Taxonomic status of the zoanthid genera Palythoa and Protopalythoa has been in question for almost a century. Separation of the two genera has been based on traditional morphological methods (colony and polyp form, nematocyst size and form, and number of septa), with Palythoa polyps embedded in a well developed coenenchyme and Protopalythoa polyps standing free and clear of the coenenchyme. Here we sequenced two mitochondrial regions, the cytochrome oxidase I (COI) gene and 16S ribosomal DNA (16S rDNA) genes, from Palythoa and Protopalythoa samples from various parts of the world and performed phylogenetic analyses of the sequence data. The phylogenetic trees for both COI and 16S rDNA from Palythoa and Protopalythoa show four monophyletic groups (designated Palythoa tuberculosa, Palythoa heliodiscus, Palythoa mutuki 1, and Palythoa mutuki 2), with levels of sequence divergence (COI and 16S rDNA divergence approximately 0.0~1.1%) similar to or lower than that previously found among congeneric species within the closely related genus Zoanthus. Surprisingly, sequence differences among Palythoa tuberculosa, Palythoa mutuki 1, and Palythoa mutuki 2 were negligible (0.0~0.2% for both COI and 16S rDNA), potentially indicating relationships below the species level. Our sequences align well with the few Palythoa and Protopalythoa sequences reported to date. These findings strongly indicate that our samples represent a minimum of two and possibly up to four species (the Palythoa tuberculosa -P. mutuki 1 - P. mutuki 2 group, and P. heliodiscus) within the genus Palythoa, and that the genus Protopalythoa is erroneous nomenclature.
Zoanthid taxonomy has long been in a state of chaos, due to the morphological plasticity of species as well as a lack of research establishing reliable criteria to identify and delineate species and genera (Fossa and Nilsen, 1998). A striking example of this taxonomic problem is the status of the two genera Palythoa and Protopalythoa; arguments for and against the separation of these two genera have been ongoing for almost a century (see Verrill, 1900; Pax, 1910; Ryland and Lancaster, 2003). Morphologically, both genera are generally well described (for a review see Ryland and Lancaster, 2003). The major criteria for the delineation of Palythoa and Protopalythoa have been the structure and shape of polyps and the coenenchyme (Fig. 1), with Palythoa having polyps virtually immersed in a well developed coenenchyme (deemed “immersae” in form by Pax ), and Protopalythoa having polyps standing free and clear of the coenenchyme, which forms basal lamellae (“intermediae” and “liberae” forms [Pax, 1910]). Other morphological characters cited include septa number, polyp column diameter, and nematocyst characters, as well as reproductive characters (Ryland and Lancaster, 2003; 2004), but all of these are extremely difficult to properly investigate, especially in the field, and may vary according to environment (Ryland and Lancaster, 2003).
A recent genetic study in the zoanthid genus Zoanthus has grouped Z. kuroshio (embedded polyps, “intermediae”) and Z. gigantus and Z. sansibaricus (free-standing polyps, “liberae”) (Reimer et al., 2006) into three monophyletic con-generic groups. These Zoanthus species differ from each other by 0.7~1.3% in mitochondrial cytochrome oxidase I (COI) gene sequence, while sequence differences between the related genera Zoanthus and Palythoa (both in the suborder Brachycnemina) are approximately 3–4%. Additionally, genetic results show individual species in Zoanthus comprising a wide range of morphotypes, with Z. sansibaricus encompassing four previously presumed species (Reimer et al., 2004). Similarly, 16S and 12S rDNA sequences from a single sample each of Palythoa and Protopalythoa (Sinniger et al., 2005) showed differences below the genus level (<1% for both genes), indicating that Palythoa and Protopalythoa might indeed be one genus.
Here we use mitochondrial COI and 16S ribosomal DNA (16S rDNA) sequences from samples identified morpholgically as putative Palythoa and Protopalythoa from southern Japan, Saipan, Madagascar, and the Caribbean to clarify the phylogenetic relationship of these two genera. We also compare our sequences with previously obtained Palythoa and Protopalythoa sequences.
