et al., 1997; Faivovich, 2002; Gomes and Peixoto, 2002; Faivovich, personal obs.) noticed in several species: Aplastodiscus perviridis, Hyla albofrenata, H. albomarginata, H. albopunctata, H. albosignata, H. alemani, H. andina, H. aromatica, H. charazani, H. balzani, H. carvalhoi, H. circumdata, H. faber, H. fasciata, H. granosa, H. inparquesi, H. jahni, H. leucopygia, H. multifasciata, H. palaestes, H. platydactyla, H. punctata, H. raniceps, H. sibleszi, and an undescribed species of the H. aromatica group. Although not explicitly mentioned in the descriptions, this character state seems evident in illustrations of other larvae: H. goiana, H. joaquini, H. marginata, H. polytaenia, and H. pulchella (Eterovick et al., 2002; Gallardo, 1964; Garcia et al., 2001b, 2003).

    Aplastodiscus A. Lutz in B. Lutz, 1950

    Type Species:

    Aplastodiscus perviridis A. Lutz in B. Lutz, 1950, by original designation.

    Diagnosis:

    This genus is diagnosed by 72 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Other apparent synapomorphies of this clade are the particular reproductive modes, where the male constructs a subterranean nest in the muddy side of streams and ponds, and where larvae spend early stages of development; subsequent to flooding, the exotrophic larvae live in ponds or streams (Haddad and Sawaya, 2000; Hartmann et al., 2004, Haddad et al., 2005). The presence of proportionally very developed metacarpal and metatarsal tubercles is a possible morphological synapomorphy of the genus (Garcia et al., 2001).

    Comments:

    Our results imply a clade composed of Aplastodiscus and two complexes of the former Hyla albomarginata group, as defined by Cruz and Peixoto (“1985” [1987]): the H. albofrenata and H. albosignata complexes, which are here included in Aplastodiscus.

    Garcia et al. (2001) suggested four synapomorphies for Aplastodiscus as then understood (that is, containing only A. cochranae and A. perviridis): (1) lack of webbing between toes I and II, and very reduced webbing in the remaining toes, (2) bicolored iris, (3) females with unpigmented eggs, and (4) highly developed inner metacarpal and metatarsal tubercles. The lack of webbing between toes I and II, the reduction of webbing among the remaining toes, and the bicolored iris occur only in the two species originally contained in Aplastodiscus. These species, A. cochranae and A. perviridis, are here included in the A. perviridis group, and therefore these two character states are possibly synapomorphic only of this group, not of Aplastodiscus as redefined here. The very developed inner metacarpal and metatarsal tubercles are also present in all species of the Hyla albofrenata and H. albosignata complexes (Cruz and Peixoto “1984” [1985], “1985” [1987]; Cruz et al., 2003), and we consider this feature as a putative synapomorphy of Aplastodiscus as redefined here. The presence of unpigmented eggs is known to occur in all species of the former H. albofrenata and H. albosignata complexes with known eggs (Haddad and Sawaya, 2000; Garcia et al., 2001; Hartmann et al., 2004; Haddad et al., 2005). However, the taxonomic distribution of egg pigmentation within Cophomantini is not well known. It is possible that unpigmented eggs are actually a synapomorphy of a more inclusive clade, as they are known to occur in at least some species of Hyloscirtus (H. jahni, H. larinopygion, H. palmeri, and H. platydactyla; La Marca, 1985, and Faivovich, personal obs.), Hypsiboas (H. lemai, Duellman [1997], and the undescribed species here called Hyla sp. 2), and Myersiohyla new genus (Hyla inparquesi; Faivovich, Myers, and McDiarmid, in prep.; eggs of M. kanaima are pigmented, Duellman and Hoogmoed, 1992); the only known eggs of species of Bokermannohyla, new genus have a pigmented animal pole (Sazima and Bokermann, 1977; Eterovick and Brandão, 2001).

    Most species of Aplastodiscus, as redefined here, possess a white parietal peritoneum (Garcia and Faivovich, personal obs.), as it occurs in some other Cophomantini (Hyla bogotensis, H. granosa, and H. punctata groups, H. marginata; Ruiz-Carranza and Lynch [1991: 4]; Garcia [2003]; Faivovich, personal obs.). While this could be a possible synapomorphy of Aplastodiscus, the taxonomic distribution of this character state is still poorly known in various components of the Cophomantini, so we prefer to await further research on the issue, before hypothesizing polarities.

    Bokermann (1967c) pointed out the overall similarity among advertisement calls of the Hyla albofrenata and H. albosignata complexes and Aplastodiscus perviridis. Future research will define whether any character state related to the advertisement calls could be considered as a synapomorphy of Aplastodiscus as redefined here, or of any of its internal clades.

    Contents:

    Fourteen species included in three species groups.

    Aplastodiscus albofrenatus Group

    Diagnosis:

    This species group is diagnosed by 114 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these transformations. We are not aware of any morphological synapomorphies for this group.

    Contents:

    Six species. Aplastodiscus albofrenatus (A. Lutz, 1924), new comb.; Aplastodiscus arildae (Cruz and Peixoto, “1985” [1987]), new comb.; Aplastodiscus ehrhardti (Müller, 1924), new comb.; Aplastodiscus musicus (B. Lutz, 1948), new comb.; Aplastodiscus weygoldti (Cruz and Peixoto, “1985” [1987]), new comb.

    Aplastodiscus albosignatus Group

    Diagnosis:

    This species group is diagnosed by 42 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. A possible morphological synapomorphy of this group is the presence of elaborate tubercles and ornamentation around the cloacal region (Cruz and Peixoto, “1985” [1987]).

    Contents:

    Seven species. Aplastodiscus albosignatus (A. Lutz and B. Lutz, 1938), new comb.; Aplastodiscus callipygius (Cruz and Peixoto, “1984” [1985]), new comb.; Aplastodiscus cavicola (Cruz and Peixoto, “1984” [1985]), new comb.; Aplastodiscus flumineus (Cruz and Peixoto, “1984” [1985]), new comb.; Aplastodiscus ibirapitanga (Cruz, Pimenta, and Silvano, 2003), new comb.; Aplastodiscus leucopygius (Cruz and Peixoto, “1984” [1985]), new comb.; Aplastodiscus sibilatus (Cruz, Pimenta, and Silvano, 2003), new comb.

    Aplastodiscus perviridis Group

    Diagnosis:

    This species group is diagnosed by 58 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Apparent morphological synapomorphies of this group include the bicolored iris and the absence of webbing between toes I and II (known instances of homoplasy within hylids occur in some Scinax and in various groups of Lophiohylini) and reduction of webbing between the other toes (Garcia et al., 2001).

    Contents:

    Two species. Aplastodiscus cochranae (Mertens, 1952); Aplastodiscus perviridis A. Lutz in B. Lutz, 1950.

    Bokermannohyla, new genus

    Type Species:

    Hyla circumdata Cope, “1870” [1871].

    Diagnosis:

    This genus is diagnosed by 65 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy.

    Etymology:

    This genus is dedicated to Werner Carlos Augusto Bokermann (1929– 1995), as homage to his contribution to the knowledge of Brazilian anurans. He also described several species now included in the new genus. The name derives from Bokermann + connecting -o + Hyla. We are adopting the ending -hyla for several of the new genera described here, most of which contain species groups formerly placed in Hyla. The gender is feminine.

    Comments:

    Bokermannohyla includes all species previously allocated in the Hyla circumdata, H. martinsi, and H. pseudopseudis groups. We include tentatively the H. claresignata group pending the inclusion of its species in the analysis, because it was associated to the H. circumdata group by Bokermann (1972) and Jim and Caramaschi (1979). Hyla alvarengai is also included because our analysis shows that Hyla sp. 9 (aff. H. alvarengai) is nested within this new genus. These species groups should be maintained within Bokermannohyla until their monophyly is rigorously tested.

    Contents:

    Twenty-three species, placed in four species groups.

    Bokermannohyla circumdata Group

    Diagnosis:

    This species group is diagnosed by 52 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. A putative morphological synapomorphy of this group is the presence of (usually thin) dark vertical stripes on the posterior surface of the thigh (Heyer, 1985).

    Contents:

    Fifteen species. Bokermannohyla ahenea (Napoli and Caramaschi, 2004), new comb.; Bokermannohyla astartea (Bokermann, 1967), new comb.; Bokermannohyla caramaschii (Napoli, 2005) new comb.; Bokermannohyla carvalhoi (Peixoto, 1981), new comb.; Bokermannohyla circumdata (Cope, “1870” [1871]), new comb.; Bokermannohyla feioi (Napoli and Caramaschi, 2004), new comb.; Bokermannohyla gouveai (Peixoto and Cruz, 1992), new comb.; Bokermannohyla hylax (Heyer, 1985), new comb.; Bokermannohyla ibitipoca (Caramaschi and Feio, 1990), new comb.; Bokermannohyla izeckshoni (Jim and Caramaschi, 1979), new comb.; Bokermannohyla lucianae (Napoli and Pimenta, 2003), new comb.; Bokermannohyla luctuosa (Pombal and Haddad, 1993), new comb.; Bokermannohyla nanuzae (Bokermann and Sazima, 1973), new comb.; Bokermannohyla ravida (Caramaschi, Napoli and Bernardes, 2001), new comb.; Bokermannohyla sazimai (Cardoso and Andrade, “1982” [1983]), new comb.

    Bokermannohyla claresignata Group

    Diagnosis:

    We are not aware of any synapomorphy supporting the monophyly of this group; see below.

    Comments:

    We did not include any exemplar of this group, and as such we did not test its monophyly. See the earlier discussion regarding its position in the South American I clade. Considering the topology of Cophomantini, synapomorphies suggested for the Bokermannohyla claresignata group can only be maintained if assumed to be reversals (i.e., a enlarged larval oral disc, complete marginal papillae, and large number of labial tooth rows are also present in Myersiohyla new genus and the Hyloscirtus armatus group; complete marginal papillae and large number of labial tooth rows are also present in the H. bogotensis and H. larinopygion groups; the marginal papillae are also complete or with an extremely reduced gap in other species of Bokermannohyla). This would be unproblematic if it were a result of the analysis, but we prefer not to assume it a priori. While these character states cannot be considered at this stage to support the monophyly of the B. claresignata group, considering that its two species are barely distinguishable from each other, we think its nonmonophyly is unlikely and we continue to recognize the group as a hypothesis to be tested.

    Contents:

    Two species. Bokermannohyla claresignata (A. Lutz and B. Lutz, 1939), new comb.; Bokermannohyla clepsydra (A. Lutz, 1925), new comb.

    Bokermannohyla martinsi Group

    Diagnosis:

    Apparent morphological synapomorphies of this group are the development of the humeral crest into a hook-like projection, and a bifid prepollex (Bokermann, 1965b).

    Comments:

    We included a single exemplar of this group, and as such we did not test its monophyly. We continue recognizing it on the basis of the morphological evidence noted above.

    Contents:

    Two species. Bokermannohyla langei (Bokermann, 1965), new comb.; Bokermannohyla martinsi (Bokermann, 1964), new comb.

    Bokermannohyla pseudopseudis Group

    Diagnosis:

    This group is diagnosed by 48 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy for this group.

    Comments:

    Considering our results, which show the undescribed species of the Hyla pseudopseudis group (Hyla sp. 6) to be the sister taxon of an undescribed species similar with H. alvarengai (Hyla sp. 9), we are tentatively including the H. alvarengai in this species group. Eterovick and Brandão (2001) characterized this group on the basis of the presence of short, lateral irregular tooth rows and for having more tooth rows (between six and eight rows) in the oral discs of the larvae than do those of the Bokermannohyla circumdata group. However, the tadpole of H. ibitiguara, included in this group by Caramaschi et al. (2001), has a labial tooth formula of 2/4 (Cardoso, 1983) and seems to lack the short, lateral irregular tooth rows, as do tadpoles of H. alvarengai (Sazima and Bokermann, 1977).

    Contents:

    Four species. Bokermannohyla alvarengai (Bokermann, 1956), new comb.; Bokermannohyla ibitiguara (Cardoso, 1983), new comb.; Bokermannohyla pseudopseudis (Miranda-Ribeiro, 1937), new comb.; Bokermannohyla saxicola (Bokermann, 1964), new comb.

    Hyloscirtus Peters, 1882

    Type Species:

    Hyloscirtus bogotensis Peters, 1882.

  • Hylonomus Peters, 1882. Type species: Hylonomus bogotensis Peters, 1882, by monotypy. Primary homonym of Hylonomus Dawson, 1860.

  • Diagnosis:

    This genus is diagnosed by 56 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. The only putative morphological synapomorphy that we are aware of for this genus is the wide dermal fringes on fingers and toes.

    Comments:

    This genus contains all species included in the Hyla armata, H. bogotensis, and H. larinopygion species groups. The groups are maintained unchanged within Hyloscirtus until the monophyly of each of them is properly tested with denser taxon sampling.

    While the wide dermal fringes in fingers and toes are present in the three species groups, in the H. armatus group they are more obvious in the first manual digit. In the H. bogotensis and H. larinopygion groups the fringes look even wider, apparently due to a combination of proportionally smaller discs and wider fringe, which gives the finger or toe the appearance of being almost as wide as the disc.

    Contents:

    Twenty-eight species placed in three species groups.

    Hyloscirtus armatus Group

    Diagnosis:

    This species group is diagnosed by 103 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Duellman et al. (1997) suggested four synapomorphies of the H. armatus group: the presence of keratin-covered bony spines on the proximal ventral surface of the humerus, on the expanded distal element of the prepollex, and on the first metacarpal; forearms hypertrophied; tadpole tail long with low fins and bluntly rounded tip; and the presence of a “shelf” on the larval upper jaw sheath.

    Comments:

    Our observations of breeding males of the two species of this group indicate the presence of darkly pigmented, keratinized spicules in the dorsum, head (particularly lips), forelimbs, undersides of forelimbs, and pectoral and abdominal region. As the breeding biology of this and the other two species groups of the genus becomes better known, it will be possible to understand if the presence of these spicules are a putative synapomorphy of the H. armatus group.

    Contents:

    Two species. Hyloscirtus armatus (Boulenger, 1902), new comb.; Hyloscirtus charazani (Vellard, 1970), new comb.

    Hyloscirtus bogotensis Group

    Diagnosis:

    This species group is diagnosed by 95 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these transformations. The only morphological synapomorphy that has been proposed for this group is the presence of a mental gland in adult males (Duellman, 1972b). See appendix 4 for a justification of the inclusion of “Hyalinobatrachium” estevesi (Rivero, 1968) in this species group.

    Comments:

    Species of the Hyloscirtus bogotensis group have a white parietal peritoneum (Ruiz-Carranza and Lynch, 1991: 4), as do some other Cophomantini (see comments for Aplastodiscus). Further research on the taxonomic distribution of this character state in the other species groups of Hyloscirtus, and in the other genera of Cophomantini, would clarify which group or groups are diagnosed by this synapomorphy.

    Contents:

    Sixteen species. Hyloscirtus albopunctulatus (Boulenger, 1882), new comb.; Hyloscirtus alytolylax (Duellman, 1972), new comb.; Hyloscirtus bogotensis Peters, 1882; Hyloscirtus callipeza (Duellman, 1989), new comb.; Hyloscirtus colymba (Dunn, 1931), new comb.; Hyloscirtus denticulentus (Duellman, 1972), new comb.; Hyloscirtus estevesi (Rivero, 1968), new comb.; Hyloscirtus jahni (Rivero, 1961), new comb.; Hyloscirtus lascinius (Rivero, 1969), new comb.; Hyloscirtus lynchi (Ruiz-Carranza and Ardila-Robayo, 1991), new comb.; Hyloscirtus palmeri (Boulenger, 1908), new comb.; Hyloscirtus phyllognathus (Melin, 1941), new comb.; Hyloscirtus piceigularis (Ruiz-Carranza and Lynch, 1982), new comb.; Hyloscirtus platydactylus (Boulenger, 1905), new comb.; Hyloscirtus simmonsi (Duellman, 1989), new comb.; Hyloscirtus torrenticola (Duellman and Altig, 1978), new comb.

    Hyloscirtus larinopygion Group

    Diagnosis:

    This species group is diagnosed by 32 transformations in mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy for this group.

    Contents:

    Ten species. Hyloscirtus caucanus (Ardila-Robayo, Ruiz-Carranza, and Roa-Trujillo, 1993), new comb.; Hyloscirtus larinopygion (Duellman, 1973), new comb.; Hyloscirtus lindae (Duellman and Altig, 1978), new comb.; Hyloscirtus pacha (Duellman and Hillis, 1990), new comb.; Hyloscirtus pantostictus (Duellman and Berger, 1982), new comb.; Hyloscirtus psarolaimus (Duellman and Hillis, 1990), new comb.; Hyloscirtus ptychodactylus (Duellman and Hillis, 1990) new comb.; Hyloscirtus sarampiona (Ruiz-Carranza and Lynch, 1982) new comb.; Hyloscirtus staufferorum (Duellman and Coloma, 1993), new comb.; Hyloscirtus tapichalaca (Kizirian, Coloma, and Paredes-Recalde, 2003), new comb.

    Hypsiboas Wagler, 1830

    Type Species:

    Hyla palmata Daudin, 1802 (= Rana boans Linnaeus, 1758), by subsequent designation by implication of Duellman, 1977 (not monotypy as stated by Duellman, 1977: 24).

  • Boana Gray, 1825. Type species: Rana boans Linnaeus, 1758, by monotypy. Coined as a synonym of Hyla and never subsequently validated as available under article 11.6.1 (ICZN, 1999).

  • Auletris Wagler, 1830. Type species: Rana boans Linnaeus, 1758, by subsequent designation of Stejneger (1907).

  • Lobipes Fitzinger, 1843. Type species: Hyla palmata Daudin, 1801 (= Rana boans Linnaeus, 1758), by original designation.

  • Phyllobius Fitzinger, 1843. Type species: Hyla albomarginata Spix, 1824, by original designation. Primary homonym of Phyllobius Schoenherr, 1824.

  • Centrotelma Burmeister, 1856. Type species: Hyla infulata Wied-Neuwied, 1824 (= H. albomarginata Spix, 1824), by subsequent implication by Duellman (1977) (not monotypy as stated by Duellman, 1977: 24).

  • Hylomedusa Burmeister, 1856. Type species: Hyla crepitans Wied-Neuwied, 1825, by subsequent designation by implication of Duellman (1977) (not monotypy as stated by Duellman, 1977: 24).

  • Cinclidium Cope, 1867. Type species: Cinclidium granulatum Cope, 1867 (= Rana boans Linnaeus, 1758), by monotypy. Primary homonym of Cinclidium Blyth, 1842.

  • Cophomantis Peters, 1870. Type species: Cophomantis punctillata Peters, 1870 (= Hyla semilineata Spix, 1824) by monotypy.

  • Cincliscopus Cope, “1870” [1871]. Replacement name for Cinclidium Cope, 1867.

  • Diagnosis:

    This genus is diagnosed by 33 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy for this genus.

    Comments:

    This genus is resurrected for all species formerly included in the Hyla albopunctata, H. boans, H. geographica, H. granosa, H. pulchella, and H. punctata species groups, the H. albomarginata complex, and several species previously unassigned to any group. Most of the former species groups are retained using the new combinations and its contents are redefined in accordance with our results to avoid paraphyly.

    It is tempting to suggest that the presence of a prepollical spine is a possible synapomorphy of this group. However, as discussed earlier, further anatomical studies are necessary to determine whether this character state is homologous with that present in Bokermannohyla.

    Duellman (2001) and Savage (2002b) suggested that the name Boana Gray, 1825 could be applied to a clade of Gladiator Frogs. Gray (1825) suggested the name Boana as a synonym of Hyla, and Stejneger (1907) subsequently designated Hyla boans as its type species. Unfortunately, as far as we can determine from literature research, the name Boana has never been used in combination with an active species name, therefore failing to fulfill the criterion established by article 11.6.1 (ICZN, 1999) for the availability of names originally proposed as synonyms. Duellman (2001) further stated that if Hyla punctata were included within this group, “the generic Hylaplesia Boie would be included in the synonymy of Boana.” However, this is not the case since, as discussed by Dubois (1982), the type species of Hylaplesia is Rana tinctoria Cuvier, 1797 (= Dendrobates tinctorius), by subsequent designation of Duméril and Bibron (1841).

    Wagler (1830) did not combine Hypsiboas with any of the species included in this genus. Subsequent authors (e.g., Tschudi, 1838; Fitzinger, 1843, Cope, 1862) considered it masculine, as we are doing here.

    Contents:

    Seventy species placed in seven species groups, plus two species unassigned to group.

    Hypsiboas albopunctatus Group

    Diagnosis:

    This species group is diagnosed by 43 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy.

    Comments:

    To avoid paraphyly, we include Hyla fasciata and H. calcarata in the group, and to avoid the creation of a species group for a single species, we also include the sister taxon of the former H. albopunctata group, H. heilprini. Larvae so far known of the group (H. albopunctata, H. calcarata, H. fasciata, H. multifasciata, H. raniceps), with the exception of H. heilprini and H. lanciformis are reported to have the mediodistal portion of the internal wall of the spiracle separated from the body wall (de Sá, 1995; 1996; Faivovich, 2002, personal obs.; Peixoto and Cruz, 1983; Wild, 1992). Peixoto and Cruz (1983) reported the same character state for larvae of H. albomarginata. Additional studies on the taxonomic distribution of this peculiar character state are needed to know the limits of the clade or clades it diagnoses. Wild (1992) noticed that larvae of H. calcarata, H. fasciata, and H. lanciformis share the presence of pigmented caudal vertical bands that interconnect laterally along the musculature, with this pattern occurring as well in H. raniceps (Faivovich, personal obs.).

