Two new species of the genus Lecidella (Lecanoraceae, Ascomycota) from maritime Antarctica, southern South America and North America

Abstract Two new species of the genus Lecidella, one with a North American-maritime Antarctic distribution and one with a so far exclusively southern South American-maritime Antarctic distribution, are described using molecular and morphological tools. Lecidella ayazii is a species growing on soil and also on mosses and has so far been found on the Antarctic Peninsula, as well as in the alpine areas of the La Sal Mountains, Utah, USA and in the Kivalliq Region (Nunavut) in the north of Canada, whereas L. drakensis occurs mainly on siliceous rocks, rarely on mosses, and has been recorded on both sides of the Drake Passage in southern Patagonia and the Antarctic Peninsula. Phylogenetic analysis of the nrITS sequence data shows that both species belong in the L. elaeochroma clade, each forming a highly supported and distinct group. Furthermore, they also differ in morphological and chemical characters from the species described so far in this clade. In addition, five further accessions were recorded from the maritime Antarctic, which were placed in the cosmopolitan and heterogeneous L. stigmatea clade, of which one could be assigned to the bipolar species L. siplei.


Introduction
The southern polar regions include not only the Antarctic continent, with less than c. 1% surface area suitable for terrestrial vegetation (Peat et al. 2007), but also the climatically milder maritime Antarctic Peninsula and the islands in the South Atlantic, as well as the islands in the southern Indian Ocean, the New Zealand shelf islands (Auckland Islands and Campbell Island), Macquarie Island (Lauer et al. 1996;Brummitt et al. 2001) and the subantarctic subregions along the mountain ranges in southern South America (Morrone 2000).
These milder and often ice-free areas are characterized by the absence of arboreal vegetation and are colonized by specialized and cold-adapted pioneer vegetation, including a high density of mostly rock-dwelling crustose lichens acting as pioneer organisms (Hertel 1984;Ruprecht et al. 2020).Lichens, as the dominant elements of Antarctic terrestrial vegetation, are represented by 300-400 species in the continent, according to the BAS (British Antarctic Survey) database, and they have been recorded in many studies during the last hundred years (e.g.Øvstedal & Smith 2001;Castello 2003;Hertel 2007;Peat et al. 2007;Ruprecht et al. 2010Ruprecht et al. , 2012;;Colesie et al. 2014;Halıcı et al. 2018Halıcı et al. , 2021;;Wagner et al. 2021).An even higher diversity of lichens can be estimated for southern South America (sSA, including Falkland Islands: Fryday et al. 2019;Ruprecht et al. 2020;Etayo et al. 2021;Fryday 2022).However, the biodiversity of lichens in the Antarctic regions is still not fully known.New species are continuing to be described, which has become possible especially through the use of modern techniques such as DNA barcoding (Ruprecht et al. 2020;Halıcı et al. 2021Halıcı et al. , 2022;;Lagostina et al. 2021).These techniques also have the potential to reveal whether species for which only an endemic distribution was previously indicated are in fact bipolar, along continents (South to North America) or even globally distributed.This also applies to a small number of lecideoid lichen species such as Lecidella siplei (C.W. Dodge & G. E. Baker) May.Inoue or Lecidea polypycnidophora U. Rupr.& Türk which were previously described as endemic to Antarctica (Castello 2003;Ruprecht et al. 2010).More recent studies have demonstrated that Lecidella siplei has a bipolar distribution and Lecidea polypycnidophora an alpine (North American)-southern polar distribution (Ruprecht et al. 2012;Hale et al. 2019).These unresolved biogeographical connections between Antarctica and Svalbard (Norway; L. siplei), as well as montane regions of western North America (L.polypycnidophora, L. andersonii Filson), show that potential colonization routes seem to exist and that the timing of dispersal events may also be highly relevant (e.g.Buschbom 2007;Fernandez-Mendoza & Printzen 2013;Hale et al. 2019;Ruprecht et al. 2020).
The globally distributed genus Lecidella Körb. is represented by c. 50 recognized species and belongs to the family Lecanoraceae (Knoph & Leuckert 2004).This is not only clearly supported by molecular data (Zhao et al. 2015), but also by the quite similar ascus type.However, the Lecidella-type ascus also occurs in the genus Japewiella (Printzen 1999).It differs from the Lecanora-type ascus by an amyloid tholus and a narrow axial body converging towards the apex (Knoph & Leuckert 2004), in contrast to a thickened and also amyloid tholus at the apex, but with a broad non-amyloid axial mass (Wirth et al. 2013).The genus is better distinguished by the extremely lax and rarely capitate paraphyses (Wirth et al. 2013).Initially, Hertel & Leuckert (1967) treated Lecidella as a subgenus of the genus Lecidea, but two years later they elevated it to genus level (Hertel & Leuckert 1969) because of its secondary chemistry, which differs from Lecidea by the presence of chlorinated norlichexanthones in many species.This genus is considered taxonomically difficult because of its high variation and/or plasticity in diagnostic characters (Zhao et al. 2015).The genus Lecidella is currently represented by nine species from the subantarctic region (Knoph & Leuckert 1994;Ruprecht et al. 2020;Etayo et al. 2021;Fryday 2022) and around six for maritime and continental Antarctica (Castello 2003;Ruprecht et al. 2012;Fryday 2022).
Globally distributed genera in lichen-forming fungi repeatedly show disjunct distributions caused, for instance, by vicariance and mid-distance dispersal (Lücking et al. 2008), or by transition from the Arctic to Patagonia (sSA) in the Pleistocene, resulting in cryptic speciation (Fernandez-Mendoza & Printzen 2013).Although this is not a common occurrence, Ruprecht et al. (2012Ruprecht et al. ( , 2020) ) reported a small number of endemic taxa and locally differentiated subgroups for the southern polar regions (southern South America, continental Antarctica) for the genera Lecidea and Lecidella.
Here we describe two new species of the genus Lecidella from maritime Antarctica and southern Patagonia, one with a North American-maritime Antarctic distribution and one with a so far exclusively southern South American-maritime Antarctic distribution, as well as several other maritime Antarctic accessions.

