Two new common, previously unrecognized species in the Sticta weigelii morphodeme (Ascomycota: Peltigeraceae)

Abstract: Sticta is a subcosmopolitan genus most diverse in the tropics. Traditionally, many taxa were considered to be widespread and morphologically variable, following broadly circumscribed morphodemes. Among these is the S. weigelii morphodeme, characterized by a cyanobacterial photobiont and rather narrow, flabellate to truncate or tapering lobes producing predominantly marginal isidia. Molecular phylogenetic analyses focusing on the ITS fungal barcoding marker revealed that this morphodeme represents several species, some of which are only distantly related to each other. Here we describe two species and one subspecies of this morphodeme as new to science, based on analysis of 400 specimens, for 344 of which we generated ITS barcoding data. The two new species, S. andina and S. scabrosa, are broadly distributed in the Neotropics and also found in Hawaii, where the latter is represented by the new subspecies, S. scabrosa subsp. hawaiiensis; in the case of S. andina, the species is also found in the Azores. Sticta andina exhibits high phenotypic variation and reticulate genetic diversification, whereas the phenotypically rather uniform S. scabrosa contains two main haplotypes, one restricted to Hawaii. Sticta andina occurs in well-preserved montane to andine forests and paramos, whereas the two subspecies of S. scabrosa are found in tropical lowland to lower montane forests, tolerating disturbance and extending into anthropogenic habitats. Citation: Moncada B., Mercado-Díaz J. A., Smith C. W., Bungartz F., Sérusiaux E., Lumbsch H. T. & Lücking R. 2021: Two new common, previously unrecognized species in the Sticta weigelii morphodeme (Ascomycota: Peltigeraceae). – Willdenowia 51: 35–45. Version of record first published online on 24 February 2021 ahead of inclusion in April 2021 issue.

Molecular phylogenetic and revisionary studies showed that such broadly circumscribed taxa represent several to sometimes numerous, often only distantly related species (McDonald & al. 2013;Moncada & al. 2013Moncada & al. , 2014Magain & Sérusiaux 2015;Dal Forno & al. 2018;Simon & al. 2018;Ekman & al. 2019;Mercado-Díaz & al. 2020). The Sticta weigelii morphodeme evolved multiple times independently in various clades, resulting in the description of several new species, e.g. S. borinquensis Merc.-Díaz & Lücking and S. rhizinata B. Moncada & Lücking, and the reinstatement of some old names, such as S. beau voisii Delise (Moncada & Lücking 2012;McDonald & al. 2003;Moncada & al. 2014;Mercado-Díaz & al. 2020). These more narrowly delimited species are not only phylogenetically supported but also exhibit diagnostic phenotypic characters that had previously been considered environmental or ontogenetical variation (Swinscow & Krog 1988;Galloway 1994Galloway , 1997. As result of a broad sampling of mostly neotropical representatives of Sticta, we accumulated a large amount of data for two undescribed lineages corresponding to the S. weigelii morphodeme. Among all globally recognized clades corresponding to this morphodeme, the two newly recognized lineages were the most abundant. Yet, no names were found in the literature that could be applied to them and they are here formally described as new to science, under the names S. andina B. Moncada, Lücking & Sérus. and S. scabrosa B. Moncada, Merc.-Díaz & Bungartz, the latter with two subspecies, subsp. scabrosa from the Neotropics and subsp. hawaiiensis B. Moncada, Lücking & C. W. Sm. from Hawaii. Judging from pre-molecular treatments including specimens now assigned to these taxa, both had previously identified with the broadly delimited name S. weigelii and more recently also with the name S. beauvoisii (Benner & Vitousek 2012). However, the two species are not closely related to either S. weigelii s.str. or S. beauvoisii and both are also only distantly related to each other. The two lineages were first informally recognized using a broad ITS-based phylogeny (Moncada & al. 2014). In that study, what is now recognized as S. andina was believed to represent seven different species, labelled "andina", "colombiana", "dioica", "paramuna", "phyl lidiata", "aff. phyllidiata" and "squamifera". However, the data now available are more consistent with merging three of these ("andina", "colombiana", "paramuna") into a single lineage, with apothecia and/or cylindric to mostly flattened isidia or phyllidia (Moncada & al. 2014: 220, fig. 4, 223, fig. 8;Moncada & al. 2020). In contrast, S. scabrosa, previously recognized as a single species (Moncada & al. 2014: 218, fig. 3), even with the now much expanded data set was found to be morphologically and genetically rather uniform, except for a unique surface morphodeme occurring solely in Hawaii.