    Hyla dentei was originally associated with both H. raniceps and the former H. geographica group. We are tentatively including it in the Hypsiboas albopunctatus group in view of its overall similarities with Hyla calcarata and H. fasciata.

    Contents:

    Nine species. Hypsiboas albopunctatus (Spix, 1824), new comb.; Hypsiboas calcaratus (Troschel, 1848), new comb.; Hypsiboas dentei (Bokermann, 1967), new comb.; Hypsiboas fasciatus (Günther, 1858), new comb.; Hypsiboas heilprini (Noble, 1923), new comb.; Hypsiboas lanciformis Cope, 1871; Hypsiboas leucocheilus (Caramaschi and Niemeyer, 2003), new comb.; Hypsiboas multifasciatus (Günther, 1859), new comb.; Hypsiboas raniceps Cope, 1862.

    Hypsiboas benitezi Group

    Diagnosis:

    This species group is diagnosed by 30 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. A putative synapomorphy of this group is the presence of a flat mental gland in males (Faivovich et al., in prep.) (known instance of homoplasy in Hyla granosa).

    Comments:

    This new species group includes the clade composed of three species from the Guayana Highlands and three species from the northwestern Amazon Basin. Because one of the Guayanan and one of the Amazonian species are still undescribed, they are not further considered. Two species, Hyla microderma and H. roraima, are a fragment of the former H. geographica group. We are also including tentatively H. hutchinsi and H. rhythmicus, based on their overall similarity with H. benitezi. See appendix 4 for a justification of the inclusion of Hyla pulidoi (Rivero, 1968) in this species group.

    Contents:

    Seven species. Hypsiboas benitezi (Rivero, 1961), new comb.; Hypsiboas hutchinsi (Pyburn and Hall, 1984), new comb.; Hypsiboas lemai (Rivero, 1971), new comb.; Hypsiboas microderma (Pyburn, 1977), new comb.; Hypsiboas pulidoi (Rivero, 1968), new comb.; Hypsiboas rhythmicus (Señaris and Ayarzagüena. 2002), new comb.; Hypsiboas roraima (Duellman and Hoogmoed, 1992), new comb.

    Hypsiboas faber Group

    Diagnosis:

    This species group is diagnosed by 28 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy for this group.

    Comments:

    We are including in this group a clade of some species resulting from the fragmentation of the Hyla boans group. Our results indicate that H. crepitans, H. faber, H. lundii, and H. pardalis form, together with H. albomarginata (a component of the Hyla albomarginata group), a monophyletic group only distantly related to H. boans, the species that gives the name to the former group. For this reason, we recognize this clade as the Hypsiboas faber species group. We tentatively include Hyla exastis in this group because Caramaschi and Rodrigues (2003) related it to H. lundii and H. pardalis on the basis of the lichenous color pattern and the rugose dorsal skin texture.

    A likely behavioral synapomorphy of most species of this group, with the exception of Hyla albomarginata, is the construction of nests by males (with two instances of homoplasy within Hylinae, some species of the now called Hypsiboas semilineatus group (see p. 88), and the Bokermannohyla circumdata group.) The inclusion of Hyla pugnax and H. rosenbergi is tentative, based on the fact that males construct nests, but they lack the reticulated palpebral membrane, a likely synapomorphy present in most species of the now called Hypsiboas semilineatus group (see below). Future research will test this bold hypothesis.

    Contents:

    Eight species. Hypsiboas albomarginatus (Spix, 1824), new comb.; Hypsiboas crepitans (Wied-Neuwied, 1824), new comb.; Hypsiboas exastis (Caramaschi and Rodrigues, 2003), new comb.; Hypsiboas faber (Wied-Neuwied, 1821), new comb.; Hypsiboas lundii (Burmeister, 1856), new comb.; Hypsiboas pardalis (Spix, 1824), new comb.; Hypsiboas pugnax (O. Schmidt, 1857), new comb.; Hypsiboas rosenbergi (Boulenger, 1898), new comb.

    Hypsiboas pellucens Group

    Diagnosis:

    This species group is diagnosed by 115 transformations in mitochondrial ribosomal genes. See appendix 5 for a complete list of these transformations. We are not aware of any morphological synapomorphy for the group.

    Comments:

    We recognize this new species group to include the clade composed of the fragment of the former Hyla albomarginata complex that includes H. pellucens and H. rufitela. The inclusion of H. rubracyla is tentative, based on its previous association with H. pellucens.

    Contents:

    Three species. Hypsiboas pellucens (Werner, 1901), new comb.; Hypsiboas rubracylus (Cochran and Goin, 1970), new comb.; Hypsiboas rufitelus (Fouquette, 1958), new comb.

    Hypsiboas pulchellus Group

    Diagnosis:

    This species group is diagnosed by 55 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Observations by Faivovich and Garcia (unpubl.) suggest that the absence of the slip of the m. depressor mandibulae that originates on the dorsal fascia at the level of the m. dorsalis scapularis (present in all other exemplars so far studied of Hypsiboas, and also of Aplastodiscus, Hyloscirtus, and Bokermannohyla) is a possible synapomorphy of the group.

    Comments:

    We continue to recognize within this species group a Hypsiboas polytaenius clade that includes Hyla beckeri, H. buriti, H. cipoensis, H. goiana, H. latistriata, H. leptolineata, H. phaeopleura, H. polytaenia, and H. stenocephala. Besides molecular data, a likely morphological synapomorphy that supports this clade is the dorsally striped pattern (homoplastic in Hyla bischoffi where a striped pattern occurs on some individuals).

    Contents:

    Thirty species. Hypsiboas alboniger (Nieden, 1923), new comb.; Hypsiboas andinus (Müller, 1926), new comb.; Hypsiboas balzani (Boulenger, 1898), new comb.; Hypsiboas beckeri (Caramaschi and Cruz, 2004), new comb.; Hypsiboas bischoffi (Boulenger, 1887), new comb.; Hypsiboas buriti (Caramaschi and Cruz, 1999), new comb.; Hypsiboas caingua (Carrizo, “1990” [1991]), new comb.; Hypsiboas callipleura (Boulenger, 1902), new comb.; Hypsiboas cipoensis (B. Lutz, 1968), new comb.; Hypsiboas cordobae (Barrio, 1965), new comb.; Hypsiboas cymbalum (Bokermann, 1963), new comb.; Hypsiboas ericae (Caramaschi and Cruz, 2000), new comb.; Hypsiboas freicanecae (Carnaval and Peixoto, 2004), new comb.; Hypsiboas goianus (B. Lutz, 1968), new comb.; Hypsiboas guentheri (Boulenger, 1886), new comb.; Hypsiboas joaquini (B. Lutz, 1968), new comb.; Hypsiboas latistriatus (Caramaschi and Cruz, 2004), new comb.; Hypsiboas leptolineatus (P. Braun and C. Braun, 1977), new comb.; Hypsiboas marginatus (Boulenger, 1887), new comb.; Hypsiboas marianitae (Carrizo, 1992), new comb.; Hypsiboas melanopleura (Boulenger, 1912), new comb.; Hypsiboas palaestes (Duellman, De la Riva, and Wild, 1997), new comb.; Hypsiboas phaeopleura (Caramaschi and Cruz, 2000), new comb.; Hypsiboas polytaenius (Cope, 1870), new comb.; Hypsiboas prasinus (Burmeister, 1856), new comb.; Hypsiboas pulchellus (Duméril and Bibron, 1841), new comb.; Hypsiboas riojanus (Koslowsky, 1895), new comb.; Hypsiboas secedens (B. Lutz, 1963), new comb.; Hypsiboas semiguttatus (A. Lutz, 1925), new comb.; Hypsiboas stenocephalus (Caramaschi and Cruz, 1999), new comb.

    Hypsiboas punctatus Group

    Diagnosis:

    This species group is diagnosed by 30 transformations in mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy for the group.

    Comments:

    We do not see any reason to keep the former Hyla granosa and H. punctata groups separated, as our analysis shows that the two nominal species form a monophyletic group and they are phenotypically similar, so we include all the species in the Hypsiboas punctatus group. The inclusion of Hyla alemani, H. atlantica, H. hobbsi, and H. ornatissima is tentative, based on their previous association with the former H. granosa and H. punctata groups. The inclusion of Hyla picturata is based on our analysis.

    Contents:

    Eight species. Hypsiboas alemani (Rivero, 1964), new comb.; Hypsiboas atlanticus (Caramaschi and Velosa, 1996), new comb.; Hypsiboas granosus (Boulenger, 1882), new comb.; Hypsiboas hobbsi (Cochran and Goin, 1970), new comb.; Hypsiboas ornatissimus (Noble, 1923), new comb.; Hypsiboas picturatus (Boulenger, 1882), new comb.; Hypsiboas punctatus (Schneider, 1799), new comb.; Hypsiboas sibleszi (Rivero, 1971), new comb.

    Hypsiboas semilineatus Group

    Diagnosis:

    This species group is diagnosed by 128 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. A possible morphological synapomorphy of this species group is the presence of a reticulated palpebral membrane (with instances of homoplasy in other species of Hypsiboas: H. hutchinsi and H. microderma).

    Comments:

    We include in this group the fragments of the former Hyla boans and H. geographica groups that form a monophyletic group. We prefer to call it the Hypsiboas semilineatus group because the use of either the H. boans or H. geographicus groups would only cause confusion regarding its contents. The inclusion of Hyla wavrini is based on the combination of the same reproductive mode of H. boans (eggs deposited in a basin built by the male) and a reticulated palpebral membrane (Hoogmoed, 1990). Hyla pombali is tentatively included based on comments by Caramaschi et al (2004a) stressing its similarities with H. semilineata, but with the caveat that it lacks the reticulated palpebral membrane. Schooling behavior has been reported for tadpoles of H. geographica (Caldwell, 1989) and H. semilineata (D'Heursel and Haddad, 2002), and this may be a synapomorphy for at least these two species.

    Contents:

    Six species. Hypsiboas boans (Linnaeus, 1758), new comb.; Hypsiboas geographicus (Spix, 1824), new comb.; Hypsiboas pombali (Caramaschi, Silva, and Feio, 2004); Hypsiboas semilineatus (Spix, 1824), new comb.; Hypsiboas wavrini (Parker, 1936), new comb.

    Species of Hypsiboas Unassigned to Group

    There are two species of Hypsiboas that we do not assign to any group because we do not have evidence favoring a relationship with any of the species groups that we are recognizing for the genus. These species are Hypsiboas fuentei (Goin and Goin, 1968), new comb., and Hypsiboas varelae (Carrizo, 1992), new comb.

    Myersiohyla, new genus

    Type Species:

    Hyla inparquesi Ayarzagüena and Señaris (“1993” [1994]).

    Diagnosis:

    This genus is diagnosed by 48 transformations in mitochondrial protein and ribosomal genes. See appendix 5 for complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy for this group.

    Etymology:

    Dedicated to Charles W. Myers in recognition of his contributions to herpetology, particularly to the herpetofauna of the Guayana Highlands. The name derives from Myersius (latinized Myers) + connecting -o + Hyla. The gender is feminine (Myers and Stothers, MS).

    Comments:

    This new genus includes the species of Hyla aromatica group and H. kanaima, a former member of the H. geographica group. Ayarzagüena and Señaris (“1993” [1994]) included the presence of a strong odor in the definition of the Hyla aromatica group; this could be a possible synapomorphy of Myersiohyla. The presence of a strong odor has yet to be recorded in H. kanaima. It should be noted that the sample we included of H. inparquesi was not collected in the type locality, but in Cerro de la Neblina, ca. 300 km southward.

    Contents:

    Four species. Myersiohyla aromatica (Ayarzagüena and Señaris, “1993” [1994]), new comb.; Myersiohyla inparquesi (Ayarzagüena and Señaris, “1993” [1994]), new comb.; Myersiohyla loveridgei (Rivero, 1961), new comb.; Myersiohyla kanaima (Goin and Wodley, 1961), new comb.

    Dendropsophini Fitzinger, 1843

  • Dendropsophi Fitzinger, 1843. Type genus: Dendropsophus Fitzinger, 1843.

  • Diagnosis:

    This tribe is diagnosed by 23 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Apparent morphological synapomorphies of this tribe are the absence of lingual papillae in the larvae (known instances of reversal in Lysapsus and Pseudis) and the absence of nuptial excrescences (with instances of homoplasy in some species of Sphaenorhynchus and several Cophomantini).

    Comments:

    This tribe contains the genera Dendropsophus, Lysapsus, Pseudis, Scarthyla, Scinax, Sphaenorhynchus, and Xenohyla. The absence of lingual papillae in the larvae is the condition reported in all species of Dendropsophus, Scarthyla, and Scinax, whose larvae have been studied (Wassersug, 1980; Duellman and de Sá, 1988; Echeverria, 1997; Faivovich, 2002; Vera Candioti et al., 2004); a reversal occurs in Lysapsus and Pseudis (de Sá and Lavilla, 1997; Vera Candioti, 2004). This character state is still unknown in Sphaenorhynchus and Xenohyla. Another possible morphological synapomorphy is the absence of keratinized nuptial excrescences. Duellman et al. (1997) and Duellman (2001) suggested that the absence of nuptial excrescences was a synapomorphy of the 30-chromosome Hyla. Nuptial excrescences are also absent in Lysapsus, Pseudis, Scarthyla, Scinax, some species of Sphaenorhynchus, and Xenohyla (Caramaschi, 1989; Duellman and Wiens, 1992; Faivovich, personal obs.; Rodriguez and Duellman, 1994). Note that, while pigmented keratinized structures are absent in all these groups, nuptial pads are present at least in some species of Dendropsophus, Scarthyla, and Scinax (Faivovich, personal obs.).

    Dendropsophus Fitzinger, 1843

    Type Species:

    Hyla frontalis Daudin, 1800 (= Rana leucophyllata Beireis, 1783), by original designation.

  • Lophopus Tschudi, 1838. Type species: Hyla marmorata Daudin (= Bufo marmoratus Laurenti, 1768), by monotypy. Primary homonym of Lophopus Duméril, 1837.

  • Hylella Reinhardt and Lütken, “1861” [1862]. Type species: Hylella tenera Reinhardt and Lütken, 1862 (= Hyla bipunctata Spix, 1824), by subsequent designation of Smith and Taylor (1948).

  • Güntheria Miranda-Ribeiro, 1926. Type species: Hyla dasynota Günther, 1869 (= Hyla senicula Cope, 1868), by monotypy.

  • Diagnosis:

    This genus is diagnosed by 33 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Karyological evidence is the presence of 30 chromosomes. Morphological synapomorphies of this clade are possibly the extreme reduction in the quadratojugal (also occurs in some Cophomantini and Hylini) and a 1/2 labial tooth row formula (known instance of homoplasy in Hyla anceps; subsequent reductions in the formula in some clades) (Duellman and Trueb, 1983; Wogel et al., 2000).

    Comments:

    This genus contains all species formerly placed in Hyla that are known or suspected to have 30 chromosomes. However, the fact that the karyotype of its sister taxon, Xenohyla, is still unknown, precludes the 30-chromosome condition to be considered a synapomorphy of Dendropsophus, because it could be a synapomorphy of Dendropsophus + Xenohyla. A similar situation occurs with two muscle characters. Burton (2004) suggested that the m. contrahentis hallucis reduced or absent and the presence of m. flexor teres hallucis are synapomorphies of this group. Unfortunately, both transformations optimize ambiguously because corresponding character states are still unknown in Xenohyla.

    While we consider the extreme reduction of the quadratojugal to be a possible morphological synapomorphy of Dendropsophus, we warn that the condition requires further study, because the quadratojugal is reduced as well in Sphaenorhynchus and Xenohyla (Caramaschi, 1989; Duellman and Wiens, 1992; Izecksohn, 1996), although apparently not to the level seen in Dendropsophus.

    Bogart (1973), Gruber (2002), Skuk and Langone (1991), and Kaiser et al. (1996) described variation in chromosome morphology for several species of Dendropsophus.

    Contents:

    Eighty-eight species, most of them placed in nine species groups, and seven unassigned to group.

    Dendropsophus columbianus Group

    Diagnosis:

    The only morphological synapomorphy suggested for this group is the presence of two close, triangular lateral spaces between the cricoid and arytenoids at the posterior part of the larynx (Kaplan, 1999).

    Comments:

    We included a single exemplar of this group, and as such we did not test its monophyly, but following Kaplan (1999) we recognize it on the basis of the evidence mentioned above.

    Contents:

    Three species. Dendropsophus bogerti (Cochran and Goin, 1970), new comb.; Dendropsophus carnifex (Duellman, 1969), new comb.; Dendropsophus columbianus (Boettger, 1892), new comb.

    Dendropsophus garagoensis Group

    Diagnosis:

    A possible morphological synapomorphy of this group is the internal surface of the arytenoids with a small medial depression (Kaplan, 1999).

    Comments:

    We did not include any exemplar of this group in the analysis. We recognize it following Kaplan (1999), who considered Hyla praestans to be the sister taxon of the H. garagoensis group on the basis of them sharing the aforementioned putative synapomorphy. We find it more informative at this stage to include it in the group than to consider it as a species unassigned to any group.

    Contents:

    Four species. Dendropsophus garagoensis (Kaplan, 1991), new comb.; Dendropsophus padreluna (Kaplan and Ruiz-Carranza, 1997), new comb.; Dendropsophus praestans (Duellman and Trueb, 1983), new comb.; Dendropsophus virolinensis (Kaplan and Ruiz-Carranza, 1997), new comb.

    Dendropsophus labialis Group

    Diagnosis:

    We are not aware of any synapomorphy for this group.

    Comments:

    We included a single exemplar of this group in the analysis, and as such we did not test its monophyly. Following Duellman and Trueb (1983) and Duellman (1989), we continue to recognize the group pending a rigorous test of its monophyly.

    Contents:

    Three species. Dendropsophus labialis (Peters, 1863), new comb.; Dendropsophus meridensis (Rivero, 1961) new comb.; Dendropsophus pelidna (Duellman, 1989), new comb.

    Dendropsophus leucophyllatus Group

    Diagnosis:

    This species group is diagnosed by 35 transformations in mitochondrial ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies.

    Comments:

    On the basis of our molecular results, we are including Hyla anceps in this group. With the exception of this species, all other members of the group share the presence of pectoral glands in males and females (Duellman, 1970).

    Contents:

    Eight species. Dendropsophus anceps (A. Lutz, 1929), new comb.; Dendropsophus bifurcus (Andersson, 1945), new comb.; Dendropsophus ebraccatus (Cope, 1874), new comb.; Dendropsophus elegans (Wied-Neuwied, 1824), new comb.; Dendropsophus leucophyllatus (Beireis, 1783), new comb.; Dendropsophus rossalleni (Goin, 1959), new comb.; Dendropsophus sarayacuensis (Shreve, 1935), new comb.; Dendropsophus triangulum (Günther, “1868” [1869]), new comb.

    Dendropsophus marmoratus Group

    Diagnosis:

    This species group is diagnosed by 73 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Possible morphological synapomorphies of this group are the warty skin around the margin of the lower lip, the crenulated margin of limbs, and the dorsal marbled pattern (Bokermann, 1964b) (instances of homoplasy in other Hylinae). Furthermore, as it is discernible from the illustrations presented by Gomes and Peixoto (1991b) and Peixoto and Gomes (1999), and confirmed by Peixoto (personal commun. cited in Altig and McDiarmid, 1999a), known larvae of this species group share the presence of a thick sheath of tissue in the basal portion of the tail muscle and adjacent fins, another likely morphological synapomorphy.

    Comments:

    Bokermann (1964b) diagnosed this group as having large vocal sacs. While this could be a synapomorphy, we are hesitant to consider it as such until more anatomical and comparative studies are done in Dendropsophus. In connection with the large vocal sacs of the species of this group, Tyler (1971) mentioned that in Hyla marmorata the pectoral lymphatic septum is modified in a way that permits the inflated sac to intrude into sub-humeral spaces.

    Contents:

    Eight species. Dendropsophus acreanus (Bokermann, 1964), new comb.; Dendropsophus dutrai (Gomes and Peixoto, 1996), new comb.; Dendropsophus marmoratus (Laurenti, 1768), new comb.; Dendropsophus melanargyreus (Cope, 1887), new comb.; Dendropsophus nahdereri (B. Lutz and Bokermann, 1963), new comb.; Dendropsophus novaisi (Bokermann, 1968) new comb.; Dendropsophus seniculus (Cope, 1868), new comb.; Dendropsophus soaresi (Caramaschi and Jim, 1983), new comb.

    Dendropsophus microcephalus Group

    Diagnosis:

    This species group is diagnosed by 42 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Morphological synapomorphies include the lack of labial tooth rows and marginal papillae (Duellman and Trueb, 1983) (a reversal occurs in the Dendropsophus decipiens clade).

    Comments:

    This group now includes all species from the Hyla decipiens, H. microcephala, and H. rubicundula groups. We did not test the monophyly of the H. decipiens or H. rubicundula groups. We continue recognition of the Dendropsophus microcephalus group and within it, pending a rigorous test, a D. decipiens clade (including H. berthalutzae, H. decipiens, H. haddadi, and H. oliveirai), and a D. rubicundulus clade (including H. anataliasiasi, H. araguaya, H. cachimbo, H. cerradensis, H. elianeae, H. jimi, H. rhea, H. rubicundula, and H. tritaeniata). Putative synapomorphies of the D. decipiens clade are the oviposition on leaves overhanging water (homoplastic with the D. leucophyllatus group and some species of the now D. parviceps group) and the presence of a posterior row of marginal papillae (a reversal). A putative synapomorphy of the D. rubicundulus clade is the green dorsum in life that changes to pinkish or violet when preserved (Napoli and Caramaschi, 1998).