MATERIALS AND METHODS
Samples of Palythoa spp. (mostly appearing to be Palythoa tuberculosa as described by Uchida and Soyama, 2001) and Protopalythoa spp. were collected from various sites in southern Japan and Saipan between August 2003 and April 2005 and stored in 80–100% ethanol at −20°C. As samples were collected in situ photographs were taken to assist in identification and for collection of morphological data (colony and polyp form, in situ coloration, etc.), and sampling data (depth, environment, date) were recorded. Samples of Palythoa cf. caribaeorum (Utila, Honduras, collected February 2004), Protopalythoa sp. (Madagascar, February 2004), and Palythoa sp. (NW Madagascar, January 2004) were kindly supplied by Frederic Sinniger of the University of Geneva. A sample of Parazoanthus gracilis (Jogasaki, Izu, Japan) was used to obtain out-group sequences for phylogenetic analyses.
DNA extraction, PCR amplification, and sequencing
DNA was extracted from samples (5–25 mg) following procedures outlined in Reimer et al. (2004). The COI gene and 16S rDNA were amplified following procedures outlined in Reimer et al. (2004) and Sinniger et al. (2005), respectively. Amplification products were checked by 1.5% agarose gel electrophoresis and sequenced with an ABI PRISM TM 3700 DNA Analyzer (PE Biosystems, Foster City, CA, USA) using a BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems). The sequences were analyzed using GENETYX-MAC version 8.0 (Software Development, Tokyo, Japan) and DNASIS Mac v3.6 (Hitachi Software Engineering Company, Ltd., Tokyo, Japan).
Our new nucleotide sequences were deposited in GenBank (accession numbers AB219195–AB219225). COI and 16S rDNA sequences were aligned separately, using CLUSTAL X version 1.8 (Thompson et al. 1997), with sequences from Zoanthus spp. (COI sequences AB214172, AB214177 [J. Reimer, unpublished data] and AB219182 [Reimer et al., 2006]; 16S rDNA sequences AB219187, AB219191, AB219192 [Reimer et al. 2006]) and from Parazoanthus gracilis (COI, AB214178 [J. Reimer, unpublished data]; 16S rDNA, AB219194 [Reimer et al., 2006]). Two putative Palythoa and Protopalythoa spp. COI sequences obtained from a previous study (AB128895, AB128896; Reimer et al., 2004) were also included in the COI alignment. The alignments were inspected by eye and manually edited. All ambiguous sites in the alignments were removed from the data sets for phylogenetic analyses. We generated two aligned data sets comprising 533 sites from 29 taxa for the COI gene, and 870 sites from 12 taxa for 16S rDNA. These alignments are available on request from the corresponding author.
The same methods were independently applied for phylogenetic analyses of the COI and 16S rDNA sequences. Maximum-likelihood (ML) analyses were performed using PhyML (Guindon and Gascuel, 2003). An input tree for PhyML was generated by BIONJ with the general time-reversible model (Rodriguez et al., 1990) of nucleotide substitution incorporating invariable sites and a discrete gamma distribution (eight categories) (GTR + I +Γ). The proportion of invariable sites, a discrete gamma distribution, and base frequencies were estimated from the data set. PhyML bootstrap trees (500 replicates) were constructed using the same parameters as the individual ML trees.
The neighbour-joining (NJ) method (Saitou and Nei, 1987) was performed using PAUP* Version 4.0 (Swofford, 1998), with the Kimura-2 parameter model (Kimura, 1980). NJ bootstrap trees (1000 replicates) were constructed using the same model.
Maximum parsimony (MP) analyses were performed by PAUP* Version 4.0 (Swofford, 1998), using heuristic searches with closest stepwise addition of taxa and tree-bisection-reconnection (TBR) branch-swapping. A bootstrap analysis of 1000 replicates was conducted by the heuristic search method to assess the confidence of branches in the MP tree.
Sequences and phylogeny of the COI gene
All samples of presumed Palythoa spp. and Protopalythoa spp. (Fig. 2, Table 1) formed a monophyletic group separate from Zoanthus and Parazoanthus, with relatively strong ML bootstrap support (80%). Protopalythoa spp. samples did not form a separate clade from Palythoa. Instead, some Protopalythoa samples (PpAT1, PpAT2, AmamiPaly, PpYS1, PpMiI1, PpBA1) formed a cluster with the Palythoa clade. Samples of putative Palythoa from Madagascar (PM1, PM2) and Honduras (PcH1) were also in the Palythoa clade. Protopalythoa samples from Madagascar and Saipan (PpSaiLL1, PpM1) constituted another distinct monophyletic group (ML bootstrap support 100%). The resulting ML tree is shown in Fig. 3.