Material and methods
ITS barcoding sequences of the genus Sticta were assembled for a much expanded data set of ingroup 677 OTUs (Suppl. File S1; Moncada & al. 2020), as compared to 370 OTUs published previously (Moncada & al. 2014). The S. andina complex initially comprised 19 OTUs, all sampled in Colombia, as mentioned above corresponding to three OTUs informally labelled "andina", "colombiana" and "paramuna" (Moncada & al. 2014). For an updated ITS-based phylogeny, we assembled a total of 164 OTUs from Central America, Colombia, Ecuador, Brazil, the Azores and Hawaii (Moncada & al. 2020). Sticta scabrosa was initially based on nine OTUs from Colombia and the Dominican Republic (Moncada & al. 2014), while the updated set included 180 OTUs from Mexico, Costa Rica, the Dominican Republic, Puerto Rico, Colombia, Brazil, Argentina, Galapagos and Hawaii (Moncada & al. 2020).
The updated alignment was assembled in BIOEDIT 7 (Hall 1999) and sequences were aligned with MAFFT 7 (Katoh & Standley 2013) using the [ -auto] option. The alignment included 677 ingroup OTUs and was 626 bases long. We did not detect critical alignment ambiguity and so included all sites, in order to maximize terminal resolution. Phylogenetic analysis was performed using maximum likelihood in RAxML 8.2.12 on the CIPRES Science Gateway (Stamatakis 2015;Miller & al. 2010), applying the GTR-Gamma model and 98 bootstrap pseudoreplicates according to an automated saturation criterion. The resulting tree were visualized in FigTree 1.4.2 (Rambaut 2016).
Morphological characters of specimens of Sticta andina and S. scabrosa were assessed at the Universidad Distrital Francisco José de Caldas (Bogotá), the Field Museum (Chicago), the Université de Liège and the Botanischer Garten und Botanisches Museum, Freie Universität Berlin using standard microscopical techniques described in Moncada (2012) and Ranft & al. (2018).

Results and Discussion
Sticta andina and S. scabrosa form two large clades in the terminal portion of a global ITS-based Sticta phylog-Willdenowia 51 -2021 eny ( Fig. 1; Suppl. File S2). Both species are geographically largely overlapping, exhibiting similar distribution ranges, predominantly across the Neotropics and Hawaii, with S. scabrosa also including the Caribbean (Dominican Republic and Puerto Rico) and the Galapagos Islands, whereas S. andina is further present in the Azores (Moncada & al. 2020). Sticta andina includes apotheciate and isidiate to phyllidiate specimens, as well as sun and shade forms (Fig. 2). Sticta scabrosa is uniformly phyllidiate but, besides most specimens having an uneven lobe surface, those from Hawaii may also present an unique surface with foveolate-pitted lobe tips (Fig. 3).
Until recently, representatives of Sticta with a cyanobacterial photobiont and predominantly marginal isidia were considered a single species, S. weigelii, presumably with a subcosmopolitan distribution (Joshi & Awasthi 1982;Swinscow & Krog 1988;Galloway 1994Galloway , 2001Galloway , 2007Galloway & al. 1995;Büdel & al. 2000;Brodo & al. 2001;Farkas 2003;Galloway & Thomas 2004). However, like other morphodemes in the genus (Moncada & al. 2014;Magain & Sérusiaux 2015;Simon & al. 2018), the S. weigelii morphodeme appears to have evolved multiple times within the genus, and most of the sequences previously deposited under this name do not represent this taxon (Moncada & al. 2014). Sticta weigelii s.str. is widely distributed in the Neotropics including the Caribbean ( Fig. 1; Suppl. File S2). Material identified with this name from other regions does not represent this taxon. For instance, the ITS accession identified as S. weigelii from Taiwan (AB245124) is an undescribed relative of the latter, whereas the sample from South Korea (KF730791) represents the cosmopolitan S. fuliginosa s.str. Another specimen from Guyana (AF524905) is an undescribed relative of S. scabrosa (see Suppl. File S2). Sticta weigelii s.str. is a species of tropical climates, similar in altitudinal zonation to S. scabrosa, but more affine to well-preserved forest, characterized by dark to blackened, marginal isidia, a rather thin and rather dark lower tomentum and partly yellow cyphellae (Galloway 2006;Moncada 2012;Mercado-Díaz & al. 2020). The two species newly recognized here, S. andina and S. scabrosa, represent the S. weigelii morphodeme in having a cyanobacterial photobiont and elongate lobes with marginal isidia and/or phyllidia, but are phylogenetically unrelated to each other and to S. weigelii ( Fig. 1; Suppl. File S2). Sticta andina agrees with S. weigelii s.str. in the slightly shiny thallus surface and rather dark vegetative propagules, but it differs in the frequent formation of phyllidia; it also has a much thicker lower tomentum, persistently white cyphellae and frequently produces apothecia, and further differs in its preference for upper montane to andine habitats. Sticta scabrosa has a similar ecology as S. weigelii, although it is more commonly found in disturbed and anthropogenic habitats, but its thallus surface is opaque and thinly scabrose, especially toward the margins. The lower tomentum is grey-brown and much thicker, and its cyphellae are consistently white to at most cream-coloured but never yellow. The three species are therefore not only phylogenetically distinct but also morphologically and ecologically well distinguished.