Material and Methods
Site descriptions (Fig. 1) James Ross Island, located in the North-East Antarctic Peninsula region, has a cold, polar-continental climate (Martin & Peel 1978) because of the Trinity Peninsula Mountains (Antarctic Peninsula) that shield the island from precipitation (Davies et al. 2013).Precipitation estimates range from 200 to 500 mm per year (van Lipzig et al. 2004) and, therefore, James Ross Island is considered a semi-arid environment.
Horseshoe Island is located in Marguerite Bay within the West Antarctic Peninsula.Gaul Cove is located on the east, while Lystad Bay is on the west coast of the island.The glacier-free regions are mainly composed of plutonic rocks consisting of granite and gabbro, banded gneiss and granitic gneiss belonging to the metamorphic complex.Sediments and moraines are also formed because of glacial movements on the island.The north of the island consists of rocks of a more diverse origin, whereas the mountains in the south are ponderous granites.
The two areas in southern Patagonia (Chile, sSA) are located in the Región de Magallanes y de la Antártica Chilena: Laguna Blanca at the volcanic Rock Morro Chico and the western part of the Torres del Paine National Park.Both areas are dominated by siliceous substrata and are influenced by the unique climate conditions for Patagonia, caused by strong westerly winds from the Pacific Ocean (Silva et al. 2009).
The specimens are deposited at the Erciyes University Herbarium Kayseri, Turkey (ERCH), at the herbarium of the University of Salzburg, Austria (SZU) and at the herbarium of Michigan State University (MSC; Table 1).

Morphological analyses
Light microscopic investigations were carried out with a Leica DVM6 digital microscope, a LEITZ Laborlux S and an Olympus BX53 (with an Olympus OM-D, E-M1 mark II camera).Photographic images were made of thallus morphology and anatomical features of asci and ascospores.Cross-sections of apothecia (12 μm thick) were prepared using a Leitz Kryomat 1703 freezing microtome.Measurements of anatomical structures always refer to water mounts, with at least 20 measurements made for all investigated anatomical structures.Spore measurements are given as: (minimum) most frequent (80%) (maximum).

DNA amplification, sequencing and phylogenetic analyses
Total DNA was extracted from individual thalli using the DNeasy Plant Mini Kit (Qiagen) following manufacturer instructions.The internal transcribed spacer (ITS) regions of the mycobionts' nuclear ribosomal DNA (nrITS) were sequenced and amplified using the primers ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. 1990) with standard conditions.PCR products were sent to Eurofins Genomics (Germany) for sequencing.
The sequences were assembled and edited using Geneious Pro v. 6.1.8(www.geneious.com),aligned with MAFFT v. 7.017 (Katoh et al. 2002) and placed in context of the most recently published phylogenies of Ruprecht et al. (2020) and Fayyaz et al. (2022) based on the concept of Zhao et al. (2015).The maximum likelihood analysis (ML) was performed using the IQ-TREE web server (Trifinopoulos et al. 2016) with default settings (ultrafast bootstrap analyses (Hoang et al. 2017), 1000 BT alignments, 1000 max.iterations, min.correlation coefficient: 0.99, SH-aLRT branch test with 1000 replicates) and presented as a consensus tree.The best-fit model according to BIC, Tne + I + G4, was selected with the implemented model finder (Kalyaanamoorthy et al. 2017) of the program IQ-TREE.Bayesian phylogenies were inferred using the Markov chain Monte Carlo (MCMC) procedure as implemented in MrBayes v. 3.2.(Ronquist & Huelsenbeck 2003).The analysis was performed assuming the general time reversible model of nucleotide substitution including estimation of invariant sites and a discrete gamma distribution with six rate categories (GTR + I + Γ; Rodriguez et al. 1990).Two runs with 2 million generations each starting with a random tree and employing four simultaneous chains were executed.Every 1000th tree was saved into a file.Subsequently, the first 25% of trees was deleted as the 'burn-in' of the chain.A consensus topology with posterior probabilities for each clade was calculated from the remaining 1501 trees.The phylogenies were visualized with the program FigTree v. 1.4.3 (Rambaut 2014).