    It seems likely that additional synapomorphies for at least some species of Dendropsophus microcephalus group will be hypothesized as larval anatomy is carefully studied. For example, the four species of the group studied by Spirandeli Cruz (1991) and Wassersug (1980) (H. microcephala, H. nana, H. phlebodes, H. sanborni) show knob-like vestiges of the filter rows in larvae. It also remains to be seen whether the peculiarities of the mannicoto glandulare described by Lajmanovich et al. (2000) for H. nana are common to other larvae of the group.

    Contents:

    Thirty-three species. Dendropsophus anataliasiasi (Bokermann, 1972), new comb.; Dendropsophus araguaya (Napoli and Caramaschi, 1998), new comb.; Dendropsophus berthalutzae (Bokermann, 1962), new comb.; Dendropsophus bipunctatus (Spix, 1824), new comb.; Dendropsophus branneri (Cochran, 1948), new comb.; Dendropsophus decipiens (A. Lutz, 1925), new comb.; Dendropsophus cachimbo (Napoli and Caramaschi, 1999), new comb.; Dendropsophus cerradensis (Napoli and Caramaschi, 1998) new comb.; Dendropsophus cruzi (Pombal and Bastos, 1998), new comb.; Dendropsophus elianeae (Napoli and Caramaschi, 2000), new comb.; Dendropsophus gryllatus (Duellman, 1973), new comb.; Dendropsophus haddadi (Bastos and Pombal, 1996), new comb.; Dendropsophus jimi (Napoli and Caramaschi, 1999), new comb.; Dendropsophus joannae (Köhler and Lötters, 2001), new comb.; Dendropsophus leali (Bokermann, 1964), new comb.; Dendropsophus mathiassoni (Cochran and Goin, 1970), new comb.; Dendropsophus meridianus (B. Lutz, 1954), new comb.; Dendropsophus microcephalus (Cope, 1886), new comb.; Dendropsophus minusculus (Rivero, 1971), new comb.; Dendropsophus nanus (Boulenger, 1889), new comb.; Dendropsophus oliveirai (Bokermann, 1963), new comb.; Dendropsophus phlebodes (Stejneger, 1906), new comb.; Dendropsophus pseudomeridianus (Cruz et al., 2000), new comb.; Dendropsophus rhea (Napoli and Caramaschi, 1999), new comb.; Dendropsophus rhodopeplus (Günther, 1858), new comb.; Dendropsophus robertmertensi (Taylor, 1937), new comb.; Dendropsophus rubicundulus (Reinhardt and Lütken, “1861” [1862]), new comb.; Dendropsophus sanborni (Schmidt, 1944), new comb.; Dendropsophus sartori (Smith, 1951), new comb.; Dendropsophus studerae (Carvalho e Silva, Carvalho e Silva, and Izecksohn, 2003), new comb.; Dendropsophus tritaeniatus (Bokermann, 1965), new comb.; Dendropsophus walfordi (Bokermann, 1962), new comb.; Dendropsophus werneri (Cochran, 1952), new comb.

    Dendropsophus minimus Group

    Diagnosis:

    No synapomorphy is known for this group.

    Comments:

    We included a single species of this group in the analisis, and as such we did not test its monophyly and we are not aware of any evidence supporting it. Following Duellman (1982), we continue to recognize it pending a rigorous test of its monophyly.

    Contents:

    Four species. Dendropsophus aperomeus (Duellman, 1982), new comb.; Dendropsophus minimus (Ahl, 1933), new comb.; Dendropsophus miyatai (Vigle and Goberdhan-Vigle, 1990), new comb.; Dendropsophus riveroi (Cochran and Goin, 1970), new comb.

    Dendropsophus minutus Group

    Diagnosis:

    No synapomorphy is known for this group

    Comments:

    We included a single species of this group in the analysis, and as such we did not test its monophyly. Following Martins and Cardoso (1987), we continue to recognize the species group pending a rigorous test of its monophyly. Considering similarities between Hyla minuta and H. limai (Haddad, personal obs.), we tentatively include the latter in the group.

    Contents:

    Four species. Dendropsophus delarivai (Köhler and Lötters, 2001), new comb.; Dendropsophus limai (Bokermann, 1962), new comb.; Dendropsophus minutus (Peters, 1872), new comb.; Dendropsophus xapuriensis (Martins and Cardoso, 1987), new comb.

    Dendropsophus parviceps Group

    Diagnosis:

    This species group is diagnosed by 27 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy supporting the monophyly of this group.

    Comments:

    We expressed our scepticism regarding the monophyly of this group, as currently defined, but found no evidence to reject monophyly. We recognize the group pending a rigorous test of its monophyly.

    Contents:

    Fifteen species. Dendropsophus allenorum (Duellman and Trueb, 1989), new comb.; Dendropsophus bokermanni (Goin, 1960), new comb.; Dendropsophus brevifrons (Duellman and Crump, 1974), new comb.; Dendropsophus gaucheri (Lescure and Marty, 2001), new comb.; Dendropsophus giesleri (Mertens, 1950), new comb.; Dendropsophus grandisonae (Goin, 1966), new comb.; Dendropsophus koechlini (Duellman and Trueb, 1989), new comb.; Dendropsophus luteoocellatus (Roux, 1927), new comb.; Dendropsophus microps (Peters, 1872), new comb.; Dendropsophus parviceps (Boulenger, 1882), new comb.; Dendropsophus pauiniensis (Heyer, 1977), new comb.; Dendropsophus ruschii (Weygoldt and Peixoto, 1987), new comb.; Dendropsophus schubarti (Bokermann, 1963), new comb.; Dendropsophus subocularis (Dunn, 1934), new comb.; Dendropsophus timbeba (Martins and Cardoso, 1987), new comb.

    Species of Dendropsophus Unassigned to Group

    There are several species of Dendropsophus that have not been associated with any group. These are: Dendropsophus amicorum (Mijares-Urrutia, 1998), new comb.; Dendropsophus battersbyi (Rivero, 1961), new comb.; Dendropsophus haraldschultzi (Bokermann, 1962), new comb.; Dendropsophus stingi (Kaplan, 1994), new comb.; Dendropsophus tintinnabulum (Melin, 1941), new comb.; Dendropsophus yaracuyanus (Mijares-Urrutia and Rivero, 2000), new comb.

    Lysapsus Cope, 1862

    Type species:

    Lysapsus limellum Cope, 1862, by monotypy.

  • Podonectes Steindachner, 1864. Type species: Podonectes palmatus Fitzinger, 1864 (= Lysapsus limellum Cope, 1862), by monotypy.

  • Diagnosis:

    This genus is diagnosed by 47 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these transformations. A possible morphological synapomorphy of this genus is the near absence of subacrosomal cone in the sperm (Garda et al., 2004).

    Contents:

    Three species. Lysapsus caraya Gallardo, 1964; Lysapsus laevis Parker, 1935; Lysapsus limellum Cope, 1862.

    Pseudis Wagler, 1830

    Type species:

    Rana paradoxa Linnaeus, 1758, by monotypy.

  • Batrachychthys Pizarro, 1876. Type species: not designated; based on larvae of Pseudis paradoxa (Linnaeus, 1758), according to Caramaschi and Cruz (1998).

  • Diagnosis:

    This genus is diagnosed by 28 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these transformations. We are not aware of any morphological synapomorphy for this genus.

    Comments:

    Garda et al. (2004) distinguished Lysapsus and Pseudis on the basis of the ultrastructure of the sperm acrosome complex, but they stated that the morphology present in Lysapsus (near absence of subacrosomal cone) is the apomorphic condition, with only the plesiomorphic condition being found in Pseudis and therefore not providing evidence of its monophyly.

    Contents:

    Six species. Pseudis bolbodactyla A. Lutz, 1925; Pseudis cardosoi Kwet, 2000; Pseudis fusca Garman, 1883; Pseudis minuta Günther, 1858; Pseudis paradoxa (Linnaeus, 1758); Pseudis tocantins Caramaschi and Cruz, 1998.

    Scarthyla Duellman and de Sá, 1988

    Type Species:

    Scarthyla ostinodactyla (= Hyla goinorum Bokermann, 1962), by original designation.

    Diagnosis:

    Molecular autapomorphies include 227 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular autapomorphies. Apparent morphological autapomorphies include the ability of its tadpoles to propel themselves out of the water, elongated tadpoles (Duellman and Wiens, 1992) and the presence of a labial arm on the oral disc (McDiarmid and Altig, 1990).

    Comments:

    The oral structure known as the labial arm has also been reported for the Scinax rostratus group (McDiarmid and Altig, 1990; Faivovich, 2002) and for four other species of Scinax (Heyer et al., 1990; Alves and Carvalho e Silva, 2002; Alves et al., 2004). Suarez Mayorga and Lynch (2001b) recently reported a similar structure in the larvae of Sphaenorhynchus dorisae, adding that as yet unpublished studies suggest that the structures present in Scinax, Scarthyla, and Sphaenorhynchus are not homologs.

    Contents:

    Monotypic. Scarthyla goinorum (Bokermann, 1962)

    Scinax Wagler, 1830

    Type Species:

    Hyla aurata Wied-Neuwied 1821, by subsequent designation of Stejneger (1907).

  • Ololygon Fitzinger, 1843. Type species: Hyla strigilata, Spix, 1824, by original designation.

  • Garbeana Miranda-Ribeiro, 1926. Type species: Garbeana garbei Miranda-Ribeiro, 1926, by monotypy.

  • Diagnosis:

    This genus is diagnosed by 83 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Morphological synapomorphies include webbing between toes I and II that does not extend beyond the subarticular tubercle of toe I, ability to bend finger I and toe I, origin of the m. pectoralis abdominalis through well-defined tendons, and m. pectoralis abdominalis overlapping m. obliquus externus (da Silva, 1998; Faivovich, 2002).

    Comments:

    Besides the first five character states mentioned above, Faivovich (2002) considered as synapomorphies of Scinax the round or poorly expanded sacral diapophyses, the occluded frontoparietal fontanelle, single origin of the m. extensor brevis superficialis digiti III from the ulnare, and the presence of the m. lumbricalis longus digiti V that originates from the lateral corner of the aponeurosis palmaris. As mentioned earlier, the position of Scinax within Hylinae suggests that outgroups employed by Faivovich (2002) are phylogenetically distant from Scinax. Because of this, we contend that the taxonomic distribution of the aforementioned character states needs to be reassessed, at least among the other Dendropsophini, before considering them synapomorphies of Scinax. For example, it seems evident that the round or poorly expanded sacral diapophyses are not a synapomorphy of Scinax, but of a more inclusive group (also present, at least, in Scarthyla and Sphaenorhynchus; Duellman and Wiens, 1992), whose limits are still unclear. In the same way, the absence of the lingual papillae, as mentioned earlier, might be a synapomorphy of Dendropsophini. The truncated discs of the digits were considered a synapomorphy of Scinax by Duellman and Wiens (1992) and Faivovich (2002). The phylogenetic position of the single exemplar of the H. uruguaya group in the analysis, as sister group of the S. ruber clade, complicates this interpretation. Discs in the two species of the group, H. uruguaya and H. pinima, are proportionally reduced in size with respect to most species of Scinax, cannot be considered truncated, and therefore determine an ambiguous optimization of this character state.

    Burton (2004) considered that m. flexor ossis metatarsus IV with insertions on both metatarsi IV and V was a synapomorphy of Scinax. Because this character state is still unknown in our two exemplars of the S. catharinae clade, and in the exemplar of Hyla uruguaya group, it optimizes ambiguously in our analysis; it is unclear if it is a synapomorphy of Scinax or of a more exclusive clade.

    The Hyla uruguaya group is being included in Scinax to avoid rendering Scinax paraphyletic. Larvae of the two species of the H. uruguaya group share with members of the S. ruber clade some synapomorphies (the proctodeal tube not reaching the free margin of the lower fin, and the presence of keratinized spurs behind the lower jaw sheath and over the infralabial papillae [Kolenc et al., “2003” [2004]]). However, preliminary observations on H. uruguaya indicate that adults show at least one conflicting character state, the m. depressor mandibulae without an origin from the dorsal fascia at the level of the m. dorsalis scapulae (Faivovich, personal obs.)—a character state that optimized as a synapomorphy of the S. catharinae clade in the analysis of Faivovich (2002). This controversy may be resolved when all the conflicting evidence is analyzed, including a much denser sampling of Scinax. In the meantime, since the molecular evidence indicates affinities with the S. ruber clade, we tentatively include the two species of the H. uruguaya group in this clade, where they are recognized as a separate group.

    Contents:

    Eighty-eight species placed in two major clades.

    Scinax catharinae Clade

    Diagnosis:

    This clade is diagnosed by 90 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Morphological synapomorphies suggested for this clade by Faivovich (2002) are absence of the anterior process of the suprascapula, internal vocal sac, distal division of the middle branch of the m. extensor digitorum comunis longus, and insertion of the medial side of this branch on the tendon of the m. extensor brevis medius digiti IV.

    Comments:

    Regardless of problems imposed by the present results to interpretation of the possible synapomorphies of Scinax resulting from Faivovich's (2002) analysis, the sparse available knowledge on the taxonomic distribution of the transformations supporting the monophyly of the very distinctive S. catharinae clade suggests that most of them still hold in the present analysis. An exception is the m. depressor mandibulae without an origin from the dorsal fascia at the level of the m. dorsalis scapulae, which also occurs in Hyla uruguaya, rendering its optimization ambiguous in our analysis.

    Scinax catharinae Group

    Diagnosis:

    Because we only included two species of the Scinax catharinae group as exemplars of the S. catharinae clade, the molecular transformations that diagnose this group are redundant with those diagnosing the S. catharinae clade. Presumed morphological synapomorphies of this group include the posterior part of the cricoid ring extensively elongated and curved, the partial mineralization of intercalary elements between ultimate and penultimate phalanges, and the laterodistal origin of the m. extensor brevis distalis digiti III (Faivovich, 2002).

    Contents:

    Twenty-seven species. Scinax agilis (Cruz and Peixoto, 1983); Scinax albicans (Bokermann, 1967); Scinax angrensis (B. Lutz, 1973); Scinax argyreornatus (Miranda-Ribeiro, 1926); Scinax ariadne (Bokermann, 1967); Scinax aromothyella Faivovich, 2005; Scinax berthae (Barrio, 1962); Scinax brieni (De Witte, 1927); Scinax canastrensis (Cardoso and Haddad, 1982); Scinax carnevalli (Caramaschi and Kisteumacher, 1989); Scinax catharinae (Boulenger, 1888); Scinax centralis (Pombal and Bastos, 1996); Scinax flavoguttatus (A. Lutz and B. Lutz, 1939); Scinax heyeri (Peixoto and Weygoldt, 1986); Scinax hiemalis (Haddad and Pombal, 1987); Scinax humilis (B. Lutz, 1954); Scinax jureia (Pombal and Gordo, 1991); Scinax kautskyi (Carvalho e Silva and Peixoto, 1991); Scinax littoralis (Pombal and Gordo, 1991); Scinax longilineus (B. Lutz, 1968); Scinax luizotavioi (Caramaschi and Kisteumacher, 1989); Scinax machadoi (Bokermann and Sazima, 1973); Scinax obtriangulatus (B. Lutz, 1973); Scinax ranki (Andrade and Cardoso, 1987); Scinax rizibilis (Bokermann, 1964); Scinax strigilatus (Spix, 1824); and Scinax trapicheiroi (B. Lutz, 1954).

    Scinax perpusillus Group

    Diagnosis:

    Presumed synapomorphies of this group are the oviposition in bromeliads and the extreme reduction of webbing between toes II and III (Peixoto, 1987; Faivovich, 2002).

    Comments:

    The monophyly of this group was not tested by Faivovich (2002) because only one species of the group was available for his analysis, where it obtained as the sister taxon of all exemplars of the S. catharinae clade. For these two reasons, we continue recognizing this group until its monophyly is rigorously tested.

    Contents:

    Seven species. Scinax alcatraz (B. Lutz, 1973); Scinax arduous Peixoto, 2002; Scinax atratus (Peixoto, 1989); Scinax littoreus (Peixoto, 1988); Scinax melloi (Peixoto, 1989); Scinax perpusillus (A. Lutz and B. Lutz, 1939); Scinax v-signatus (B. Lutz, 1968).

    Scinax ruber Clade

    Diagnosis:

    This clade is supported by 53 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. A morphological synapomorphy suggested for this clade by Faivovich (2002) is the proctodeal tube positioned above the margin of the lower fin.

    Comments:

    Faivovich (2002) was skeptical about the monophyly of the S. ruber clade; however, the present analysis recovers it as monophyletic, with a considerable number of transformations supporting its monophyly.

    As in the case of several of the synapomorphies suggested by Faivovich's (2002) analysis for Scinax, we are unsure as to whether the suggested morphological synapomorphies are optimized identically in our analysis. In particular, we do not know the taxonomic distribution within Dendropsophini for two other synapomorphies proposed for this clade (Faivovich, 2002): the arytenoids with a dorsal prominence developed over the pharyngeal margin, and absence of the lateral m. extensor brevis distalis digiti V (pes). Preliminary observation on the larvae of some species of Sphaenorhynchus (Sphaenorhynchus bromelicola, S. orophilus, S. pauloalvini, and S. prasinus; Faivovich, personal obs.) indicate that their proctodeal tubes are attached to the free margin of the lower fin, similar to the S. catharinae clade, instead of having the characteristic position seen in larvae of the S. ruber clade.

    Scinax megapodius and S. trachythorax are considered here to be junior synonyms of S. fuscovarius for reasons discussed in appendix 4. There are two species, Hyla dolloi and H. karenanneae, that upon examination of their type series we consider to be species of Scinax (see appendix 4 for further comments on them).

    Contents:

    Fifty-six species. Eleven assigned to two groups, 43 unassigned to any group.

    Scinax rostratus Group

    Diagnosis:

    Putative morphological synapomorphies of this group include the juxtaposed inner margins of the vomers; overlap of the otic plate of the crista parotica due to a broad otic plate; nonfenestration of the cartilaginous plate of the squamosal with the oblique cartilage; pointed tubercle on heel; absence of the m. extensor brevis distalis digiti II; presence of m. extensor brevis distalis digiti I (pes); discontinuity of lateral margins with the posterior portion of the oral disc; third posterior labial tooth row placed on a labial arm; reduction of the third posterior labial tooth to one-quarter the length of the second row; absence of keratinized spurs behind the lower jaw sheath; and head-down calling position (Faivovich, 2002).

    Contents:

    Nine species. Scinax boulengeri (Cope, 1887); Scinax garbei (Miranda-Ribeiro, 1926); Scinax jolyi (Lescure and Marty, 2001); Scinax kennedyi (Pyburn, 1973); Scinax nebulosus (Spix, 1824); Scinax pedromedinae (Henle, 1991); Scinax proboscideus (Brongersma, 1933); Scinax rostratus (Peters, 1870); Scinax sugillatus (Duellman, 1973).

    Scinax uruguayus Group

    Diagnosis:

    Putative morphological synapomorphies of this group include the bicolored iris and the presence of two keratinized and pigmented plates on the sides of the lower jaw sheath (Kolenc et al., “2003” [2004]).

    Comments:

    The marginal papillae of the posterior margin of the oral disc being larger than those of the lateral margins (Kolenc et al., “2003” [2004]) and the reduction in toe webbing could be other synapomorphies of the group.

    Contents:

    Two species. Scinax pinima (Bokermann and Sazima, 1973) new comb.; Scinax uruguayus (Schmidt, 1944) new comb.

    Species of the Scinax ruber Clade Unassigned to a Species Group

    We follow Faivovich (2002) in not recognizing the former Scinax ruber and S. staufferi groups, as both were not monophyletic on his analysis. We are considering all species formerly included in these groups as members of the S. ruber clade, although we consider them as unassigned to any group. These species are Scinax acuminatus (Cope, 1862); Scinax altae (Dunn, 1933); Scinax alter (B. Lutz, 1973); Scinax auratus (Wied-Neuwied, 1821); Scinax baumgardneri (Rivero, 1961); Scinax blairi (Fouquette and Pyburn, 1972); Scinax boesemani (Goin, 1966); Scinax caldarum (B. Lutz, 1968); Scinax cardosoi (Carvalho e Silva and Peixoto, 1991); Scinax castroviejoi De la Riva, 1993; Scinax chiquitanus (De la Riva, 1990); Scinax crospedospilus (A. Lutz, 1925); Scinax cruentommus (Duellman, 1972); Scinax curicica Pugliese, Pombal, and Sazima, 2004; Scinax cuspidatus (A. Lutz, 1925); Scinax danae (Duellman, 1986); Scinax dolloi (Werner, 1898) new comb.; Scinax duartei (B. Lutz, 1951); Scinax elaeochrous (Cope, 1875); Scinax eurydice (Bokermann, 1964); Scinax exiguus (Duellman, 1986); Scinax flavidus La Marca, 2004; Scinax funereus (Cope, 1874); Scinax fuscomarginatus (A. Lutz, 1925); Scinax fuscovarius (A. Lutz, 1925); Scinax granulatus (Peters, 1871); Scinax hayii (Barbour, 1909); Scinax ictericus Duellman and Wiens, 1993; Scinax karenanneae (Pyburn, 1992) comb. nov.; Scinax lindsayi Pyburn, 1992; Scinax manriquei Barrio-Amorós, Orellana, and Chacon, 2004; Scinax maracaya (Cardoso and Sazima, 1980); Scinax nasicus (Cope, 1862); Scinax oreites Duellman and Wiens, 1993; Scinax pachycrus (Miranda-Ribeiro, 1937); Scinax parkeri (Gaige, 1926); Scinax perereca Pombal, Haddad, and Kasahara, 1995; Scinax quinquefasciatus (Fowler, 1913); Scinax ruber (Laurenti, 1768); Scinax similis (Cochran, 1952); Scinax squalirostris (A. Lutz, 1925); Scinax staufferi (Cope, 1865); Scinax trilineatus (Hoogmoed and Gorzula, 1977); Scinax wandae (Pyburn and Fouquette, 1971); Scinax x-signatus (Spix, 1824).