Although Sticta andina and S. scabrosa have broadly overlapping geographic ranges, they exhibit different evolutionary histories, which may be explained by their autecology. As a species largely confined to undisturbed andine forests and paramos, S. andina underwent fragmentation and partial isolation in the recent past, due to the insular nature of these habitats both in space and time (Moncada & al. 2020). Similar effects on genetic diversification have been shown for andine orchids, bromeliads and paramo plants (Küper & al. 2004;Givnish 2010;Madriñan & al. 2013;Givnish & al. 2014Givnish & al. , 2015. The level of genetic and morphological diversification in S. andina is indeed much higher than in other widespread species of the genus, particularly S. fuliginosa s.str. and S. limbata s.str. (Moncada 2012;Moncada & al. 2014;Magain & Sérusiaux 2015). Based on an earlier ITSbased phylogeny of Sticta (Moncada & al. 2014), the clade now recognized as S. andina, at that time with 19 accessions from Colombia, had been tentatively divided into three taxa ("andina", "colombiana", "paramuna"). In our much-expanded dataset, with more than eight times as many accessions, the original "andina" corresponds to a small group in one of the subclades, whereas "colombiana" and "paramuna" largely represent two other subclades. Despite the observed phylogenetic structure and morphological variation, separating these subclades at species level is not warranted, because the internal topology is not supported and there is no clearcut correlation between the subclades and the observed morphological variation.
In contrast, Sticta scabrosa was found to be phylogenetically less complex, with a distinctive haplotype present only in Hawaii, and morphologically more uniform, except for a deviating morphodeme with foveolate surface in part of the Hawaiian material. Because this species is frequent in tropical settings and tolerates disturbances, it may retain more effective genetic interchange between populations (Moncada & al. 2020). Stic ta scabrosa is thus rather well-defined as a taxon and the Hawaiian metapopulation is formally best classified as a subspecies. Diagnosis -Differing from Sticta weigelii in the formation of mostly flattened or dorsiventral isidia and phyllidia instead of cylindric isidia, a thicker lower tomentum, consistently white cyphellae, and the upper montane versus lower montane-tropical habitat preferences.
Vegetative propagules (flattened) isidia to phyllidia, formed densely along lobe margins and extending onto lamina, richly branched and becoming palmate to coralloid, up to 1 mm long and 0.5 mm broad, with terminal parts dorsiventrally flattened to squamiform and basal part forming a short, cylindric stipe, often with cyphellae primordia; phyllidia of same colour as thallus or often somewhat darker on upper side, somewhat paler on underside, nitidous.
Etymology -The epithet was selected among the names originally considered for this complex (Moncada & al. 2014), because it best fits the centre of distribution and the ecology of this species.
Distribution and ecology -Naturally distributed across the Neotropics, with its centre in the northern Andes. Given that the records from Hawaii and the Azores represent the same haplotype as one of the common haplotypes present in the Andes, these disjunct records may be the result of recent, perhaps anthropogenic long-distance dispersal. Biogeographic connections between the Neotropics, Hawaii and the Azores have been reported for other lichens and bryophytes, e.g. the Sticta ciliata Taylor complex (Magain & Sérusiaux 2015;Mercado-Díaz & al. 2020;Moncada & al. 2021) and the liverworts Lepto scyphus azoricus Grolle (Vanderpoorten & Long 2006;Devos & Vanderpoorten 2009) and Syzygiella rubricau lis (Nees) Steph. (Maciel-Silva & al. 2016). However, in these cases, the material found in different regions represents distinct lineages and not identical haplotypes as in S. andina.
In South America, Sticta andina is an upper montane to andine species mostly found above 2500 m elevation and usually confined to well-preserved forest and paramo habitats, typically growing epiphytically on trunks and branches of trees and shrubs. In Hawaii and in the Azores, the species is also found in humid montane forests, although generally at lower altitudes due to a mass elevation effect.