Zoanthid samples used in this study
Sequences and phylogeny of 16S rDNA
All samples of presumed Palythoa spp. and Protopalythoa spp. (Fig. 2, Table 1) formed a monophyletic group separate from Zoanthus and Parazoanthus, with strong MP bootstrap support (86%). The Protopalythoa spp. samples did not form a separate clade from Palythoa. Instead, four Protopalythoa samples (PpYS1, PpMiI1, PpAT1, PpAT2) were within the Palythoa clade (ML bootstrap support 90%). Sequence AF398920 (Burnett, unpublished data) of Protopalythoa sp. from Bali was also in the Palythoa clade, but was not included in the 16S rDNA alignment, as the sequence was too short. Sequences AF282931 from Palythoa caesia and AF282932 from Palythoa caribaeorum (Burnett, unpublished data) similarly belonged to the Palythoa clade, but as with Protopalythoa from Bali, were not included in the 16S rDNA alignment asthe sequences were too short. Protopalythoa samples from Erabu, Japan and Saipan (PpEK1, PpM1) constituted another distinct clade (ML bootstrap support 99%). The resulting ML tree is shown in Fig. 4.
Diversity of Palythoa/Protopalythoa species
As shown in Figs. 3 and 4, all of our samples of presumed Palythoa (but not presumed Protopalythoa), regardless of the species identification and collection area, belonged to the same monophyletic group, with very little divergence among samples (0.0~0.1% for COI and 0.0% for 16S rDNA). Similar results on a smaller scale were obtained by Burnett et al. (1994) when they examined Palythoa caesia on the Great Barrier Reef and found that this single species encompassed a wide range of morphotypes. Based on our genetic results from worldwide samples, it appears that our samples of presumed Palythoa from the Caribbean, Indian Ocean, and Pacific may all be conspecific. Similarly, our samples of putative Protopalythoa species only formed three non-monophyletic groups, indicating that many of the 217 currently described species of Palythoa/Protopalythoa (Fautin, 2004) in all likelihood constitute varying morphotypes of the same species. This result is similar to that reported for Zoanthus spp. (Reimer et al., 2004).
Species of the Palythoa/Protopalythoa group have often been described simply on the basis of color, or polyp shape and size (e.g., Zunan, 1998), but as is shown in Fig. 2 (compare 2a and 2b, or 2e, 2f, and 2g), Palythoa/Protopalythoa colonies can vary greatly in polyp form and color with variations in environment (e.g., current strength and duration, degree of wave action, sediment color). The most striking examples of this were the samples from Miyakejima, Japan (PpMiI1, shown to be Palythoa mutuki 1 [Fig. 2f], and PtMiI1, P. tuberculosa, not shown), which were dark gray in polyp color due to volcanic ash incorporated into their tissue, in stark contrast to all other samples from all other sites, which were white, light tan, or brown in color. Additionally, P. tuberculosa from Shin's Reef, Yoron, Japan (sample PtYoS1), had a unique colony and polyp form (Fig. 2a), much more “intermediae” than all other P. tuberculosa samples, and yet was unambiguously within the P. tuberculosa clade for both the COI and 16S rDNA data sets. Similarly, sample PaIsK2, which appeared to be Palythoa anthoplax as described by Zunan (1998), with smaller polyps than Palythoa tuberculosa samples, was unambiguously within the COI P. tuberculosa clade.
Are Palythoa and Protopalythoa separate genera?