    Sphaenorhynchus Tschudi, 1838

    Type Species:

    Hyla lactea Daudin, 1801, by original designation.

  • Dryomelictes Fitzinger, 1843. Type species: Hyla lactea Daudin, 1802, by original designation.

  • Dryomelictes Cope, 1865. Type species: Hyla aurantiaca Daudin, 1802, by original designation. Junior homonym of Dryomelictes Fitzinger, 1843.

  • Hylopsis Werner, 1894. Type species: Hylopsis platycephalus Werner, 1894, by monotypy.

  • Sphoenohyla Lutz and Lutz, 1938. Substitute name (explicit subgenus of Hyla) for Sphaenorhynchus thought erroneously to be preoccupied by Sphenorhynchus Lichtenstein, 1823.

  • Diagnosis:

    This genus is diagnosed by 157 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Duellman and Wiens (1992) proposed the following synapomorphies for Sphaenorhynchus: posterior ramus of pterygoid absent; zygomatic ramus of squamosal absent or reduced to a small knob; pars facialis of maxilla and alary process of premaxilla reduced; postorbital process of maxilla reduced, not in contact with quadratojugal; neopalatine reduced to a sliver or absent; pars externa plectri entering tympanic ring posteriorly (rather than dorsally); pars externa plectri round; hyale curved medially; coracoids and clavicle elongated; and prepollex ossified, bladelike. Other likely synapomorphies include the differentiation of the m. intermandibularis into a small apical supplementary element, and the extreme development of the m. interhyoideus (Tyler, 1971).

    Comments:

    Duellman and Wiens (1992) considered that the transverse process of presacral vertebra IV elongate, oriented posteriorly is a synapomorphy of Sphaenorhynchus. The presence of this character state in Xenohyla (Izecksohn, 1996) suggests that in the context of our topology, its optimization is ambiguous. There are also some larval features that could be considered synapomorphies of at least some species of Sphaenorhynchus, such as the morphology and position of the nostrils and the presence of some notably large marginal papillae (see Kenny, 1969; Bokermann, 1973; Cruz, 1973; Cruz and Peixoto, 1980; Suarez-Mayorga and Lynch, 2001b). The presence of a white peritoneum in five species (S. carneus, S. lacteus, S. planicola, S. prasinus, and S. surdus; Haddad and Faivovich, personal obs.) may be another synapomorphy of this genus (with several instances of homoplasy within Hylinae). Observations on six species (S. carneus, S. dorisae, S. lacteus, S. planicola, S. prasinus, and S. surdus) suggest that they are ant specialists (Duellman, 1978; Rodriguez and Duellman, 1994; Parmalee, 1999; Haddad, personal obs.), another likely synapomorphy whose taxonomic distribution within the group deserves additional study.

    Contents:

    Eleven species. Sphaenorhynchus bromelicola Bokermann, 1966; Sphaenorhynchus carneus (Cope, 1868); Sphaenorhynchus dorisae (Goin, 1967); Sphaenorhynchus lacteus (Daudin, 1801); Sphaenorhynchus orophilus (A. Lutz and B. Lutz, 1938); Sphaenorhynchus palustris Bokermann, 1966; Sphaenorhynchus pauloalvini Bokermann, 1973; Sphaenorhynchus planicola (A. Lutz and B. Lutz, 1938); Sphaenorhynchus platycephalus (Werner, 1894); Sphaenorhynchus prasinus Bokermann, 1973; Sphaenorhynchus surdus (Cochran, 1953).

    Xenohyla Izecksohn, 1996

    Type Species:

    Hyla truncata Izecksohn, 1959, by original designation.

    Diagnosis:

    For the purposes of this paper, we consider that the 128 transformations in mitochondrial protein and ribosomal genes autapomorphic of Xenohyla truncata are synapomorphies of this genus. See appendix 5 for a complete list of these molecular synapomorphies. Although species of Xenohyla are very distinctive, we are aware of only three putative morphological synapomorphies: the retention in adults of the scars of the windows of forelimbs emergence (but see Comments below); the presence of a small, transverse process in the urostyle; and frugivorous habits (reported for X. truncata by da Silva et al. [1989] and Izecksohn [1996]; unknown in X. eugenioi).

    Comments:

    We included a single species of this genus in the analysis, and as such we did not test its monophyly, but consider it very likely on the basis of the evidence noted above and its unique external aspect. Izecksohn (1996) and Caramaschi (1998) noticed that adults of Xenohyla retain scars of the large windows of forelimb emergence that are evident in recently metamorphosed individuals. Each of these scars actually corresponds to a thick pectoral patch of glands that is macroscopically evident upon superficial dissection (Faivovich, personal obs.).

    Contents:

    Two species. Xenohyla eugenioi Caramaschi, 2001; Xenohyla truncata (Izecksohn, 1959).

    Hylini Rafinesque, 1815

  • Hylarinia Rafinesque, 1815. Type genus: Hylaria Rafinesque, 1814 (an unjustified emendation of Hyla Laurenti, 1768).

  • Hylina Gray, 1825. Type genus: Hyla Laurenti, 1768.

  • Dryophytae Fitzinger, 1843. Type genus: Dryophytes Fitzinger, 1843.

  • Acridina Mivart, 1869. Type genus: Acris Duméril and Bibron, 1841.

  • Triprioninae Miranda-Ribeiro, 1926. Type genus: Triprion Cope, 1866.

  • Diagnosis:

    This tribe is diagnosed by 107 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. The only known morphological synapomorphy is the undivided tendon of the m. flexor digitorum brevis superficialis (there are several instances of homoplasy within Hylidae including at least Scinax, Scarthyla + Pseudis, and a reversal within Hylini).

    Comments:

    The tribe Hylini is proposed for the clade of Middle American/Holarctic hylids. It includes Acris, Anotheca, Duellmanohyla, Exerodonta, Hyla, Pseudacris, Ptychohyla, Smilisca (including Pternohyla), Triprion, and six new genera, Bromeliohyla new gen., Charadrahyla new gen., Ecnomiohyla new gen., Isthmohyla new gen., Megastomatohyla new gen., and Tlalocohyla new gen. Morescalchi (1973) recognized the tribe Hylini in which he included most genera currently placed in Hylinae. Subsequent authors have not used Hylini in the sense that Morescalchi (1973) used it.

    Acris Duméril and Bibron, 1841

    Type Species:

    Rana gryllus LeConte, 1825, by subsequent designation of Fitzinger (1843).

    Diagnosis:

    This genus is diagnosed by 138 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Other apparent synapomorphies include the differentiation of the m. intermandibularis into an apical supplementary element (Tyler, 1971), and diploid chromosome number of 22 (Bushnell et al., 1939; Cole, 1966; Duellman, 1970).

    Contents:

    Two species. Acris crepitans Baird, 1854; Acris gryllus (LeConte, 1825).

    Anotheca Smith, 1939

    Type Species:

    Gastrotheca coronata Stejneger, 1911 (= Hyla spinosa Steindachner, 1864), by original designation.

    Diagnosis:

    This monotypic genus is diagnosed by 219 transformations of nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these transformations. Morphological autapomorphies include the tendo superficialis hallucis that tapers from an expanded corner of the aponeurosis plantaris; with fibers of the m. transversus plantae distalis originating on distal tarsal 2–3 inserting on the lateral side of the tendon (several of homoplasy, see appendix 1); the unique skull ornamentation composed of sharp, dorsally pointed spines in the margins of frontoparietal, maxilla, nasal (including canthal ridge), and squamosal, and character states that result in its reproductive mode, including maternal provisioning of trophic eggs to tadpoles (see Jungfer, 1996).

    Contents:

    Monotypic. Anotheca spinosa (Steindachner, 1864).

    Bromeliohyla, new genus

    Type Species:

    Hyla bromeliacia Schmidt, 1933.

    Diagnosis:

    For the purposes of this paper we consider that the 141 transformations in nuclear and mitochondrial protein and ribosomal genes autapomorphic of Bromeliohyla bromeliacia are synapomorphies of this genus. See appendix 5 for a complete list of these molecular synapomorphies. Possible nonmolecular synapomorphies of this genus are the reproductive mode, where eggs are laid in water accumulated in bromeliads (several instances of homoplasy, e.g., two species of Isthmohyla, Phyllodytes, some species Osteopilus, and the Scinax perpusillus group), and tadpoles with dorsoventrally flattened bodies and elongated tails.

    Etymology:

    From Bromelia + Hyla, in reference to the bromeliad breeding habits of its species. The gender is feminine.

    Comments:

    We included a single species of this genus, and as such we did not test its monophyly. We consider it likely based on the evidence noted above.

    Contents:

    Two species. Bromeliohyla bromeliacia (Schmidt, 1933), new comb.; Bromeliohyla dendroscarta (Taylor, 1940), new comb.

    Charadrahyla, new genus

    Type Species:

    Hyla taeniopus Günther, 1901.

    Diagnosis:

    This genus is diagnosed by 56 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy supporting this genus.

    Etymology:

    Derived from the Greek word charadra- (ravine) + Hyla. In reference to the habits of these frogs. The gender is feminine.

    Comments:

    This new genus includes the species formerly placed in the Hyla taeniopus group.

    Contents:

    Five species. Charadrahyla altipotens (Duellman, 1968), new comb.; Charadrahyla chaneque (Duellman, 1961), new comb.; Charadrahyla nephila (Mendelson and Campbell, 1999), new comb.; Charadrahyla taeniopus (Günther, 1901), new comb.; Charadrahyla trux (Adler and Denis, 1972), new comb.

    Duellmanohyla Campbell and Smith, 1992

    Type Species:

    Hyla uranochroa Cope, 1876, by original designation.

    Diagnosis:

    This genus is diagnosed by 48 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Likely morphological synapomorphies of this group are the red iris, the labial stripe expanded below orbit, the lack of nuptial excrescences, the ventrally oriented funnel-shaped oral disc in larvae, labial tooth rows reduced in length, and absence of lateral processes on upper jaw sheath (Duellman, 2001).

    Contents:

    Eight species. Duellmanohyla chamulae (Duellman, 1961); Duellmanohyla ignicolor (Duellman, 1961); Duellmanohyla lythrodes (Savage, 1968); Duellmanohyla rufioculis (Taylor, 1952); Duellmanohyla salvavida (McCranie and Wilson, 1986); Duellmanohyla schmidtorum (Stuart, 1954); Duellmanohyla soralia (Wilson and McCranie, 1985); Duellmanohyla uranochroa (Cope, 1876).

    Ecnomiohyla, new genus

    Type Species:

    Hypsiboas miliarius Cope, 1886.

    Diagnosis:

    This genus is diagnosed by 37 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy supporting this genus.

    Etymology:

    From the Greek, ecnomios, meaning marvelous, unusual; an obvious reference to the incredible frogs of the Hyla tuberculosa group. The gender is feminine.

    Comments:

    This new genus contains the Hyla tuberculosa group, excluding H. dendrophasma, and including one species of the H. miotympanum group as well. Erecting a new genus for this clade is the only way of being consistent with the new monophyletic taxonomy that is proposed for hylids. Although naming the former H. tuberculosa group as a genus constitutes a testable claim of monophyly, we expect that it will ultimately be found to be two or three different clades, with one of these being the one named here.

    Contents:

    Ten species. Ecnomiohyla echinata (Duellman, 1962), new comb.; Ecnomiohyla fimbrimembra (Taylor, 1948), new comb.; Ecnomiohyla miliaria (Cope, 1886), new comb.; Ecnomiohyla minera (Wilson, McCranie, and Williams, 1985), new comb.; Ecnomiohyla miotympanum (Cope, 1863), new comb.; Ecnomiohyla phantasmagoria (Dunn, 1943); Ecnomiohyla salvaje (Wilson, McCranie, and Williams, 1985), new comb.; Ecnomiohyla thysanota (Duellman, 1966), new comb.; Ecnomiohyla tuberculosa (Boulenger, 1882), new comb.; Ecnomiohyla valancifer (Firschein and Smith, 1956), new comb.

    Exerodonta Brocchi, 1879

    Type Species:

    Exerodonta sumichrasti Brocchi, 1879, by monotypy.

    Diagnosis:

    This genus is diagnosed by 80 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy supporting this genus.

    Comments:

    Exerodonta is resurrected for the species previously placed in the Hyla sumichrasti group and a fragment of the former H. miotympanum group as defined by Duellman (2001) that corresponds to the traditionally recognized H. pinorum group (Duellman 1970). Although we did not include the type species, E. sumichrasti, in the analysis, but only H. chimalapa and H. xera, we consider that these two species and H. sumichrasti and H. smaragdina are so similar that we are not hesitant to consider them closely related. Although we are not aware of any synapomorphy supporting the monophyly of the former H. pinorum group (Duellman, 1970), and our results suggest it is paraphyletic with respect to the H. sumichrasti group, we are tentatively including the other species associated with it, H. abdivita, H. bivocata, H. catracha, and H. juanitae by Snyder (1972), Porras and Wilson (1987), and Campbell and Duellman (2000), in this resurrected genus.

    Contents:

    Eleven species, four placed in one species group, seven unassigned to group.

    Exerodonta sumichrasti Group

    Diagnosis:

    This species group is diagnosed by 76 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Putative morphological synapomorphies of this group are the massive nasals (Duellman, 1970) and, in the species with a known tadpole, the enlarged larval oral disc (homoplastic with the former Hyla mixomaculata group), and the 3/6 to 7 labial tooth row formula (Canseco-Márquez et al., 2003; Duellman, 1970).

    Comments:

    The illustrations of the oral discs of Hyla smaragdina, H. sumichrasti (Duellman, 1970), and H. xera (Canseco-Márquez et al., 2003) show that they share the multiple interruption of the last posterior labial tooth row into shorter rows, possibly another synapomorphy.

    Contents:

    Four species. Exerodonta chimalapa (Mendelson and Campbell, 1994), new comb.; Exerodonta smaragdina (Taylor, 1940), new comb.; Exerodonta sumichrasti Brocchi, 1879; Exerodonta xera (Mendelson and Campbell, 1994), new comb.

    Species of Exerodonta Unassigned to Group

    Considering that in our analysis Hyla melanomma and H. perkinsi are a grade leading to the E. sumichrasti group, we are not assigning to any group these and the other species associated with the former Hyla pinorum group (Duellman, 1970; Snyder, 1972; Porras and Wilson, 1987; Campbell and Duellman, 2000). These species are: Exerodonta abdivita (Campbell and Duellman, 2000), new comb.; Exerodonta bivocata (Duellman and Hoyt, 1961), new comb.; Exerodonta catracha (Porras and Wilson, 1987), new comb.; Exerodonta juanitae (Snyder, 1972), new comb.; Exerodonta melanomma (Taylor, 1940), new comb.; Exerodonta perkinsi (Campbell and Brodie, 1992), new comb.; Exerodonta pinorum (Taylor, 1937), new comb.

    Hyla Laurenti, 1768

    Type Species:

    Hyla viridis Laurenti, 1768 (= Rana arborea Linnaeus, 1758), by subsequent designation of Stejneger (1907).

  • Calamita Schneider, 1799. Type species: Rana arborea Linnaeus, 1758, by subsequent designation of Stejneger (1907).

  • Hylaria Rafinesque, 1814. Unjustified emendation for Hyla.

  • Hyas Wagler, 1830. Type species: Rana arborea Linnaeus, 1758, by monotypy. Junior homonym of Hyas Leach, 1815.

  • Dendrohyas Wagler, 1830. Substitute name for Hyas Wagler, 1830.

  • Dryophytes Fitzinger, 1843. Type species: Hyla versicolor LeConte, 1825, by original designation.

  • Epedaphus Cope, 1885. Type species: Hyla gratiosa LeConte, 1856, by monotypy.

  • Diagnosis:

    This genus is diagnosed by 25 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy for the genus.

    Comments:

    Hyla is restricted to all species previously placed in the H. arborea, H. cinerea, H. eximia, and H. versicolor groups, which are redefined herein.

    Contents:

    Thirty-two species, with 31 placed in four species groups and one species unassigned to group.

    Hyla arborea Group

    Diagnosis:

    This species group is diagnosed by 37 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy supporting this group.

    Comments:

    The contents of the Hyla arborea group are restricted to avoid its paraphyly. The inclusion of the species that were not included in the present analysis and do not show the NOR in chromosome 6 is tentative, because no evidence, other than the molecular data presented here, is known to support its monophyly.

    Contents:

    Fourteen species. Hyla annectans (Jerdon, 1870); Hyla arborea (Linnaeus, 1758); Hyla chinensis Günther, 1858; Hyla hallowellii Thomson, 1912; Hyla immaculata Boettger, 1888; Hyla intermedia Boulenger, 1882; Hyla meridionalis Boettger, 1874; Hyla sanchiangensis Pope, 1929; Hyla sarda (De Betta, 1853); Hyla savignyi Audouin, 1827; Hyla simplex Boettger, 1901; Hyla tsinlingensis Liu and Hu, 1966; Hyla ussuriensis Nikolsky, 1918; Hyla zhaopingensis Tang and Zhang, 1984.

    Hyla cinerea Group

    Diagnosis:

    This species group is diagnosed by 35 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy supporting this group.

    Comments:

    Hyla femoralis is excluded from the H. cinerea group to avoid the paraphyly of the group.

    Contents:

    Three species. Hyla cinerea (Schneider, 1799); Hyla gratiosa LeConte, “1856” [1857]; Hyla squirella Bosc, 1800.

    Hyla eximia Group

    Diagnosis:

    This species group is diagnosed by 17 transformations in mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy supporting this group.

    Comments:

    The inclusion of Hyla suweonensis is tentative, based on the fact that Anderson (1991) reported a NOR in chromosome 6, a character state shared by H. femoralis and the H. eximia and H. versicolor groups.

    Contents:

    Eleven species. Hyla andersonii Baird, 1854; Hyla arboricola Taylor, 1941; Hyla arenicolor Cope, 1886; Hyla bocourti (Mocquard, 1889); Hyla euphorbiacea Günther, 1859; Hyla eximia Baird, 1854; Hyla japonica Günther, “1858” [1859]; Hyla plicata Brocchi, 1877; Hyla suweonensis Kuramoto, 1980; Hyla walkeri Stuart, 1954; Hyla wrightorum Taylor, 1939.

    Hyla versicolor Group

    Diagnosis:

    This species group is diagnosed by 51 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy supporting this group.

    Comments:

    Hyla andersonii is transferred to the H. eximia group to avoid the paraphyly of the H. versicolor group.

    Contents:

    Three species. Hyla avivoca Viosca, 1928; Hyla chrysoscelis Cope, 1880; Hyla versicolor LeConte, 1825.

    Species of Hyla Unassigned to Group

    Considering that relationships of Hyla femoralis Bosc, 1800 with the H. versicolor and H. eximia groups are unresolved, we prefer to keep this species unassigned as a more stable alternative to merging the H. versicolor and the H. eximia groups into a single group.

    Isthmohyla, new genus

    Type Species:

    Hyla pseudopuma Günther, 1901.

    Diagnosis:

    This genus is diagnosed by 42 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy for the genus.

    Etymology:

    From Isthmo, Greek, in reference to the mostly isthmian distribution of these frogs (the only exception is Hyla insolita) + Hyla. The gender is feminine.

    Comments:

    This new genus includes all species of the Hyla pseudopuma and H. pictipes groups, as defined by Duellman (2001), with the exception of H. thorectes, which is transferred to Plectrohyla. Our taxon sampling of the relevant species groups was too sparse to result in a test of their respective monophyly. We tentatively recognize these species groups as reviewed by Duellman (2001), with the exception that H. thorectes is excluded from the former H. pictipes group and not included in Isthmohyla.

    Contents:

    Fourteen species placed in two species groups.

    Isthmohyla pictipes Group

    Diagnosis:

    We are not aware of any synapomorphy supporting the monophyly of this group.

    Comments:

    We included a single exemplar of this group, and as such we did not test its monophyly. It is being tentatively recognized following Duellman (2001) until a rigorous test is performed. This species group is formed by the former Hyla lancasteri, H. pictipes, H. rivularis, and H. zeteki groups (Duellman, 1970, 2001). The monophyly of the former H. zeteki group does not seem to be controversial, as its two species share massive temporal musculature, bromeliad dwelling larvae, a terminal oral disc, and a labial tooth row formula of 1/1. The monophyly of the group composed of the former H. rivularis and H. pictipes groups (as defined by Duellman, 1970) is supported by the presence of an enlarged oral disc (Duellman, 2001) with a broad band of conic submarginal papillae on the posterior part of the disc, about three rows on the anterior part, and an M-shaped upper jaw sheath27 (Faivovich, personal obs.; see also illustrations in Duellman, 2001). The monophyly of the former H. lancasteri group seems to be supported by the presence of granular dorsal skin (Duellman, 2001; known homoplastic instance in H. debilis), a short snout, and the presence of dark ventral pigmentation (Wilson et al., 1994b, known homoplastic instance in H. thorectes.)