Remarks -Sticta andina is one of a number of partially unrelated species that share the S. weigelii morphodeme, i.e. associated with a cyanobacterial photobiont and forming marginally isidiate-phyllidiate lobes. From S. weigelii s.str. (Galloway 2006;Moncada 2012;Mercado-Díaz & al. 2020), S. andina can be differentiated by the often flattened to dorsiventral isidia and phyllidia (vs. consistently cylindric isidia in S. weigelii), the much thicker lower tomentum, the uniformly white cyphellae (vs. partially yellow in S. weigelii) and the upper montane versus lower montane-tropical habitat. Sticta rhizinata is also similar but can be distinguished by the narrower lobes, the more or less cylindric, rather dark isidia, the formation of conspicuous, large rhizines on the underside, and the predominant growth on the ground between bryophytes (Moncada Willdenowia 51 -2021& Lücking 2012. Sticta scabrosa (see below) differs by the marginally thinly scabrose lobe surface, the lighter brown phyllidia that are concolorous with the thallus, and the light grey-brown lower tomentum, together with a preferred growth in tropical, often disturbed or anthropogenic habitats.
Additional specimens examined -See Suppl. File S1. Diagnosis -Differing from Sticta beauvoisii in the thinly scabrose versus glabrous lobe surface, the formation of dorsiventrally flattened phyllidia instead of cylindric isidia, and the more greyish lower tomentum.
Vegetative propagules phyllidia, formed densely along lobe margins but sometimes extending onto lamina, richly branched and becoming palmate to coralloid, up to 0.5 mm long and 0.3 mm broad, with terminal parts dorsiventrally flattened to squamiform and basal part forming a short, flattened stipe, without cyphellae primordia; phyllidia of same colour as thallus or somewhat darker on upper side, somewhat paler on underside, slightly nitidous. Apothecia not observed. Pycnidia immersed.
Etymology -The epithet refers to the often thinly scabrose lobe surface toward the tips.
Distribution and ecology -Sticta scabrosa is widely distributed in the Neotropics, including the Caribbean and the Galapagos Islands (subsp. scabrosa) and also found in Hawaii (subsp. hawaiiensis, see below). In contrast to S. andina, it is a lowland to lower montane, tropical species found both in forest habitats and in disturbed or anthropogenic situations, e.g. on planted trees along roads, sometimes with a weedy character. Both subspecies have a similar ecology (Moncada & al. 2021).
Remarks -Like Sticta andina, S. scabrosa also corresponds to the S. weigelii morphodeme, but is more similar to S. beauvoisii than to S. weigelii s.str., a species only recently removed from synonymy under S. weigelii (McDonald & al. 2003). Sticta scabrosa is not closely related to either S. weigelii s.str. or S. beauvoisii (Moncada & al. 2014; this paper) and differs from the latter in the thinly scabrose versus glabrous lobe surface, the formation of dorsiventrally flattened phyllidia instead of mostly cylindric isidia, and the more greyish lower tomentum. The two subspecies, subsp. scabrosa and subsp. hawai iensis, are morphologically mostly identical, but the latter includes a distinctive lobe surface morphodeme (see below). Willdenowia 51 -2021 Sticta scabrosa subsp. scabrosa - Fig. 3A -L.
Diagnosis -Differing from subsp. hawaiiensis in two positions in the ITS (see Suppl. File S3), namely position 143 (T > C) and position 401 (T > C). Upper lobe surface smooth to uneven, never pitted.
Description -See above.
Distribution and ecology -Widely distributed in the Neotropics, lowlands to lower montane zones, often in exposed situations (Moncada & al. 2021).

Remarks -See above.
Additional specimens examined -See Suppl. File S1. Diagnosis -Differing from subsp. scabrosa in two positions in the ITS (see Suppl. File S3), namely position 143 (C > T) and position 401 (C > T). Upper lobe surface variable, smooth to uneven but in some forms distinctly foveolate-pitted.
Etymology -The subspecific epithet refers to the geographic distribution of this subspecies.
Distribution and ecology -Restricted to the Hawaiian archipelago, where it has been found on all five major islands (Hawaii or Big Island, Oahu, Molokai, Maui and Kauai). In lowlands to lower montane zones, often in exposed microhabitats (Moncada & al. 2021). Its ecology is the same as in the species as a whole. It is the only taxon of the genus present in Hawaii consistently found in disturbed habitats.
Remarks -Sticta scabrosa subsp. hawaiiensis is here separated as a formal taxon due to its consistent phylo genetic differences with subsp. scabrosa, with a clear geographic correlation. Because both subspecies cannot be separated morphologically (with exception of the additional lobe surface morphodeme present in Hawaii) and the phylogenetic differences are small (two substitutions out of 550 in a pairwise ITS alignment, i.e. 99.6 % similarity; see Suppl. File S3), we consider the rank of subspecies appropriate, to reflect the phylogenetic distinctiveness of this lineage and its geographic distribution in an isolated archipelago distant from the geographic range of subsp. scabrosa.