The COI and 16S rDNA trees clearly show that the Palythoa and Protopalythoa species groups are no more divergent than species groups within the genus Zoanthus (Table 2, Figs. 3, 4). COI sequences of Zoanthus congeners are divergent by 0.8~1.1%, while our assumed Palythoa and Protopalythoa samples are divergent by 0.0~1.1%. Similarly, 16S rDNA results show Zoanthus congeners divergent by 0.7~1.3% and Palythoa and Protopalythoa sequences divergent by a maximum of 0.9%. These results are congruent with those of Sinniger et al. (2005), who saw 0.5% divergence between Palythoa tuberculata and Protopalythoa sp. utilizing 16S rDNA, and 0.2% utilizing 12S rDNA. On the basis of our COI and 16S rDNA phylogenies and past examinations of COI data in Anthozoa (Medina et al., 1999; Reimer et al., 2004; van Oppen et al., 2004), as well as morphological characteristics described earlier, we interpreted our presumed Palythoa spp. and Protopalythoa spp. samples to be congeners (Table 2).
Comparison of cytochrome oxidase I (COI) and mitochondrial 16S ribosomal DNA (16S rDNA) sequence difference levels in zoanthids
It is true that when polyps are expanded, many Zoanthus species look strikingly similar, but often when polyps are closed, colony and coenenchyme structure appear different (J. Reimer, personal observation). Many Palythoa/Protopalythoa species' polyps are closed during the daytime, making coenenchyme and polyp differences more striking to most observers (in the daytime) than in Zoanthus spp. Overall, however, morphological differences between Palythoa and Protopalythoa are not as pronounced as other generic-level comparisons (e.g., Zoanthus [no sand incorporated into polyps] and Palythoa [sand incorporated into polyps], or Palythoa [colonial] and Sphenopus [non-colonial], the latter two both members of family Sphenopidae).
Species examined in the current study
All samples of presumed Palythoa/Protopalythoa species investigated during the course of this study were placed genetically within one of four clades. Examination of in situ photographs and collection data (depth, etc.) showed that no clear morphological divisions could be made that were congruent with the majority of our genetic data. Morphological variation (colony shape, colony size, polyp shape, polyp size, oral disk and polyp color) existed within three of the following four clades (excepting Palythoa heliodiscus).
1. Palythoa tuberculosa (Fig. 2a, b) as described in Uchida and Soyama (2001). This group includes all presumed Palythoa samples (YakuPaly, PtAT1, PtAT2, PtBA1, PtWK1, PtMiI1, PtYS1, PtSaiLL1, PtIsO1, PtIsK3, PtYoS1, PtEO1, PtKK1, PaIsK2, PcH1, PM1, PM2), with colonies generally “immersae” in form (Pax 1910), but occasionally “intermediae” (Fig. 2a, b). Samples from this clade were found worldwide (Japan, Saipan, Madagascar, Honduras) and correspond to Palythoa tuberculosa from Japan described in Uchida and Soyama (2001). One sample from Kataguwa Reef, Ishigaki Is., Japan, had smaller polyps and more closely resembled Palythoa anthoplax (as described in Zunan, 1998), but the COI and 16S rDNA sequences were identical to our P. tuberculosa sequences. It should be noted that P. tuberculosa (and P. mutuki 1) samples were found on the north end of Miyakejima Island, Japan, and we believe that this is the farthest north Palythoa has been recorded in the western Pacific.
2. Palythoa heliodiscus Ryland and Lancaster, 2003 (Fig. 2c, d). Samples PpSaiLL1, PpEK1, and PpM1, which formed a monophyletic group in both our COI and 16S rDNA analyses, correspond to Protopalythoa heliodiscus (Ryland and Lancaster, 2003) and Palythoa (Protopalythoa) lesueuri (as described in Uchida and Soyama, 2001). The level of sequence difference of this clade from Palythoa tuberculosa (1.1% COI, 0.9% 16S rDNA) and its very high bootstrap support (100% for ML, both COI and 16S rDNA) clearly show that this group is a separate Palythoa species from all other observed Palythoa/Protopalythoa samples in this study. We have chosen here to use the name Palythoa heliodiscus, as the species description in Ryland and Lancaster (2003) is highly detailed. Similarly to observations made by Burnett et al. (1997) and Ryland and Lancaster (2003), this species was generally found to be subtidal (Table 1), and to occur in areas of lower light (i.e., on rock/coral ledges and shelves under other coral colonies, away from direct light exposure) than the other putative Palythoa species described here. Palythoa heliodiscus specimens had shorter tentacles than Palythoa mutuki 1–2 (see below), as well as a white mouth opening. Polyps were “liberae” (Pax, 1910) in form (Fig. 2c, d). For a morphological description separating P. heliodiscus and P. mutuki (Protopalythoa in the text), refer to Ryland and Lancaster (2003).