    Contents:

    Ten species. Isthmohyla calypsa (Lips, 1996), new comb.; Isthmohyla debilis (Taylor, 1952), new comb.; Isthmohyla insolita (McCranie, Wilson, and Williams, 1993), new comb.; Isthmohyla lancasteri (Barbour, 1928), new comb.; Isthmohyla picadoi (Dunn, 1937), new comb.; Isthmohyla pictipes (Cope, 1876), new comb.; Isthmohyla rivularis (Taylor, 1952), new comb.; Isthmohyla tica (Starret, 1966), new comb.; Isthmohyla xanthosticta (Duellman, 1968), new comb.; Isthmohyla zeteki (Gaige, 1929), new comb.

    Isthmohyla pseudopuma Group

    Diagnosis:

    We are not aware of any synapomorphy supporting the monophyly of this group.

    Comments:

    We included a single exemplar of this group, and as such we did not test its monophyly. It is being tentatively recognized following Duellman (2001) until a rigorous test is performed.

    Contents:

    Four species. Isthmohyla angustilineata (Taylor, 1952), new comb.; Isthmohyla graceae (Myers and Duellman, 1982), new comb.; Isthmohyla infucata (Duellman, 1968), new comb.; Isthmohyla pseudopuma (Günther, 1901), new comb.

    Megastomatohyla, new genus

    Type Species:

    Hyla mixe Duellman, 1965.

    Diagnosis:

    For the purposes of this paper, we consider that the 209 transformations in nuclear and mitochondrial protein and ribosomal genes autapomorphic of Hyla mixe are synapomorphies of this genus. See appendix 5 for a complete list of these molecular synapomorphies. A possible morphological synapomorphy of this genus is the greatly enlarged oral disc of the known larvae bearing 7–10 anterior rows and 10–11 posterior rows.

    Etymology:

    From the Greek, mega, large, plus the stem of the genitive stomatos, mouth, in reference to the enlarged oral disc of the larvae + Hyla. The gender is feminine.

    Comments:

    We included a single species of this genus, and as such we did not test its monophyly, but consider it very likely on the basis of the evidence noted above. As mentioned earlier, the sequenced sample comes from a tadpole that was assigned to the Hyla mixomaculata group based on the enlarged oral disc and the labial tooth row formula and tentatively assigned to H. mixe for being the only species of the group known from the region where it was collected. Considering the uncertainty in its determination, its position in the tree should be viewed cautiously. This is not a situation we feel most comfortable with, but for a matter of being consistent with the general approach of this contribution, we consider that it is better to describe a new genus for the H. mixomaculata group than to leave it as incerta sedis. Although we did not test the monophyly of the group, as stated earlier, we consider it based on the morphological synapomorphy for larvae mentioned above. Another possible synapomorphy of this group could be the lack of vocal slits (Duellman, 1970), but this is contingent on the internal relationships of the nearby Charadrahyla; C. chaneque is the only species of that genus known to lack vocal slits (Duellman, 2001). If future studies show it to be the sister group of the remaining species of Charadrahyla, it could render the optimization of the lack of vocal slits as ambiguous for both Charadrahyla and Megastomatohyla. Males of species included in Megastomatohyla lack nuptial excrescences on the thumb (Duellman, 1970). The polarity of this character state is unclear because it also occurs in Charadrahyla altipotens and in Hyla godmani and H. loquax (Duellman, 1970).

    Contents:

    Four species. Megastomatohyla mixe (Duellman, 1965), new comb.; Megastomatohyla mixomaculata (Taylor, 1950), new comb.; Megastomatohyla nubicola (Duellman, 1964), new comb.; Megastomatohyla pellita (Duellman, 1968), new comb.

    Plectrohyla Brocchi, 1877

    Type Species:

    Plectrohyla guatemalensis Brocchi, 1877, by original designation.

  • Cauphias Brocchi, 1877. Replacement name for Plectrohyla Brocchi, 1877.

  • Diagnosis:

    This genus is supported by 43 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy supporting this genus as redefined here.

    Comments:

    We are including in Plectrohyla all species formerly placed in the Hyla bistincta group and some of the members of the former H. miotympanum (H. cyclada and H. arborescandens) and H. pictipes (H. thorectes) groups. H. thorectes is being tentatively included because a still undescribed species, very similar to H. thorectes (Hyla sp. 5) is nested within this clade. Hyla hazelae is tentatively included because of its similarities with H. thorectes. Technically our results are certainly compatible with the recognition of a separate genus for the members of the H. bistincta group and the few species from other groups associated with them. However, we are particularly concerned that the present, clean separation between Plectrohyla and these exemplars probably will not hold when more species of the two clades, particularly from the H. bistincta group, are added. The facts that no apparent morphological synapomorphies are known for the H. bistincta group and that some authors raised doubts regarding the limits between it and Plectrohyla support the conservative stance of including all these species in Plectrohyla. We preserve a Plectrohyla guatemalensis group for all the species of Plectrohyla as defined in the past and tentatively recognize a group that contains all members of the H. bistincta group plus the species of other groups shown to be related with it in this analysis.

    The reasons why we are not considering some of the characters states shared by Plectrohyla and the Hyla bistincta group that were advanced by Duellman (2001) as synapomorphies of the redefined Plectrohyla were discussed earlier in this paper (p. 68). The only character state that seems to be inclusive of Plectrohyla and the H. bistincta group is the long medial ramus of the pterygoid in contact with the otic capsule. However, both H. arborescandens and H. cyclada were reported by Duellman (2001) to have a short medial ramus that does not contact the prootic. In a more densely sampled context, this character state could probably be interpreted as a reversal; however, in the present context it optimized ambiguously, so we do not consider it a morphological synapomorphy of the redefined Plectrohyla.

    Contents:

    Thirty-nine species placed in two species groups.

    Plectrohyla bistincta Group

    Diagnosis:

    Exemplars of this species group in our analysis are diagnosed by 16 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these transformations. We are not aware of any morphological synapomorphy supporting this group.

    Contents:

    Twenty-one species. Plectrohyla ameibothalame (Canseco-Márquez, Mendelson, and Gutiérrez-Mayén et al., 2002), new comb.; Plectrohyla arborescandens (Taylor, “1938”[1939]), new comb.; Plectrohyla bistincta (Cope, 1877), new comb.; Plectrohyla calthula (Ustach, Mendelson, McDiarmid, and Campbell, 2000), new comb.; Plectrohyla calvicollina (Toal, 1994), new comb.; Plectrohyla celata (Toal and Mendelson, 1995), new comb.; Plectrohyla cembra (Caldwell, 1974), new comb.; Plectrohyla charadricola (Duellman, 1964), new comb.; Plectrohyla chryses (Adler, 1965), new comb.; Plectrohyla crassa (Brocchi, 1877), new comb.; Plectrohyla cyanomma (Caldwell, 1974), new comb.; Plectrohyla cyclada (Campbell and Duellman, 2000) new comb.; Plectrohyla hazelae (Taylor, 1940), new comb.; Plectrohyla labedactyla (Mendelson and Toal, 1996), new comb.; Plectrohyla mykter (Adler and Dennis, 1972), new comb.; Plectrohyla pachyderma, (Taylor, 1942), new comb.; Plectrohyla pentheter (Adler, 1965), new comb.; Plectrohyla psarosema (Campbell and Duellman, 2000), new comb.; Plectrohyla robertsorum (Taylor, 1940), new comb.; Plectrohyla sabrina (Caldwell, 1974), new comb.; Plectrohyla siopela, (Duellman, 1968), new comb.; Plectrohyla thorectes (Adler, 1965), new comb.

    Plectrohyla guatemalensis Group

    Diagnosis:

    This group is diagnosed by 34 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these transformations. Possible morphological synapomorphies of this species group are bifurcate alary process of the premaxilla; sphenethmoid with anterior part ossified; frontoparietals abbuting posteriorly, exposing only small part of the frontoparietal fontanelle; humerus having well-developed flanges; hypertrophied forearm; prepollex enlarged and ossified in both sexes; prepollex truncate; and absence of lateral labial folds in larvae (Duellman and Campbell, 1992; Duellman, 2001).

    Contents:

    Eighteen species. Plectrohyla acanthodes Duellman and Campbell, 1992; Plectrohyla avia Stuart, 1952; Plectrohyla chrysopleura Wilson, McCranie, and Cruz-Díaz, 1994; Plectrohyla dasypus McCranie and Wilson, 1981; Plectrohyla exquisitia McCranie and Wilson, 1998; Plectrohyla glandulosa (Boulenger, 1883); Plectrohyla guatemalensis Brocchi, 1877; Plectrohyla hartwegi Duellman, 1968; Plectrohyla ixil Stuart, 1942; Plectrohyla lacertosa Bumhanzen and Smith, 1954; Plectrohyla matudai Hartweg, 1941; Plectrohyla pokomchi Duellman and Campbell, 1984; Plectrohyla psiloderma McCranie and Wilson, 1992; Plectrohyla pycnochila Rabb, 1959; Plectrohyla quecchi Stuart, 1942; Plectrohyla sagorum Hartweg, 1941; Plectrohyla tecunumani Duellman and Campbell, 1984; Plectrohyla teuchestes Duellman and Campbell, 1992.

    Pseudacris Fitzinger, 1843

    Type Species:

    Rana nigrita LeConte, 1825, by monotypy.

  • Chorophilus Baird, 1854. Type species: Rana nigrita LeConte, 1825, by original designation.

  • Helocaetes Baird, 1854. Type species: Hyla triseriata Wied-Neuwied, 1839, by subsequent designation of Schmidt (1953).

  • Hyliola Mocquard, 1899. Type species: Hyla regilla Baird and Girard, 1852, by subsequent designation of Stejneger (1907).

  • Limnaoedus Mittleman and List, 1953. Type species: Hyla ocularis Bosc and Daudin, 1801, by original designation.

  • Parapseudacris Hardy and Borrough, 1986. Type species: Hyla crucifer Wied-Neuwied, 1838, by original designation.

  • Diagnosis:

    This genus is diagnosed by 37 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. The m. transversus metatarsus II broad, occupying the entire length of metatarsus II optimizes in this analysis as a morphological synapomorphy of this genus.

    Contents:

    Fourteen species placed in four clades.

    Pseudacris crucifer Clade

    Diagnosis:

    This clade is diagnosed by molecular data presented by Moriarty and Cannatella (2004).

    Contents:

    Two species. Pseudacris crucifer (Wied-Neuwied, 1838); Pseudacris ocularis (Bosc and Daudin, 1801).

    Pseudacris ornata Clade

    Diagnosis:

    This clade is diagnosed by molecular data presented by Moriarty and Cannatella (2004).

    Contents:

    Three species. Pseudacris illinoensis Smith, 1951; Pseudacris ornata (Holbrook, 1836); Pseudacris streckeri A.A. Wright and A.H. Wright, 1933.

    Pseudacris nigrita Clade

    Diagnosis:

    This clade is diagnosed by molecular data presented by Moriarty and Cannatella (2004).

    Comments:

    According to Moriarty and Cannatella (2004), this clade contains a more exclusive clade that contains all species but P. brimleyi and P. brachyphona.

    Contents:

    Seven species. Pseudacris brachyphona (Cope, 1889); Pseudacris brimleyi Brandt and Walker, 1933; Pseudacris clarkii (Baird, 1854); Pseudacris feriarum (Baird, 1854); Pseudacris maculata (Agassiz, 1850); Pseudacris nigrita (LeConte, 1825); Pseudacris triseriata (Wied-Neuwied, 1838).

    Pseudacris regilla Clade

    Diagnosis:

    This clade is diagnosed by molecular data presented by Moriarty and Cannatella (2004).

    Contents:

    Two species. Pseudacris cadaverina (Cope, 1866); Pseudacris regilla (Baird and Girard, 1852).

    Ptychohyla Taylor, 1944

    Type Species:

    Ptychohyla adipoventris Taylor, 1944 (= Hyla leonhardschultzei Ahl, 1934), by original designation.

    Diagnosis:

    This genus is diagnosed by 11 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. An apparent morphological synapomorphy of this group is the well-developed lingual flange of the pars palatina of premaxillar (Campbell and Smith, 1992).

    Comments:

    To avoid paraphyly, we are including Hyla dendrophasma in Ptychohyla. As mentioned earlier in the discussion, other synapomorphies of Ptychohyla proposed by Campbell and Smith (1992) and Duellman (2001) are also present in some species of its sister taxon (Bromeliohyla + Duellmanohyla), so we do not recognize them as synapomorphies until a phylogenetic analysis including that evidence is performed. Known males of the exemplars of Ptychohyla included in the analysis share the presence of enlarged individual nuptial spines and hypertrophied ventrolateral glands (Duellman, 2001), as do males of P. macrotympanum and P. panchoi. Discovery of males of H. dendrophasma will confirm whether this character state is an apparent synapomorphy of these species or of a less inclusive clade.

    Contents:

    Thirteen species. Ptychohyla acrochorda Campbell and Duellman, 2000; Ptychohyla dendrophasma (Campbell, Smith, and Acevedo, 2000), new comb.; Ptychohyla erythromma (Taylor, 1937); Ptychohyla euthysanota Kellogg, 1928; Ptychohyla hypomykter McCranie and Wilson, 1993; Ptychohyla legleri (Taylor, 1958); Ptychohyla leonhardschultzei (Ahl, 1934); Ptychohyla macrotympanum (Tanner, 1957); Ptychohyla panchoi Duellman and Campbell, 1982; Ptychohyla salvadorensis (Mertens, 1952); Ptychohyla sanctaecrucis Campbell and Smith, 1992; Ptychohyla spinipollex (Schmidt, 1936); Ptychohyla zophodes Campbell and Duellman, 2000.

    Smilisca Cope, 1865

    Type Species:

    Smilisca daulinia Cope, 1865 (= Hyla baudinii Duméril and Bibron, 1841), by monotypy.

  • Pternohyla Boulenger, 1882. Type species Pternohyla fodiens Boulenger, 1882, by monotypy. new synonymy.

  • Diagnosis:

    This genus is diagnosed by 38 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy.

    Comments:

    Pternohyla is included in the synonymy of Smilisca to avoid paraphyly.

    Contents:

    Eight species. Smilisca baudinii (Duméril and Bibron, 1841); Smilisca cyanosticta (Smith, 1953); Smilisca dentata (Smith, 1957), new comb.; Smilisca fodiens (Boulenger, 1882), new comb.; Smilisca phaeota (Cope, 1862); Smilisca puma (Cope, 1885); Smilisca sila (Duellman and Trueb, 1966); Smilisca sordida (Peters, 1863).

    Tlalocohyla, new genus

    Type Species:

    Hyla smithii Boulenger, 1902.

    Diagnosis:

    This genus is diagnosed by 92 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy.

    Etymology:

    From Tlaloc, the Olmec God of the rain, + connecting -o + Hyla. The gender is feminine.

    Comments:

    The inclusion of Hyla godmani and H. loquax is tentative and based on its association with H. picta and H. smithii in the former H. godmani group by Duellman (2001). The larvae of H. loquax and H. smithii share a reduction in the length of the third posterior tooth row (Caldwell, 1986; Lee, 1996). This feature is not present in the larvae of H. godmani as described by Duellman (1970).

    Contents:

    Four species. Tlalocohyla godmani (Günther, 1901), new comb.; Tlalocohyla loquax (Gaige and Stuart, 1934), new comb.; Tlalocohyla picta (Günther, 1901), new comb.; Tlalocohyla smithii (Boulenger, 1902), new comb.

    Triprion Cope, 1866

    Type Species:

    Pharyngodon petasatus Cope, 1865, by monotypy.

  • Pharyngodon Cope, 1865. Junior homonym of Pharyngodon Diesing, 1861. Type species: Pharyngodon petasatus Cope, 1865, by monotypy.

  • Diaglena Cope, 1887. Type species: Triprion spatulatus Günther, 1882, by monotypy.

  • Diagnosis:

    For the purposes of this paper we consider that the 125 transformations in nuclear and mitochondrial protein and ribosomal genes autapomorphic of Triprion petasatus are synapomorphies of this genus. See appendix 5 for a complete list of these molecular synapomorphies. In Duellman's (2001) phylogenetic analysis of Pternohyla, Smilisca, and Triprion, the monophyly of Triprion is supported by three synapomorphies28: maxilla greatly expanded laterally; prenasal bone present (known homoplastic instance in Aparasphenodon); and presence of parasphenoid odontoids.

    Comments:

    We included a single species of this genus, and as such we did not test its monophyly, but we do not consider it controversial on the basis of the morphological evidence mentioned above.

    Contents:

    Two species. Triprion petasatus (Cope, 1865); Triprion spatulatus Günther, 1882.

    Lophiohylini Miranda-Ribeiro, 1926

    Lophiohylinae Miranda-Ribeiro, 1926. Type genus: Lophyohyla Miranda-Ribeiro, 1926.

  • Trachycephalinae B. Lutz, 1969. Type genus: Trachycephalus Tschudi, 1838.

  • Diagnosis:

    This tribe is diagnosed by 63 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. A putative morphological synapomorphy of this tribe is the presence of at least four posterior labial tooth rows in the larval oral disc (e.g., Bokermann, 1966b; Duellman, 1974; de Sá, 1983; Lannoo et al., 1987; McDiarmid and Altig, 1990; Schiesari et al., 1996; da Silva in Altig and McDiarmid, 1999b; Wogel et al., 2000) (reversals in Osteopilus marianae, O. crucialis, O. wilderi [Dunn, 1926] and in Osteocephalus oophagus [Jungfer and Schiesari, 1995]).

    Comments:

    This tribe contains all South American and West Indian casque-headed frogs and related groups. It includes Aparasphenodon, Argenteohyla, Corythomantis, Osteopilus, Phyllodytes, Tepuihyla, a new monotypic genus, and the genera Osteocephalus and Trachycephalus as redefined here.

    Recently, Kasahara et al. (2003) noticed that Aparasphenodon brunoi, Corythomantis greeningi, and Osteocephalus langsdorffii share similar chromosome morphology, where there is a clear discontinuity in the chromosome lengths of the first five pairs and the remaining seven pairs. Furthermore, they share the presence of a secondary constriction in pair 10. Available information on karyotypes of other casque-headed frogs of this clade suggests that the discontinuity in chromosome lengths occurs as well in Argenteohyla (apparent from plates published by Morand and Hernando, 1996), Phrynohyas venulosa (apparent from plates published by Bogart, 1973), and some species of Osteopilus (O. brunneus, O. dominicensis, O. marianae, O. septentrionalis), but not in Osteocephalus taurinus, the only species of the genus Osteocephalus, as redefined here, whose karyotype was studied (Anderson, 1996). The position of the secondary constriction also varies, having been observed in chromosome 4 in Argenteohyla (Morand and Hernando, 1996), chromosome 9 in Osteopilus brunneus, O. dominicensis, O. septentrionalis, and O. wilderi (Anderson, 1996), chromosome 10 in Phrynohyas venulosa (apparent from plates published by Bogart, 1973), and in chromosome 12 in Osteocephalus taurinus. The taxonomic distribution of these character states needs further study to define the inclusiveness of the clades they support.

    Delfino et al. (2002) noticed that serous skin glands of Osteopilus septentrionalis and Phrynohyas venulosa produce secretory granules with a dense cortex and a pale medulla; they observed the same in a photograph of a section of skin of Corythomantis greeningi published by Toledo and Jared (1995). Very few hylid taxa were studied for serous gland histology, and these include a few species of Phyllomedusa, Holarctic Hyla, Scinax, and Pseudis paradoxa (see Delfino et al., 2001, 2002). The taxonomic distribution of these peculiar secretory granules requires additional study to assess its level of generality and the clade or clades that it diagnoses.

    Aparasphenodon Miranda-Ribeiro, 1920

    Type Species:

    Aparasphenodon brunoi Miranda-Ribeiro, 1920, by monotypy.

    Diagnosis:

    For the purposes of this paper we consider that the 83 transformations in nuclear and mitochondrial protein and ribosomal genes autapomorphic of Aparasphenodon brunoi are synapomorphies of this genus. See appendix 5 for a complete list of these molecular synapomorphies. A possible morphological synapomorphy of this genus is the presence of a prenasal bone (Trueb, 1970a.)

    Comments:

    We included a single species of this genus, and as such we did not test its monophyly, but we consider it possible based on the morphological evidence mentioned above.

    Contents:

    Three species. Aparasphenodon bokermanni Pombal, 1993; Aparasphenodon brunoi Miranda-Ribeiro, 1920; Aparasphenodon venezolanus (Mertens, 1950).

    Argenteohyla Trueb, 1970

    Type Species:

    Hyla siemersi Mertens, 1937, by original designation.

    Diagnosis:

    Molecular autapomorphies include 102 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Apparent morphological autapomorphies of this taxon include the articulation of the zygomatic ramus of the squamosal with the pars fascialis of the maxillary, and the noticeable reduction in the size of discs of fingers and toes (Trueb, 1970b.)

    Contents:

    Monotypic. Argenteohyla siemersi (Mertens, 1937).

    Corythomantis Boulenger, 1896

    Type Species:

    Corythomantis greeningi Boulenger, 1896, by monotypy.

    Diagnosis:

    Molecular autapomorphies include 132 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these transformations. Morphological autapomorphies of this monotypic genus include the absence of palatines, and nasals that conceal the alary processes of premaxillaries (Trueb, 1970a).

    Contents:

    Monotypic. Corythomantis greeningi Boulenger, 1896.

    Itapotihyla, new genus

    Type Species:

    Hyla langsdorffii Duméril and Bibron, 1841.

    Diagnosis:

    Molecular autapomorphies include 122 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these transformations. A possible morphological autapomorphy is the presence of a prominent subcloacal flap.

    Etymology:

    From Itapoti + -Hyla. The generic name is an allusion to the resemblance of the unique known species of this genus with lichens and mosses. Itapoti is a Tupi-Guarani term, a composition of “itá” (= rock) with “poti” (= flower or to flourish), which means lichen or moss.

    Contents:

    Monotypic. Itapotihyla langsdorffii (Duméril and Bibron, 1841), new comb.

    Nyctimantis Boulenger, 1882

    Type Species:

    Nyctimantis rugiceps Boulenger, 1882, by monotypy.

    Diagnosis:

    Molecular autapomorphies include 139 transformations in mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Possible morphological autapomorphies are the development of an irregular orbital flange in the frontoparietal, and the sphenethmoid almost completely concealed dorsally by the frontoparietals and nasals (Duellman and Trueb, 1976).

    Contents:

    Monotypic. Nyctimantis rugiceps Boulenger, 1882.

    Osteocephalus Steindachner, 1862

    Type Species:

    Osteocephalus taurinus Steindachner, 1862, by subsequent designation of Kellogg (1932).

    Diagnosis:

    This genus is diagnosed by 34 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy supporting this genus.

    Contents:

    Seventeen species. Osteocephalus buckleyi (Boulenger, 1882); Osteocephalus cabrerai (Cochran and Goin, 1970); Osteocephalus deridens Jungfer, Ron, Seipp, and Almendáriz, 2000; Osteocephalus elkejungingerae (Henle, 1981); Osteocephalus exophthalmus Smith and Noonan, 2001; Osteocephalus fuscifacies Jungfer, Ron, Seipp, and Almendáriz, 2000; Osteocephalus heyeri Lynch, 2002; Osteocephalus leoniae Jungfer and Lehr, 2001; Osteocephalus leprieurii (Duméril and Bibron, 1841); Osteocephalus mutabor Jungfer and Hödl, 2002; Osteocephalus oophagus Jungfer and Schiesari, 1995; Osteocephalus pearsoni (Gaige, 1929); Osteocephalus planiceps Cope, 1874; Osteocephalus subtilis Martins and Cardoso, 1987; Osteocephalus taurinus Steindachner, 1862; Osteocephalus verruciger (Werner, 1901); Osteocephalus yasuni Ron and Pramuk, 1999.

    Osteopilus Fitzinger, 1843

    Type Species:

    Trachycephalus marmoratus Duméril and Bibron, 1841 (= Hyla septentrionalis Duméril and Bibron, 1841).

  • Calyptahyla Trueb and Tyler, 1974. Type species: Trachycephalus lichenatus Gosse, 1851 (= Hyla crucialis Harlan, 1826), by original designation.

  • Diagnosis:

    This genus is diagnosed by 43 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. No morphological synapomorphies are known for this genus.

    Contents:

    Eight species. Osteopilus brunneus (Gosse, 1851); Osteopilus crucialis (Harlan, 1826); Osteopilus dominicensis (Tschudi, 1838); Osteopilus marianae (Dunn, 1926); Osteopilus pulchrilineatus (Cope “1869” [1870]); Osteopilus septentrionalis (Duméril and Bibron, 1841); Osteopilus vastus (Cope, 1871); Osteopilus wilderi (Dunn, 1925).

    Phyllodytes Wagler, 1830

    Type Species:

    Hyla luteola Wied-Neuwied, 1824, by monotypy.

  • Amphodus Peters, “1872” [1873]. Type species: Amphodus wuchereri Peters, “1872” [1873], by original designation.

  • Lophyohyla Miranda-Ribeiro, 1923. Type species: Lophyohyla piperata Miranda-Ribeiro, 1923 (= Hyla luteola Wied-Neuwied, 1824), by original designation.

  • Diagnosis:

    This genus is diagnosed by 174 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Two morphological synapomorphies of this taxon are the presence of odontoids on the mandible and on the cultriform process of the parasphenoid (Noble, 1931).

    Contents:

    Eleven species placed in four species groups (Caramaschi et al., 2004a), one of which is monotypic.

    Phyllodytes auratus Group

    Diagnosis:

    We are not aware of any possible synapomorphy for this group.

    Comments:

    We did not include any exemplar of this group, but we continue to recognize it following Caramaschi et al. (2004b) pending a rigorous test. Caramaschi et al. (2004b) diagnosed the different species groups based on color patterns; it is unclear if any of these patterns could be considered synapomorphic.

    Contents:

    Two species. Phyllodytes auratus (Boulenger, 1917); Phyllodytes wuchereri (Peters, “1872” [1873]).

    Phyllodytes luteolus Group

    Diagnosis:

    We are not aware of any possible synapomorphy for this group.

    Comments:

    We included a single exemplar of this group and thus did not test its monophyly, but we continue to recognize it following Caramaschi et al. (2004b) pending a rigorous test. See comments for the P. auratus group.

    Contents:

    Six species. Phyllodytes acuminatus Bokermann, 1966; Phyllodytes brevirostris Peixoto and Cruz, 1988; Phyllodytes edelmoi Peixoto, Caramaschi, and Freire, 2003; Phyllodytes kautskyi Peixoto and Cruz, 1988; Phyllodytes luteolus (Wied-Neuwied, 1824); Phyllodytes melanomystax Caramaschi, Silva, and Britto-Pereira, 1992.

    Phyllodytes tuberculosus Group

    Diagnosis:

    We are not aware of any possible synapomorphy for this group.

    Comments:

    We did not include any exemplar of this group, but we continue to recognize it following Caramaschi et al. (2004b) pending a rigorous test. See comments for the P. auratus group.

    Contents:

    Two species. Phyllodytes punctatus Caramaschi and Peixoto, 2004; Phyllodytes tuberculosus Bokermann, 1966.

    Species of Phyllodytes Unassigned to Group

    Peixoto et al. (2003) assigned Phyllodytes gyrinaethes Peixoto, Caramaschi, and Freire, 2003 to its own species group. As stated earlier in this paper, we consider that monotypic species groups are not informative.

    Tepuihyla Ayarzagüena and Señaris, “1992” [1993]

    Type Species:

    Hyla rodriguezi Rivero, 1968, by original designation.

    Diagnosis:

    For the purposes of this paper we consider that the 90 transformations in nuclear and mitochondrial protein and ribosomal genes autapomorphic of Tepuihyla edelcae are synapomorphies of this genus. See appendix 5 for a complete list of these molecular synapomorphies. In the context of our results, the reduction of webbing between toes I and II is a putative morphological synapomorphy of this genus (Ayarzagüena et al., “1992” [1993b]; several instances of homoplasy within Lophiohylini, in Phyllodytes, and in the clade composed of Corythomantis, Argenteohyla, Aparasphenodon, and Nyctimantis).

    Comments:

    We included a single species of Tepuihyla, and as such we did not test its monophyly. We continue to recognize it following Ayarzagüena and Señaris (“1992” [1993b]) until its monophyly is rigorously tested.

    Contents:

    Eight species. Tepuihyla aecii (Ayarzagüena, Señaris, and Gorzula, “1992” [1993]); Tepuihyla celsae Mijares-Urrutia, Manzanilla-Pupo, and La Marca, 1999; Tepuihyla edelcae (Ayarzagüena, Señaris, and Gorzula, “1992” [1993]); Tepuihyla galani (Ayarzagüena, Señaris, and Gorzula, “1992” [1993]); Tepuihyla luteolabris (Ayarzagüena, Señaris, and Gorzula, “1992” [1993]); Tepuihyla rimarum (Ayarzagüena, Señaris, and Gorzula, “1992” [1993]); Tepuihyla rodriguezi (Rivero, 1968); Tepuihyla talbergae Duellman and Yoshpa, 1996.

    Trachycephalus Tschudi, 1838

    Type Species:

    Trachycephalus nigromaculatus Tschudi, 1838, by monotypy.

  • Phrynohyas Fitzinger, 1843. Type species: Hyla zonata Spix, 1824 (= Rana venulosa Laurenti, 1768). new synonymy.

  • Acrodytes Fitzinger, 1843. Type species: Hyla venulosa Daudin, 1802 (= Rana venulosa Laurenti, 1768), by original designation.

  • Scytopis Cope, 1862. Type species: Scytopis hebes Cope, 1862, by monotypy.

  • Tetraprion Stejneger and Test, 1891. Type species: Tetraprion jordani Stejneger and Test, 1891, by original designation.

  • Diagnosis:

    This genus is diagnosed by 37 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. The only possible morphological synapomorphy that we are aware of for this genus is the presence of paired vocal sacs protruding posterior to the angles of the jaws when inflated (Trueb and Duellman, 1971; see also Tyler, 1971).

    Comments:

    We are including Phrynohyas in the synonymy of Trachycephalus to avoid the nonmonophyly of the two genera. There are other alternatives to resolve this situation, such as restricting Trachycephalus to the southeastern Brazilian taxa, including P. mesophaea, while retaining Phrynohyas for the remaining species currently placed in that genus, and resurrecting Tetraprion to accommodate T. jordani. We consider that the action taken here is the most conservative.

    Contents:

    Ten species. Trachycephalus atlas Bokermann, 1966; Trachycephalus coriaceus (Peters, 1867), new comb.; Trachycephalus hadroceps (Duellman and Hoogmoed, 1992), new comb.; Trachycephalus imitatrix (Miranda-Ribeiro, 1926), new comb.; Trachycephalus lepidus (Pombal, Haddad, and Cruz, 2003), new comb.; Trachycephalus mesophaeus (Hensel, 1867), new comb.; Trachycephalus nigromaculatus Tschudi, 1838; Trachycephalus resinifictrix (Goeldi, 1907), new comb.; Trachycephalus venulosus (Laurenti, 1768), new comb.

    Incertae Sedis and Nomina Dubia

    The taxonomic scheme introduced above comprises most of the valid species of Hylinae. However, there are a number of species of former Hyla whose position in this new taxonomy is uncertain. There are two likely reasons for this. (1) The species have known type material and/or are known from multiple specimens, but the available information is not sufficient to allow even the tentative assignment to any of the taxonomic groups, so they are here considered as incerta sedis. (2) The species are known mostly from their original descriptions or type materials are reported to be lost or lack clear locality data. These are considered nomina dubia. See appendix 4 for additional comments on some of these species. Within the first category fall Hyla alboguttata Boulenger, 1882, Hyla chlorostea Reynolds and Foster, 1992, Hyla helenae Ruthven, 1919, Hyla imitator (Barbour and Dunn, 1921), Hyla inframaculata Boulenger, 1882, Hyla vigilans Solano, 1971, and Hyla warreni Duellman and Hoogmoed, 1992. In the second category we include (those with extant type material are followed by an asterisk) Calamita melanorabdotus Schneider, 1799, Calamita quadrilineatus Schneider, 1799, Hyla auraria* Peters, 1873, Hyla fusca Laurenti, 1768, Hypsiboas hypselops Cope, 1871, Hyla molitor* Schmidt, 1857, Hyla palliata Cope, 1863, Hyla roeschmanni De Grys, 1938, Hyla surinamensis Daudin, 1802, and Litoria americana* Duméril and Bibron, 1841.

    PHYLLOMEDUSINAE GÜNTHER, 1858

  • Phyllomedusidae Günther, 1858. Type genus: Phyllomedusa Wagler, 1830.

  • Pithecopinae B. Lutz, 1969. Type genus: Pithecopus Cope, 1866.

  • Diagnosis:

    The monophyly of this subfamily is supported by 95 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. A possible morphological synapomorphy is the pupil constricting to vertical ellipse (Duellman, 2001; known instance of homoplasy in Nyctimystes). There are several larval character states that may be synapomorphies, such as: the ventrolateral position of the spiracle; arcus subocularis of larval chondrocranium with distinct lateral processes; ultralow suspensorium; secondary fenestrae parietales; and absence of a passage between ceratohyal and ceratobranchial I (Haas, 2003).

    Comments:

    Duellman (2001) considered the presence of a process on the medial surface of metacarpal II a synapomorphy of Phyllomedusinae, with a known instance of homoplasy in Centrolenidae. However, because Phyllomedusinae appears to be the sister taxon of Pelodryadinae, the situation is more complex. As noticed by Tyler and Davies (1978b), this character state is also present in some species groups of Litoria, so the internal topology of Pelodryadinae will determine whether this character state is indeed a synapomorphy of Phyllomedusinae, with homoplastic instances in Pelodryadinae, or if it is a synapomorphy of Phyllomedusinae + Pelodryadinae, with subsequent reversals in the latter taxon. The supplementary posterolateral elements of the m. intermandibularis have been considered a synapomorphy of Phyllomedusinae (Duellman, 2001; Tyler, 1971). As mentioned earlier, because it is more parsimonious to interpret the sole presence of supplementary elements of the m. intermandibularis as a synapomorphy of Pelodryadinae + Phyllomedusinae, at this point it is ambiguous which of the positions (apical as present in Pelodryadinae or posterolateral as in Phyllomedusinae) is the plesiomorphic state of this clade.

    The absence of the slip of the m. depressor mandibulae that originates from the dorsal fascia at the level of the m. dorsalis scapulae (which subsequently reverses in Hylomantis and Phyllomedusa, see below) could also be a synapomorphy of Phyllomedusinae; however, its taxonomic distribution among non-phyllomedusines needs to be assessed. This is most needed in Pelodryadinae, where as far as we are aware, all observations on this muscle are limited to Starrett's (1968) unpublished dissertation where she commented on its morphology in 2 of the 172 known valid species of the subfamily. Oviposition on leaves out of water could also be another synapomorphy of Phyllomedusinae, but this is dependent on the position of Phrynomedusa within Phyllomedusinae (species of this genus do not oviposit on leaves but on rock crevices or fallen trunks) and on the topology of Pelodryadinae (however, only two species of Pelodryadinae, Litoria iris and L. longirostris, are known to lay eggs out of water, and not necessarily on leaves; Tyler, 1963; McDonald and Storch, 1993).

    Several transformations that resulted as synapomorphies of Phyllomedusinae in Burton's (2004) analysis optimize ambiguously in our trees because their distribution is unknown in Cruziohyla new genus. Consequently, it is unclear which transformations are synapomorphic of the subfamily and which ones support the monophyly of internal clades. These transformations are: two insertions of the m. flexor digitorum brevis superficialis; the tendon of the m. flexor digitorum brevis superficialis divided along its length into a medial tendon, from which arise tendo superficialis IV and m. lumbricalis longus digiti V, and a lateral tendon from which arise tendo superficialis V and m. lumbricalis longus digiti IV; tendo superficialis pro digiti II arising from a deep, triangular muscle, which originates on the distal tarsal 2–3; tendo superficialis pro digiti III arising entirely from the margin of the aponeurosis plantaris; two tendons of insertion of m. lumbricalis longus digiti V arising from two equal muscle slips; pennate insertion of the lateral slip of the medial m. lumbricalis brevis digiti V; m. transversus metatarsus II broad, occupying the entire length of metatarsal II; m. transversus metatarsus III broad, occupying more than 75% of the length of metatarsal III; m. extensor brevis superficialis digiti III with two insertions, a flat tendon onto basal phalanx III and a pennate insertion on metatarsus III; and finally the m. extensor brevis superficialis digiti IV with a single origin with belly undivided. The presence of m. flexor teres hallucis is shared with Pelodryadinae; however, Burton (2004) stressed that in that subfamily, presence or absence of this muscle is subject to great intraspecific variation, without providing information as to the states present in the particular specimens he studied, so the character was scored as missing data in our matrix.

    There are several other character systems that will likely provide additional synapomorphies for this group of frogs. Manzano and Lavilla (1995b) and Manzano (1997) described several unique character states from musculature, whose taxonomic distribution across all Phyllomedusinae needs to be assessed. Tyler and Davies (1978a) mentioned that Phyllomedusinae are the only hylids where the mandibular branch of the trigeminal nerve subdivides into two twigs after traversing the mandible. Various authors (e.g., Kenny; 1969; Cruz, 1982; Lescure et al., 1995) noticed that larvae of several species of Phyllomedusinae are usually suspended in water in an oblique or even vertical position relative to the water surface. Bagnara (1974) observed a light-sensitive tail-darkening reaction in larvae of two phyllomedusines (Pachymedusa dacnicolor and Phyllomedusa trinitatis), and we observed a similar reaction in tadpoles of Phyllomedusa tetraploidea (Faivovich, pers. obs.). Further research will determine how inclusive is the clade or clades supported by these synapomorphies.

    The presence of multiple bioactive peptides has been suggested as a distinctive character of Phyllomedusinae (Cei, 1985). Since the beginning of the biochemical prospecting, it has become evident that Phyllomedusinae have several different classes of bioactive peptides (Erspamer, 1994), some unique (e.g., sauvagine, deltorphins), some not (e.g., bombesins, caeruleins), as do the Pelodryadinae (Apponyi et al., 2004). Because there are multiple bioactive peptides, it seems reasonable to consider the different peptide families individually as potential synapomorphies of Phyllomedusinae, Pelodryadinae, or Phyllomedusinae + Pelodryadinae. More work needs to be done to better understand the taxonomic distribution of the different classes of peptides.

    Agalychnis Cope, 1864

    Type Species:

    Agalychnis callidryas Cope, 1862, by original designation.

    Diagnosis:

    The monophyly of this group is supported by 23 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Agalychnis has extensively developed webbing on hands and feet in relationship with Pachymedusa, Hylomantis, Cruziohyla new genus, Phasmahyla, and Phyllomedusa. Also, with the exception of A. annae, which has a yellow iris, the other species have either a red or a dark red iris.

    Comments:

    Considering the lack of knowledge regarding the internal structure of Pelodryadinae, where some species have extensive hand and foot webbing (e.g., Tyler, 1968), it is still unknown if these character states are plesiomorphic for Phyllomedusinae. Consequently, at this stage we do not know exactly in which point of the topology of Phyllomedusinae they are homoplastic (both hands and foot webbing are developed in Cruziohyla new genus and, somewhat less extensively, in Phrynomedusa).

    Contents:

    Six species. Agalychnis annae (Duellman, 1963); Agalychnis callidryas Cope, 1862; Agalychnis litodryas (Duellman and Trueb, 1967); Agalychnis moreletii (Duméril, 1853); Agalychnis saltator (Taylor, 1955); Agalychnis spurrelli (Boulenger, “1913” [1914].)

    Cruziohyla, new genus

    Type Species:

    Agalychnis calcarifer Boulenger, 1902.

    Diagnosis:

    For the purposes of this paper we consider that the 171 transformations in nuclear and mitochondrial protein and ribosomal genes autapomorphic of Cruziohyla calcarifer are synapomorphies of this genus. See appendix 5 for a complete list of these molecular synapomorphies. Possible morphological synapomorphies include the extensive hand and foot webbing (but see comments for Agalychnis) and the development of tadpoles in water-filled depressions on fallen trees. See comments below.

    Etymology:

    The name comes from the Latinization of Cruz, Cruzius + connecting -o + Hyla. We dedicate this new genus to our colleague and friend Carlos Alberto Gonçalves da Cruz, in recognition of his various contributions to our knowledge of Phyllomedusinae.

    Comments:

    Phrynomedusa, the only genus of Phyllomedusinae missing from our analysis, shares with Cruziohyla a bicolored iris, developed foot webbing (although more extensively developed in Cruziohyla), and oral disc with complete marginal papillae in the larvae. However, they differ in that eggs of Phrynomedusa are laid in rock crevices (A. Lutz and B. Lutz, 1939; Weygoldt, 1991) or fallen trunk cavities above streams, from where tadpoles drop and develop. The larvae of Cruziohyla, unlike those of most other known Phyllomedusinae, develop in water-filed depressions of fallen trees (Donnelly et al., 1987; Hoogmoed and Cadle, 1991; Caldwell, 1994; Block et al., 2003). Hoogmoed and Cadle (1991) reported two situations where tadpoles associated with Agalychnis craspedopus were found in small pools in the forest, without a clear indication of where the eggs were laid. This could be interpreted either as a polymorphic reproductive trait or as an indication that more than one species is involved.

    The oral disc with marginal papillae as a morphological synapomorphy of Cruziohyla + Phrynomedusa should be taken cautiously because of our general ignorance of the internal topology of Pelodryadinae. Some Pelodryadinae also have an oral disc with complete marginal papillae (see Anstis, 2002), and further analysis could show that this is actually a plesiomorphy for Phyllomedusinae. The same problem holds for the presence of foot webbing.

    Instead of creating Cruziohyla to include Agalychnis calcarifer and A. craspedopus, we could place both species in Phrynomedusa. Both alternatives imply taxonomic risks (in particular, that Cruziohyla could be shown to be nested within Phrynomedusa). Taking into account our almost complete ignorance of the relationships of Pelodryadinae, and therefore character-state polarities at its base, and that Phrynomedusa could not be included in this analysis, we consider that at this stage it is more appropriate to create Cruziohyla than to enlarge Phrynomedusa, without being certain about character polarities at the base of Phyllomedusinae.

    Agalychnis craspedopus could not be included in the analysis, but the close relationship between A. craspedopus and A. calcarifer seems uncontroversial, as both have been repeatedly associated by some authors (Duellman, 1970; Hoogmoed and Cadle, 1991; Duellman, 2001).

    Contents:

    Two species. Cruziohyla calcarifer (Boulenger, 1902), new comb., Cruziohyla craspedopus (Funkhouser, 1957), new comb.

    Hylomantis Peters, “1872” [1873]

    Type Species:

    Hylomantis aspera Peters, “1872” [1873], by monotypy.

    Diagnosis:

    The monophyly of this group is supported by 38 transformations in mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. We are not aware of any morphological synapomorphy supporting this genus.