3. Palythoa mutuki 1 (Fig. 2e, f) (as described in Ryland and Lancaster, 2003). Samples of this species (PpYS1, PpBA1, PpMiI1) were virtually indistinguishable from Palythoa mutuki 2 samples (see below), with very similar habitat, polyp size and structure, as well as polyp spacing and coenenchyme appearance. The only noticeable morphological differences observed were that P. mutuki 1 had a green oral disk with a pale green center and dark-colored tentacles, whereas P. mutuki 2 had a brown oral disk with a white oral opening and pale radii (marking septa), and comparatively lighter tentacles than P. mutuki 1. However, differences in color must be interpreted with extreme caution, as shown in the genus Zoanthus, where many different color morphotypes have been clearly shown to be conspecific (Reimer et al., 2004; 2006). Whether these samples are truly a new cryptic species, or a subspecies or different genotype of either P. tuberculosa or P. mutuki 2, is open to speculation; the COI sequences were identical to P. tuberculosa and only one base pair different from P. mutuki 2, and the 16S rDNA sequences differed from both P. tuberculosa and P. mutuki 2 by only one base pair. In the future, less conservative genetic markers more appropriate for examining interspecific relationships, such as the internal transcribed spacer (ITS) rDNA region, should be utilized to clarify the taxonomic status of the P. tuberculosa - P. mutuki 1 - P. mutuki 2 phylogenetic group.
4. Palythoa mutuki 2 (Fig. 2g) (as described in Ryland and Lancaster, 2003). Palythoa mutuki 2 samples (Amami-Paly, PpAT1, PpAT2) were found to be either “liberae” or “intermediae” in form, having much larger and thicker polyps connecting the oral disk to the coenenchyme than P. heliodiscus (Fig. 2g), but with a coenenchyme not as well-developed as that of P. tuberculosa. All P. mutuki 2 samples were found intertidally in areas experiencing strong waves and/or currents, and only at the Amami sampling location. As mentioned above, P. mutuki 1 and P. mutuki 2 are morphologically indistinguishable, and genetically very closely related (perhaps below the species level) to each other and to P. tuberculosa; thus P. mutuki 1 and/or 2 may have a subspecies-level relationship with P. tuberculosa and/or each other. Despite an apparent close taxonomic relationship to P. tuberculosa, P. mutuki 1 and 2 samples are morphologically very different from P. tuberculosa. Further investigation into the taxonomic relationship between P. tuberculosa and P. mutuki 1 and 2 is clearly needed.
Taken as a whole, our molecular genetic evidence, plus an examination of what morphologically constitutes genera in other zoanthid groups, strongly suggest that Palythoa and Protopalythoa are not truly separate genera, but instead comprise congeneric species and/or subspecies within the genus Palythoa. The sequence similarities between putative, “immersae”-form P. tuberculosa and the morphologically very similar “intermediae/liberae”-form P. mutuki 1 and P. mutuki 2 show that zoanthid species assignment based solely on morphology is uncertain speculation at best. Further genetic investigations utilizing other genetic markers and other presumed Palythoa and Protopalythoa species besides the ones examined here (and in particular the Palythoa and Protopalythoa type species, Palythoa mamillosa and Protopalythoa variabilis) will help solidify our conclusions as well as shed further light on the true level of diversity found in this genus.
We thank Mitsugu Kitada at Shin-enoshima Aquarium (Enoshima, Japan) and Frederic Sinniger (University of Geneva) for kindly supplying some of the samples used in our ongoing zoanthid research. Dr. Yasuo Furushima (JAMSTEC), Mika Reimer, Ryan Cox, Shin Hisashi, Denny Probizanski, Craig Walker, and John Bryon helped with sample collection in Japan and Saipan. Masaru Kawato and Hirokazu Kuwahara at JAMSTEC graciously lent their time and expertise with DNA sequencing.