    Comments:

    Only Phyllomedusa lemur of the P. buckleyi group was included in the analysis, and it obtains as the sister group of our exemplar of Hylomantis, H. granulosa, but with a Bremer support value of 3. While it is evident that this group should be excluded from Phyllomedusa, the possible taxonomic actions (whether to create a new genus or to include it in Hylomantis) deserve further discussion. From the definition of the group given by Cannatella (1980), the only character state that could be considered a synapomorphy is the bright orange flanks in life. The other character states included by Cannatella (1980) are either likely symplesiomorphies (absence of the slip of the m. depressor mandibulae originating from the dorsal fascia at the level of the m. dorsalis scapulae; hands and feet less than one-fourth webbed; parotoid gland not differentiated; palpebrum unpigmented; frontoparietal fontanelle exposed, large, and oval; oral discs of larvae lacking marginal papillae anteriorly) or character states whose taxonomic distribution in Phyllomedusinae makes their polarity unclear (lack of spots or pattern on flanks; cream or white iris; size; dorsum uniformly green by day; presence or absence of calcars). Like the P. buckleyi group, the two species included in Hylomantis by Cruz (1990) also lack spots or pattern on flanks, which are light yellow (instead of bright orange). At this point, we have no evidence regarding the polarity of these two character states; consequently, we consider that the morphological evidence of monophyly of the P. buckleyi group is weak. Considering that we could not test the monophyly of the P. buckleyi group, and that available morphological evidence for its monophyly is not compelling, provisionally, and with the caveat that the molecular support for this grouping is rather weak, we prefer to include all species of this group in Hylomantis, where we recognize them as a separate species group, pending a rigorous test of its monophyly when a denser taxon sampling becomes available.

    Contents:

    Eight species placed in two species groups.

    Hylomantis aspera Group

    Diagnosis:

    Possible morphological synapomorphies of this group are the lanceolate discs and presence of the slip of the m. depressor mandibulae originating from the dorsal fascia at the level of the m. dorsalis scapulae (known homoplastic instance in Phyllomedusa and several other anurans).

    Comments:

    We included a single species of this group, and as such we did not we did not test its monophyly, but we recognize it based on the aforementioned evidence.

    Contents:

    Two species. Hylomantis aspera Peters, “1872” [1873]; Hylomantis granulosa (Cruz, “1988” [1989]).

    Hylomantis buckleyi Group

    Diagnosis:

    The only apparent morphological synapomorphy of this group is the possession of bright orange flanks in life (Cannatella, 1980).

    Comments:

    We included a single species of this group, and as such we did not we did not test its monophyly. We recognize it following Cannatella (1980), pending a rigorous test of its monophyly. Ruiz-Carranza et al. (1988) tentatively included Phyllomedusa danieli in the P. buckleyi group because of the reduced webbing, absence of parotoid glands, toe I shorter than toe II, presence of a calcar, and unpigmented palpebrum. As the authors noted, these characteristics are also shared with Phasmahyla and Hylomantis (they refer to these genera using the former species groups of Phyllomedusa), but some also with Phrynomedusa. One difference they noticed was the golden iris coloration instead of white; however, in the present scenario the polarity of this state is unclear (a white iris is present in Phasmahyla and Hylomantis, as redefined here). A difference is the large snout–vent length (SVL) of P. danieli compared with the species of Hylomantis (the only reported specimen of P. danieli, a female, is 81 mm SVL; females of the other species reach a maximum of 57 mm, according to Cannatella [1980]). Phyllomedusa danieli shares with the Hylomantis buckleyi group its only apparent morphological synapomorphy (but see comments above), the bright orange flanks in life. Because of this, we tentatively include P. danieli in Hylomantis.

    Contents:

    Six species. Hylomantis buckleyi (Boulenger, 1882), new comb.; Hylomantis danieli (Ruiz-Carranza, Hernández-Camacho, and Rueda-Almonacid, 1988), new comb.; Hylomantis hulli (Duellman and Medelson, 1995), new comb.; Hylomantis lemur (Boulenger, 1882), new comb.; Hylomantis medinai (Funkhouser, 1962), new comb.; Hylomantis psilopygion (Cannatella, 1980), new comb.

    Pachymedusa Duellman, 1968

    Type Species:

    Phyllomedusa dacnicolor Cope, 1864.

    Diagnosis:

    Molecular autapomorphies include 105 transformations in nuclear and mitochondrial proteins and ribosomal genes. See appendix 5 for a complete list of these transformations. Possible morphological autapomorphies are the first toe opposable to others, reticulated palpebral membrane (homoplastic with some species of Phyllomedusa; Duellman et al., 1988b), and the iris reticulation (Duellman, 2001).

    Comments:

    Duellman (2001) also included the toes about one-fourth webbed as an autapomorphy of Pachymedusa. In the context of our results, this is probably not an autapomorphy, as the webbing is also equally or more reduced in Hylomantis (as redefined here), Phasmahyla, and Phyllomedusa.

    Contents:

    Monotypic. Pachymedusa dacnicolor (Cope, 1864).

    Phasmahyla Cruz, 1990

    Type Species:

    Phyllomedusa guttata A. Lutz, 1924, by original designation.

    Diagnosis:

    The monophyly of this genus is supported by 94 transformations in mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these molecular synapomorphies. Possible morphological synapomorphies of this genus are the absence of a vocal sac, and the modification of the larval oral disc into an anterodorsal funnel-shaped structure (Cruz, 1990).

    Comments:

    Cruz (1990) mentioned the absense of parotoid glands in Phasmahyla but stressed the presence of a pair of latero-dorsal glands. While these glands could be cosidered as possible synapomorphies of Phasmahyla, additional work is needed in order to determine if they could be considered as homologous to the parotoid glands present in Phyllomedusa.

    Contents:

    Four species. Phasmahyla cochranae (Bokermann, 1966); Phasmahyla exilis (Cruz, 1980); Phasmahyla guttata (A. Lutz, 1924); Phasmahyla jandaia (Bokermann and Sazima, 1978).

    Phrynomedusa Miranda-Ribeiro, 1923

    Type Species:

    Phrynomedusa fimbriata Miranda-Ribeiro, 1923, by subsequent designation of Miranda-Ribeiro (1926).

    Diagnosis:

    A likely synapomorphy of this taxon is the oviposition in rock crevices or fallen trunks overhanging streams (A. Lutz and B. Lutz, 1939; Weygoldt, 1991).

    Comments:

    We did not include any species of this genus in our analysis. Besides the place of oviposition, we are not aware of any other possible synapomorphy of Phrynomedusa. This is not a strong support for its monophyly, particularly if we consider that with the exception of Weygoldt's (1991) studies in captivity of Phrynomedusa marginata, reports on oviposition of Phrynomedusa are mostly anecdotal.

    The most obvious difference between Phrynomedusa and Cruziohyla is the impressive SVL difference. Although the reduction in SVL could actually be a synapomorphy of Phrynomedusa, considering how rudimentary is our knowledge of the topology of Pelodryadinae, and considering its taxonomic distribution in Phyllomedusinae (Phasmahyla, Hylomantis, and Cruziohyla also have a proportionally smaller SVL, as do some species of Phyllomedusa), the polarity of SVL as a character, if definable at all, is far from clear. Phrynomedusa could either be the sister group of Cruziohyla or the remaining Phyllomedusinae.

    Contents:

    Five species. Phrynomedusa appendiculata (A. Lutz, 1925); Phrynomedusa bokermanni Cruz, 1991; Phrynomedusa fimbriata Miranda-Ribeiro, 1923; Phrynomedusa marginata (Izecksohn and Cruz, 1976); Phrynomedusa vanzolinii Cruz, 1991.

    Phyllomedusa Wagler, 1830

    Type Species:

    Rana bicolor Boddaert, 1772 by monotypy.

  • Pithecopus Cope, 1866. Type species: Phyllomedusa azurea Cope, 1862.

  • Bradymedusa Miranda-Ribeiro, 1926. Type species: Hyla hypochondrialis Daudin, 1800, by subsequent designation of Vellard (1948).

  • Diagnosis:

    The monophyly of this taxon is supported by 49 transformations in nuclear and mitochondrial protein and ribosomal genes. See appendix 5 for a complete list of these transformations. Apparent morphological synapomorphies of Phyllomedusa are the presence of parotoid glands, toe I longer than toe II, and presence of the slip of the m. depressor mandibulae originating from the dorsal fascia at the level of the m. dorsalis scapulae (known instance of homoplasy in the Hylomantis granulosa group, and several other anurans) (Duellman et al., 1988b).

    Comments:

    The transformation from presence to absence of the m. abductor brevis plantae hallucis optimizes ambiguously in our analysis because the state of this character is unknown in Phasmahyla.

    Blaylock et al. (1976) described the peculiar wiping behavior in P. boliviana (as P. pailona), P. hypochondrialis, P. sauvagii, and P. tetraploidea (as P. iheringii). This behavior was subsequently reported in P. distincta, P. tarsius (Castanho and De Luca, 2001), and P. iheringii (Langone et al., 1985). Castanho and De Luca (2001) further noticed a peculiar daily molting behavior. Further research on the taxonomic distribution of these behaviors in Phyllomedusa will determine the limits of the group(s) they support. The presence of the so-called lipid glands has been so far been reported in the five species of Phyllomedusa that were studied (P. bicolor, P. boliviana, P. hypochondrialis, P. sauvagii, and P. tetraploidea; Blaylock et al., 1976; Delfino et al., 1998; Lacombe et al., 2000) and were noticed to be unique to the genus by Delfino et al. (1998), so they could likely be another synapomorphy. As noticed by Cruz (1982), and corroborated by most larval descriptions of Phyllomedusinae, the larvae of most species of Phyllomedusa,29 as redefined here, have the third posterior row of labial teeth reduced in relation to the first and second posterior rows.

    Contents:

    Twenty-six species, some of them included in four species groups.

    Phyllomedusa burmeisteri Group

    Diagnosis:

    We are not aware of any synapomorphy of this group.

    Comments:

    We included only a single species of this group in our analysis, and as such we did not test its monophyly, but we recognize it following Pombal and Haddad (1992), pending a rigorous test of its monophyly.

    Contents:

    Four species. Phyllomedusa burmeisteri Boulenger, 1882; Phyllomedusa distincta B. Lutz, 1950; Phyllomedusa iheringii Boulenger, 1885; Phyllomedusa tetraploidea Pombal and Haddad, 1992.

    Phyllomedusa hypochondrialis Group

    Diagnosis:

    We are not aware of any synapomorphy of this group.

    Comments:

    We included a single species of this group, and as such we did not test its monophyly, but we recognize it following Brandão (2002), pending a rigorous test of its monophyly. Manzano and Lavilla (1995b) described the muscle epicoracoideus in Phyllomedusa hypochondrialis, and Manzano (1997) noticed its absence in other species that she studied (P. atelopoides, P. boliviana, and P. sauvagii). Our observations on the only other species of the group available to us, P. rohdei (AMNH A-20263), indicate that it also has the m. epicoracoideus, so we consider the presence of this muscle a possible synapomorphy of the group. All species of this group lack vomerine teeth, as do Phasmahyla, Phyllomedusa palliata and some species of Phrynomedusa (Brandão, 2002; Cruz, 1990). The taxonomic distribution of other myological peculiarities described by Manzano and Lavilla (1995b) in P. hypochondrialis, such as the presence of thin and/or shortened muscles, and unusual insertions of some of them, needs to be assessed in other Phyllomedusinae.

    Contents:

    Six species. Phyllomedusa ayeaye (B. Lutz, 1966); Phyllomedusa centralis Bokermann, 1965; Phyllomedusa hypochondrialis (Daudin, 1800); Phyllomedusa megacephala (Miranda-Ribeiro, 1926); Phyllomedusa oreades Brandão, 2002; Phyllomedusa rohdei Mertens, 1926.

    Phyllomedusa perinesos Group

    Diagnosis:

    A possible synapomorphy of this group is the purple coloration on the hands, feet, flanks, and concealed surfaces, as well as the purple venter with white granules (Cannatella, 1982).

    Comments:

    We did not include any exemplar of this group in the analysis. Its monophyly is tentatively assumed following Cannatella (1982) and is based on the evidence mentioned above.

    Contents:

    Four species. Phyllomedusa baltea Duellman and Toft, 1979; Phyllomedusa duellmani Cannatella, 1982; Phyllomedusa ecuatoriana Cannatella, 1982; Phyllomedusa perinesos Duellman, 1973.

    Phyllomedusa tarsius Group

    Diagnosis:

    We are not aware of any synapomorphy supporting the monophyly of this group.

    Comments:

    We included a single species of this group, and as such we did not test its monophyly, but we continue to recognize it following De la Riva (1999) until its monophyly is rigorously tested.

    Contents:

    Four species. Phyllomedusa boliviana Boulenger, 1902; Phyllomedusa camba De la Riva, 2000; Phyllomedusa sauvagii Boulenger, 1882; Phyllomedusa tarsius (Cope, 1868).

    Species of Phyllomedusa Unassigned to Group

    There are several species that are currently not assigned to any group. These are: Phyllomedusa atelopoides Duellman, Cadle, and Cannatella, 1988; Phyllomedusa bicolor (Boddaert, 1772); Phyllomedusa coelestis (Cope, 1874); Phyllomedusa palliata Peters, “1872” [1873]; Phyllomedusa tomopterna (Cope, 1868); Phyllomedusa trinitatis Mertens, 1926; Phyllomedusa vaillanti Boulenger, 1882; and Phyllomedusa venusta Duellman and Trueb, 1967.

    BIOGEOGRAPHICAL COMMENTARY

    Our objective here is to comment on patterns of distribution among the major biogeographic/tectonic units and not to provide a detailed biogeographic analysis. The distribution/biogeographic units of our discussion are (1) Australia plus New Guinea, (2) continental South America, (3) Middle America (in the sense of being composed of tropical Mexico, the Chortis Block of Central America, and the Panamanian Isthmus), and (4) the temperate Holarctic. Clearly all of these regions have histories that provide clues as to movements and diversifications within these areas. Because of the enormity of the topic of biogeography for the entire Hylidae, our comments will be truncated, limited either by our taxonomic sampling, knowledge of earth history, or phylogenetic resolution. Nevertheless, there are obvious geographic patterns that warrant our attention.

    The distribution of the Hylidae strongly suggests a southern-continent origin of the taxon, a conclusion in accord with suggestions based on different lines of evidence advanced over the last 80 years (Metcalf, 1923a, 1923b, 1928; Duellman, 1970, 2001; Savage, 2002a) and supported by the observation that all the major groups of hylids have their centers of diversity in southern continents, with only phylogenetically secondary centers of diversification existing in Middle America, North America, and even more attenuated areas of radiation in Eurasia.

    Relationships Between Australia and South America

    The relationship between Australian and South American taxa has been previously noted for the Hylidae (Darst and Cannatella, 2004; Hoegg et al., 2004) and in several other groups (see Sanmartín and Ronquist [2004] for a review). In our results (fig. 13), the Australopapuan Pelodryadinae forms the sister taxon of the predominantly South American Phyllomedusinae, a distribution which we think speaks to one of the earliest patterns in the entire Hylidae, that of an Australia–Antarctica–South American connection. We cannot address any other topics of pelodryadine biogeography due to our limited sampling.

    Relationships Between South and Middle America

    Having the sister taxon of the Phyllomedusinae in Australia strongly suggests a southern (South American) origin of the Phyllomedusinae, although distribution of the most basal taxon, Cruziohyla calcarifer, is in Chocó/lower Middle America, with the remaining taxa found from northwestern Mexico to southern Brazil. This distribution suggests that the phyllomedusine biogeographic pattern is not recent. Assuming a connection between Australia and South America was by way of Antarctica, one would be driven to the conclusion that South America is the home of the phyllomedusines. The fact that we could not include any exemplar of Phrynomedusa, an Atlantic Forest genus possibly related with Cruziohyla, could possibly cloud the general picture.

    Apart from the Hylini, there are several instances of members of the other three tribes of Hylinae having a Middle American distribution (figs. 14–16), corroborating the suggestion of Duellman (2001) regarding the existence of several independent vicariance or dispersal events with hylids between South America and Middle America. Our results imply eight independent events of dispersal from South America into Middle America: (1) Dendropsophus ebraccatus, (2) D. microcephalus, (3) ancestor of Hylini, (4) Hypsiboas boans, (5) H. rufitelus, (6) Scinax boulengeri, (7) S. elaeochrous and S. staufferi, and (8) Trachycephalus venulosus. However, this number is a clear underestimation because we did not include other terminals that also have a Middle American distribution (e.g., Dendropsophus phlebodes, D. robertmertensi, D. sartori, D. subocularis, Hypsiboas pugnax, H. rosenbergi, Scinax altae, and S. rostratus) and which may represent additional entries into Middle America from South America. For some of these species (e.g., Scinax altae and S. rostratus) we consider it likely that they are related to other Middle American members of their respective phylogenetic nearest relatives (hence not adding to the number of independent biogeographic events). Other species (Dendropsophus subocularis) might imply additional events because they are probably nested within mostly South American clades. The uncertain position of the other species (e.g., Dendropsophus phlebodes, D. robertmertensi, D. sartori, Hypsiboas pugnax, H. rosenbergi) in our phylogenetic hypothesis does not allow us to suggest that their presence in Middle America either represents independent events or that they are contained within other groups of species whose ancestors moved into Middle America.

    Considering the Hylinae with a Middle American origin, the results imply two biogeographic events to explain the presence of these lineages in South America: the cases of Scinax elaeochrous (fig. 15) and Smilisca phaeota (fig. 16). Once again, this is a minimal number of events. The monophyly of Ecnomiohyla could be in error, because most species were unavailable for study and at least E. tuberculosa (not studied) is possibly unrelated to the Middle American fringe-limbed treefrogs. Duellman (2001) suggested that Smilisca sila and S. sordida together are monophyletic (apparent synapomorphies: ventral oral disc in the larvae and small inner metatarsal tubercle) and together are the sister taxon of S. puma. If this hypothesis withstands further testing, it would represent a third independent biogeographic event involving a Middle American lineage present in northern South America.

    South America

    Guayana Highlands–Andes–Atlantic Forest

    Within Cophomantini (fig. 14), the first four genera contain elements from three characteristic formations, quite distant geographically from each other: Myersiohyla is composed solely of Guayana Highlands species; Hyloscirtus is composed exclusively of Andean species; Bokermannohyla and Aplastodiscus are composed almost exclusively of species from the southeastern Brazilian Atlantic Forest and Rocky fields associated with this formation and to the Cerrado. We are not aware of any similar biogeographic pattern in any other animal group.

    Guayana Highlands

    Our analysis included 6 of the 19 hylid endemics (updated from Duellman's [1999] list by adding Hypsiboas rhythmicus) of the Guayana Highlands, plus two undescribed species. The topology suggests a minimum of four independent occurrences of endemic hylines in the Guayana Highlands (figs. 14, 16): (1) the Hypsiboas benitezi group (this group also contains three species from western Amazonia: H. hutchinsi, H. microderma, and Hypsiboas sp. 2), (2) Hypsiboas sibleszi, (3) Myersiohyla, and (4) Tepuihyla. In the H. benitezi group, it is ambiguous whether there is an origin in the Guayana Highlands with a subsequent dispersal/vicariance event into northwestern Amazonia, or two independent events that led to the presence of these species in the highlands.

    Considering the 13 taxa from the Guayana Highlands that were unavailable for this study, all but two species are members of groups represented in the analysis. Seven are species of Tepuihyla, three are species of Myersiohyla, two are species of Scinax (S. danae, and S. exiguus), one is a species tentatively associated with the Hypsiboas benitezi group (H. rhythmicus), and one is incerta sedis (“Hyla warreni”). Phylogenetic relationships of the two species of Scinax with other species of the genus are still unknown, as is the position of “Hyla warreni”. When considering relationships of the Guayana Highlands lineages with the other Hylinae, current evidence suggests they are related to elements from the Amazon Basin (Osteocephalus, Hypsiboas microderma, Hypsiboas sp. 2) and the Chocó (Hypsiboas picturatus).

    Impact of the Andes in Hyline Evolution

    The uplift of the Andes and subsequent climatic changes in the Quaternary have an impressive correlation with large radiations of anuran groups, such as certain Bufonidae, Centrolenidae, Dendrobatidae, Hemiphractinae, and Eleutherodactylinae (e.g., Lynch, 1986; Coloma, 1995; Lynch and Duellman, 1997; Lynch et al., 1997; Lynch, 1998; Duellman, 1999). Previous knowledge of hylid distribution, as well as our results, suggests a much more limited impact of the Andes in hylid radiation and speciation, with three hyline radiations in the Andes (figs. 14, 16): Hyloscirtus, the Andean clade of the Hypsiboas pulchellus group, and the Dendropsophus columbianus + D. labialis groups clade. If we consider the taxa that were not included in this analysis, there are 41 with an Andean distribution: Dendropsophus aperomeus, D. battersbyi, “Hyla chlorostea”, D. delarivai, D. praestans, D. stingi, D. yaracuyanus, “Hyla vigilans”, Osteocephalus elkejungingerae, O. leoniae, O. pearsoni, Scinax fuscovarius, S. castroviejoi, S. manriquei, S. oreites, the four species of the Dendropsophus garagoensis group, plus 23 additional members of Hyloscirtus, the Andean clade of the Hypsiboas pulchellus group, and the Dendropsophus columbianus + D. labialis groups clade (Duellman, 1999; Mijares-Urrutia and Rivero, 2000; Jungfer and Lehr, 2001; Köhler and Lötters, 2001a; Barrio-Amorós et al., 2004). If the D. garagoensis group is not related to the Dendropsophus columbianus + D. labialis groups clade, then it would represent a fourth Andean radiation of hylids. Relationships of the Andean D. aperomeus, D. battersbyi, D. delarivai, D. stingi, and D. yaracuyanus within Dendropsophus are unknown, so they may represent as many as five additional events leading to the presence of hylids in the Andes. A similar situation occurs with “Hyla chlorostea”, “Hyla vigilans”, Scinax manriquei, S. oreites, and the species of Osteocephalus. Scinax castroviejoi and S. fuscovarius are sister species (Faivovich, unpubl. data), so they are considered another independent entrance into the Andes. Adding up all these species and clades gives a maximum total of 17 independent biogeographic events, of which 5 subsequently radiated and 13 are single taxa. If the Andean species of Osteocephalus were monophyletic, then the figure could decrease to 14 independent events, 6 of which subsequently radiated.

    Considering the information outlined above, and returning to the beginning of this section, whereas now we have an upper and a lower limit for the number of independent radiations of hylids in the Andes, we have no idea as to the number of independent radiations of Bufonidae, Centrolenidae, Dendrobatidae, and Hemiphractinae in the Andes.

    A question that might arise is why there is such poor diversification of hylids in the Andes (note that its complement, Why are there so many species in extra-Andean areas?, is equally valid). There are several scenarios that could answer this question. The presence in several areas of the Andes of anuran groups with obligate aquatic life-history stages dependent on either ponds or streams (Centrolenidae, Bufonidae) appears to be a strong argument against a hypothesis of lack of appropriate habitats. The fact that hylids are found in fairly high altitudes in the Andes (e.g., species in the Andean stream-breeding clade reach up to 2400 m; Duellman et al., 1997; species of the D. labialis group reach up to 3500 m; Lüddecke and Sanchez, 2002) and other places (e.g., several Hylini living between 2000 and 3000 m; see Duellman, 2001) could indicate that there may be few physiological constraints limiting the exploitation of higher areas.30

    Atlantic Forest

    Of the several instances of hyline taxa present in the Atlantic Forest of Brazil, eight are single terminals and six are clades (figs. 14– 16). The terminals are (1) Aparasphenodon brunoi, (2) Dendropsophus anceps, (3) D. giesleri, (4) D. minutus, (5) D. seniculus, (6) Hypsiboas albopunctatus, (7) Scinax uruguayus, and (8) Xenohyla truncata. The clades are (1) Aplastodiscus, (2) the Hypsiboas faber group plus the H. pulchellus group clade, (3) Trachycephalus nigromaculatus plus T. mesophaeus, (4) Phyllodytes, (5) the Scinax catharinae clade, and (6) Bokermannohyla. Including the approximately 134 hylid species that were unavailable for this study with a distribution in eastern Brazil would certainly increase the number of clades and terminals in an unpredictable way.

    Faivovich (2002) observed that Scinax was divided in two clades, one endemic to the Atlantic Forest (the S. catharinae clade) and another that was widespread in the Neotropics (the S. ruber clade). Our results imply an ambiguous situation. The position of S. uruguayus as the sister taxon of the remaining species of the S. ruber clade suggests that Scinax could have as well originated in southeastern Brazil and colonized other areas of the Neotropics in subsequent events. A denser taxon sampling of Scinax would allow a test of this hypothesis.

    In Lophiohylini, nearly all species of Phyllodytes are from the Atlantic Forest (the only exception being P. auratus from Trinidad), as is also true for Itapotihyla langsdorffii and several other species of the tribe. However, the situation here is equivocal because it would be equally parsimonious to postulate two independent events leading to the presence of I. langsdorffii and Phyllodytes in the Atlantic Forest.

    Within Bokermannohyla, the B. circumdata species group, mostly from forested regions, is nested within a clade composed of species and species groups (B. pseudopseudis and B. martinsi groups) restricted to the highland formations of rocky fields (Bokermann and Sazima, 1973b; Eterovick and Brandão, 2001; Lugli and Haddad, in prep.). The facts that the other species included in the B. martinsi group, B. langei, is from forested areas (Bokermann, 1964a) and that two species of the B. circumdata group, B. nanuzae and B. sazimai are from rocky fields (Bokermann and Sazima, 1973b; Cardoso and Andrade “1982” [1983]) suggest that a denser taxon sampling of Bokermannohyla is necessary to better understand whether these frogs are an original element from the rocky fields that secondarily radiated in the forested areas or vice versa.

    The clade composed of the Hypsiboas faber and H. pulchellus groups could be an example of an Atlantic Forest (or at least an eastern Brazilian31) origin with subsequent radiations into other regions. Faivovich et al. (2004) found that within the H. pulchellus group, an Andean clade was nested within an Atlantic Forest clade. Exactly the same pattern for the group is corroborated by our analysis (fig. 14).

    Origin of West Indian Hylids

    Our results support assertions made by Hass et al. (2001) and Hedges (1996), based on immunological distances and unpublished sequence data, regarding a diphyletic origin of West Indian hylids. Osteopilus is the sister group of a clade composed of Osteocephalus and the montane Tepuihyla (fig. 16). While the incomplete taxon sampling precludes a careful assessment of the distribution of the most basal taxa of these genera, our results are compatible with a northern South American origin for Osteopilus; Tepuihyla is restricted to highlands in Venezuela and Guyana (Ayarzagüena et al., “1992” [1993b], Duellman and Yoshpa, 1996; Mijares-Urrutia et al., 1999), and Osteocephalus is widespread in the Amazon Basin and surrounding regions, from Venezuela and French Guiana to Bolivia (e.g., De la Riva et al., 2000; Lescure and Marty, 2000; Jungfer and Lehr, 2001; Smith and Noonan, 2001).

    Regarding the other West Indian hylid species, Hypsiboas heilprini and its sister-group relationship with the H. albopunctatus group (fig. 14) is notable in that the basal taxon of the group, H. raniceps, has a broad distribution through open areas of South America, spanning about 3500 km, from French Guiana (Lescure and Marty, 2000) to eastern central Argentina (Basso, 1995). The fact that both hylid lineages present in the West Indies clearly have a northern South American origin is coincident with the suggestions made by Hedges (1996).

    Middle America and the Holarctic

    The molecular evidence for Hylini does not support the idea of a basal split between an Isthmian–Lowlands clade and a Mexican– Nuclear Central American clade as was suggested by Duellman (2001). Instead, it suggests the existence of a clade composed primarily of, but not limited to, Mexican highlands-Nuclear Central American elements (fig. 15). Nested within this clade, there is a lineage composed of two lowland clades (Tlalocohyla, and the group composed of Anotheca, Triprion, and Smilisca), an Isthmian Highlands clade (Isthmohyla) and one North American–Eurasiatic clade (Hyla).

    Within the Mexican Highlands–Nuclear Central American clade, and besides the major lineage that diversified outside this region (Hyla, Isthmohyla), there are two other instances of terminals distributed in lower Central America (fig. 15), Duellmanohyla rufioculis and Ecnomiohyla miliaria. There are also at least three more cases that were unavailable for this study: two species of Duellmanohyla (D. lythrodes, D. uranochroa), the lower Central American species of Ecnomiohyla (E. fimbrimembra, E. thysanota), and Ptychohyla (P. legleri). The two instances implied by our results are a lower limit; the addition of further exemplars of Duellmanohyla, Ecnomiohyla, and Ptychohyla will determine if there were other events as well.

    Hylini is nested within a South American clade, supporting a South American origin for this group. It is interesting, however, to see a likely Mexican highlands–Nuclear Central American origin for the lowland lineages (Anotheca, Tlalocohyla, Smilisca, Triprion), and that the Isthmian Highlands Isthmohyla is nested within all these lineages. We see these situations as being at least partially compatible with the current paleogeographic scenario for Middle America (see Iturralde-Vinent and McPhee, 1999; Savage, 2002a).

    The topology of the Mexican Highlands– Nuclear Central American clades also shows some fine-grained patterns, like the possible sister-taxon relationship between a clade from the Mexican highlands (Plectrohyla bistincta group) and one from the Nuclear Central American highlands (Plectrohyla guatemalensis group). The problem is that considering the scarce taxon sampling of these two species groups in our analysis, and the very likely possibility of further rearrangements upon addition of more exemplars of these groups (see comments in earlier discussion), it seems risky to hypothesize about the recovered pattern.

    Most authors who have discussed the origin of Eurasiatic Hyla assumed its origin from western North American Hyla and its dispersion to Eurasia presumably through Beringia (Anderson, 1991; Borkin, 1999; Duellman, 2001; Kuramoto, 1980). The topology of Hyla (fig. 15) shows two Eurasiatic taxa. One of these, the H. arborea group, forms the sister taxon of the remaining Hyla. The other, H. japonica, is imbedded within the H. eximia group, which in turn is nested within a grade of eastern North American species. This situation implies two independent biogeographic events to explain the origin of Eurasiatic Hyla, as suggested by Anderson (1991) and Borkin (1999). While this pattern is partially compatible with the idea of a western North American origin of at least some Eurasiatic Hyla (those nested within the H. eximia group) claimed by previous authors, it remains at least equivocal for the clade that contains most exemplars of the H. arborea group. The only way of maintaining a western North American origin for this clade is to invoke a very important historical shift in the distribution of the currently eastern North American species groups or to accept an eastern North American–European (North Atlantic) vicariance or dispersal event. The position of the H. eximia group nested within eastern North American species groups requires a dispersal/vicariance event southward to explain the distribution of this lineage, as suggested by Duellman (2001).

    A Rough Temporal Framework: Clues from the Hylid Fossil Record

    The hylid fossil record is remarkably scant (Sanchiz, 1998a), and hylid remains, on most occasions, are represented by disarticulated ilia. The oldest fossil record tentatively assigned to Hylidae is remains of an ilium and a humerus from the Maastrichtian of Naskal, India (Prasad and Rage, 1995) tentatively assigned to Hylidae, although Sanchiz (1998a) considered the identification dubious. Hylids were mentioned from the Paleocene of Itaboraí (Brazil) by Estes (1970), Estes and Reig (1973), Estes and Baez (1985), and Baez (2001), but the material—also iliac remains—is stills unstudied.

    The earliest known record for North America is Hyla swanstoni, from the Late Eocene of Cypress Hill Formation (Saskatchewan, Canada), described by Holman (1968) based on eroded iliac remains; Sanchiz (1998a) also considered this taxonomic assignment to be dubious. Other iliac remains from the mid-Miocene of North America (mostly from the Hemingfordian North American Land Mammal Age) were also referred to hylids (i.e., Acris barbouri, Hyla miocenica, H. miofloridiana, Proacris mintoni, Pseudacris nordensis), as well as several Pleistocene remains that were assigned to extant species (Holman, 2003).

    The oldest known hylid record for Europe was reported by Sanchiz (1998b) from early Miocene lignite deposits of Austria. The remains consist of a fragmentary sacral vertebra and a scapula, which Sanchiz (1998b) found to be similar to Hyla arborea and H. meridionalis, and assigned to Hyla sp. Other remains from the late Miocene, as well as from the Pliocene to Holocene, were referred to Hyla sp., H. arborea, and the most recent ones to some extant species (see Sanchiz [1998a] for review).

    The oldest fossil record of hylids in Australia dates to the Lower to Middle Miocene, where some species of Litoria were described on the basis of iliac remains (e.g., Tyler, 1991). Other Pleistocene and Holocene remains were assigned to extant species (see Sanchiz [1998a] for a review).

    We are not inclined to construct far-reaching hypotheses based on such a sparse fossil record. Without taking into account Hyla swanstoni, already questioned by Sanchiz (1998a), we must consider the possibility that at least one of the North American iliac remains from the Miocene assigned to hylids is actually related to the extant groups of Holarctic hylids. If this were the case, it would imply a minimum age of approximately 15 mybp of hylid presence in North America. If we consider with the same leniency the Miocene remains from Europe, especially the approximately 17 mybp Hyla sp. reported by Sanchiz (1998b), we could consider an even earlier, though still unspecified, minimum age of hylid presence in North America. All the evidence regarding hylid fossil record and its minimum ages has been presented.

    How this information could be used depends on how much we are willing to assume about the role of known geophysical data in dating cladogenetic events. In recent studies addressing phylogenetic studies of some frog groups, geophysical information was used for molecular clock calibrations (e.g., Bossuyt and Milinkovitch, 2000; Vences et al., 2003c). Leaving aside particular objections about molecular clock estimations, the use of clade-independent information as calibration points entails assumptions about the evolutionary process and about the primacy and precision of paleogeographic reconstructions that should be taken cautiously (Page and Lydeard, 1994). It has been this clade-independent information that has classically been used to date one of the important events of the hylid radiation: the origin of the Hylini.

    Both Savage (1966, 1982, 2002a) and Duellman (1970, 2001) suggested an early colonization event of Middle America by Hylinae and Phyllomedusinae stocks in the late Cretaceous–Eocene; from these stocks, all remaining Middle American and North American elements differentiated due to several vicariant and dispersal events through the Tertiary. Considering that the evidence for the existence of a late mid-Miocene landbridge between North and South America (necessarily involving Central America) is ambiguous (Iturralde-Vinent and McPhee, 1999), a “classical” assumption of nonoverwater dispersal would imply that actually the minimum age of colonization of Middle America must have been at least about 75 mybp, during the existence of the so-called post-Cretaceous Arc landbridge (Iturralde-Vinent and McPhee, 1999). While this seems like a logical deduction, we find it to be unsupported by the available data pertaining to hylids. We only know (or better, assume) that hylids were present in North America 15 mybp and possibly in Europe 17 mybp. Accordingly, we are still uncomfortable in assigning 75 mybp as the minimum age of Hylini. Instead, this age should be the result of either a fossil record (still unknown) or a dating exercise.

    FUTURE DIRECTIONS

    In order to increase our knowledge of hylid phylogenetics, we see several lines of inquiry stemming from this project whose pursuit would be fruitful. These are:

    1. Nonmolecular data set: As stated elsewhere in this paper, an extensive, well-researched nonmolecular data set is a major gap in our analysis. Every effort should be made to complete one and to combine it with the available molecular data.

    2. Phylogeny of Pelodryadinae: By far the greatest deficiency in our knowledge of hylid relationships that still need to be solved is the relationships among Pelodryadinae. The combination of a densely sampled data set of Pelodryadinae with ours will be helpful for understanding Phyllomedusinae relationships.

    3. Inclusion of unrepresented groups and denser taxon sampling of poorly represented groups of Hylinae: No exemplars of the Bokermannohyla claresignata and the Dendropsophus garagoensis groups were available for this analysis. Furthermore, several groups have been poorly represented; such is the case for Ecnomiohyla, Exerodonta, Isthmohyla, Megastomatohyla, Plectrohyla, and Tlalocohyla. A major task in the future will be to rigorously test all the tentative associations of species not included in the analysis with the various clades that we identified.

    4. Relationships within smaller taxonomic units: Our results provide a general framework for the study of relationships within almost any of the genera or species groups of the species-rich genera of Hylidae. In particular, we think that the study of relationships through an increased taxon sampling within Bokermannohyla, Dendropsophus, Ecnomiohyla, Hyloscirtus, Hypsiboas, and Plectrohyla would result in important changes in our understanding of those groups.

    5. Resolution of the taxa herein considered insertae sedis: A careful study of available material of the taxa that we could not associate with any of clades that we identify and, if available, the inclusion of molecular data derived from them would hopefully permit their inclusion in the phylogenetic context of hylids.

    Acknowledgments

    First and foremost, we thank William E. Duellman for his major contribution to our knowledge of hylid frogs. His lifework was the foundation of this project and a permanent source of inspiration. Several of his ideas were corroborated here, while we were unable to find evidence supporting a few others. These discrepancies are immaterial in our appreciation of his monumental contribution.

    For the loan of tissue samples employed in this study, voucher specimens, help in the field, or critical material, we thank (see appendix 4 for institutional abreviations): Raoul Bain, Charles J. Cole, Linda S. Ford, and Charles W. Myers (AMNH); Angelique Corthals (AMCC-AMNH); Cornelya Klutsch (ZFMK); Diego F. Cisneros-Heredia (DFCH-USFQ); Eric Smith and Paul Ustach (UTA); Boris L. Blotto, Andres Sehinkman, Martin Ramirez, Santiago Nenda, and Gustavo R. Carrizo (MACN); Oscar Flores-Villela and Adrian Nieto Montes de Oca (MZFC); Dwight Lawson (Zoo Atlanta), David Wake, and Meredith Mahoney (MVZ); José P. Pombal Jr. (MNRJ); Márcio Martins, Miguel T. Rodrigues, and Renata Cecilia Amaro Ghilardi (Universidade de São Paulo); Hussam Zaher, Paulo Nuin, and Patricia Narvaes (MZUSP); Frederick H. Sheldon and Donna Dittmann (LSU); W. Ronald Heyer and Roy W. McDiarmid (USNM); Karen Lips (SIUC); Luis Coloma (QCAZ); Jiri Moravec (NMP6V); Steve Lougheed (Queen's University); Ignacio De la Riva (MNCN); Diego Baldo (Universidad Nacional de Misiones); George Lenglet (IRSNB); James Hanken (MCZ); Frank Glaw (ZSM); Elizabeth Scot (TMSA); Rainer Günther (ZMB); Barry Clarke (BMNH); Arnold Kluge and Greg Schnider (UMMZ); Robert Murphy (ROM); Steve Donnellan (SAMA); Jorge Williams (MLP); Göran Nilson (NHMG); Janalee P. Caldwell (Sam Noble Oklahoma Museum of Natural History, University of Oklahoma; samples collected under National Science Fundation grants DEB-9200779 and DEB-9505518); Rafael de Sá (University of Richmond); Renoud Boistel (Université Paris Sud); Luis Canseco-Márquez (Universidad Nacional Autonoma de Mexico); Esteban O. Lavilla (Fundación Miguel Lillo); Maureen A. Donnelly (Florida International University); John D. Lynch (Instituto de Ciencias Naturales, Universidad Nacional de Colombia); Magno Segalla (Museu de História Natural Capão da Imbuia); Hélio da Silva (Universidade Federal Rural do Rio de Janeiro); Ana Carolina de Queiroz Carnaval (University of Chicago); Paul Moler (Florida Fish and Wildlife Conservation Commission); Robert N. Fisher (U.S. Geological Survey, San Diego, CA); Valerie McKenzie (University of California, Santa Barbara); Aaron Bauer (Villanova University); Marcos Vaira (Museo de Ciencias Naturales, Universidad Nacional de Salta); Claude Gascon (Conservation International); Joseph Mendelsohn (Utah State University); Axel Kwet (Staatliches Museum für Naturkunde, Stuttgart); Rogério P. Bastos (Universidade Federal de Goiás); João Luis Gasparini (Universidade Federal do Espírito Santo); Marcelo Gordo (Universidade Federal do Amazonas); Sônia Cechin (Universidade Federal de Santa Maria); Ivan Sazima (Universidade Estadual de Campinas); Marília T.A. Hartmann, Paulo Hartmann, Luciana Lugli, Ariovaldo P. Cruz Neto, Cynthia P.A. Prado, Luíz O. M. Giasson, and Luís F. Toledo (Universidade Estadual Paulista–Rio Claro); Giovanni Vinciprova (Universidade Federal do Rio Grande do Sul); Phillipe Gaucher (Mission Pour la Cré ation du Parc de la Guyane); Karl-Heinz Jungfer; Sylvain Dubey; A.P. Almeida; Pedro Cacivio; Martin Henzel; and Pierrino and Sandy Mascarino. Their generosity was instrumental in the completion of this project.

    Tissues from Bolivian AMNH specimens were collected during an expedition supported by the Center for Biodiversity and Conservation at the American Museum of Natural History and the Center for Environmental Research and Conservation at Columbia University, New York, in collaboration with the Museo de Historia Natural Noel-Kempff Mercado, Santa Cruz, Bolivia, and Colección Boliviana de la Fauna, La Paz. Tissues from AMNH Vietnamese specimens were collected under National Science Foundation DEB-9870232 to the Center for Biosiversity and Conservation at the AMNH. Exportation permits of Brazilian samples were issued by Autorização de Acesso e de Remessa de Amostra de Componente do Patrimônio Genético n° 037/2004; permits for collection were issued by IBAMA/RAN, licença 057/03, processo 02001.002792/98–03.

    For discussions on hylid systematics and/ or nomenclatural problems, we thank José A. Langone, José P. Pombal, Jr., Charles W. Myers, Esteban O. Lavilla, and Roy McDiarmid. Tom Burton answered many questions and requests of clarifications regarding his important contribution (Burton, 2004). Jan De Laet and Pablo Goloboff made useful suggestions regarding the analyses. Maureen A. Donnelly, Jose P. Pombal, Jr., Charles W. Myers, Jose L. Langone, Taran Grant, Norberto P. Giannini, Raoul Bain, Diego Pol, and W. Leo Smith kindly read partially or completely the manuscript and provided useful comments and ideas.

    Julián Faivovich acknowledges the American Museum of Natural History, E3B/Columbia University, NASA/AMNH computational phylogenetic fellowship, AMNH Roosevelt Grant, and National Science Foundation grant no. DEB-0407632 for financial support. Furthermore, he thanks Diego Pol, W. Leo Smith, Taran Grant, Rafael de Sá, Charles J. Cole, Fernando Lobo, and Esteban Lavilla for their early encouragment to pursue this project.

    Celio F.B. Haddad thanks programa BIOTA/FAPESP (proc. 01/13341–3) and CNPq for financial support.

    Jonathan A. Campbell acknowledges National Science Foundation grant no. DEB-0102383 for financial support.

    Darrel R. Frost and Ward C. Wheeler acknowledge support from NASA grants NAG5–8443 and NAG5–12333. They also especially thank Lisa Gugenheim and Merrily Sterns for their efforts in support of molecular research at the AMNH.

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