Phylogenetic analyses
The final data matrix of this phylogeny contains 61 sequences of the ITS marker with a length of 538 characters, and was rooted with species of the genera Carbonea (Hertel) Hertel and Lecanora Ach.The phylogeny (Fig. 2) is divided into four main clades: L. stigmatea (Ach.)Hertel & Leuckert, L. enteroleucella (Nyl.)Hertel, L. elaeochroma (Ach.)M. Choisy (Zhao et al. 2015) and Lecidella sp.nov.(Ruprecht et al. 2020).Five accessions from the maritime Antarctic were located in the very heterogeneous clade of L. stigmatea.One of these accessions (JR._0.303) can be assigned to L. siplei.Both new species described here are part of the L. elaeochroma clade and form two strongly supported    (Fig. 3) Thallus crustose, granulose to rimose, up to 0.2 mm thick.Areoles distinct, almost squamule-like, irregular.Surface rough, pruinose.Colour greyish to chalky white.
Etymology.Named in honour of Çağan Ayaz Halıcı, dear son of the last author, who was born during his father's Antarctic expedition to James Ross Island in 2017.
Distribution and habitat.Lecidella ayazii is currently known from James Ross Island, located in the NE part of the Antarctic Peninsula, and Horseshoe Island in the SW; where it occurs on soil or sometimes on mosses from 5 to 345 m a.s.l., especially in humid habitats such as near streams.It is also known from the La Sal Mountains, Utah, USA and the Kivalliq Region, Nunavut, Canada, growing on mosses over siliceous substrata (Fig. 1).
Notes.Lecidella ayazii forms a distinct and highly supported clade of five accessions from maritime Antarctica as well as one accession from alpine areas of the La Sal Mountains, Utah, USA and two accessions from the Kivalliq Region, Nunavut in the north of Canada; it is placed in the L. elaeochroma clade (Zhao et al. 2015).According to Øvstedal & Smith (2001), one of two muscicolous species of Lecidella known from Antarctica is L. wulfenii, which is also part of the L. elaeochroma clade, but the latter species has a different chemistry, an orange-brown (McCune 2017) or reddish brown hypothecium and occurs over calcareous substrata (Knoph & Leuckert 2004;Wirth et al. 2013), in contrast to the newly described species which occurs over siliceous substrata.Furthermore, L. ayazii and L. wulfenii are phylogenetically placed in distinct clades and are not closely related.The second Antarctic species growing on mosses is L. siplei, which belongs to the L. stigmatea clade and is therefore clearly distinct from L. ayazii.Morphologically it differs with an often dark-pigmented (grey) thallus and a different chemistry (Ruprecht et al. 2012;Zhao et al. 2015).Two other phylogenetically related species, L. effugiens and L. elaeochromoides, grow on rocks, have a different chemistry and larger apothecia (Knoph & Mies 1995).The most closely related species is L. elaeochroma which shares a partially similar chemistry (atranorin, thuringione), but does not have an inspersed hymenium and typically grows only on bark (Wirth et al. 2013).

MycoBank No.: MB 851245
Differing from L. elaeochroma by having much smaller apothecia, from L. flavosorediata by the absence of soralia, from L. wulfenii by having more oblong ascospores, from L. euphorea by having a dark green to bluish green epihymenium, and from L. meiococca by having a thinner thallus.All the above-mentioned species also differ from L. drakensis in their chemistry.
Etymology.The term drakensis was chosen because the collected specimens occurred north (Chile, Región de Magallanes y de la Antártica Chilena) and south (maritime Antarctica) of the Drake Passage.
Distribution and habitat.Two specimens of Lecidella drakensis were found in the subantarctic areas of southern Patagonia (sSA, Morro Chico and Torres del Paine National Park) solely on siliceous rock and the other three in maritime Antarctica (James Ross Island) on siliceous rock and once on mosses.This species is currently recorded only from these areas.
Notes.The collections of L. drakensis form a distinct and highly supported clade containing accessions from southern Patagonia and maritime Antarctica, which is placed in the L. elaeochroma clade (Zhao et al. 2015).The species is clearly distinguished morphologically by the following characteristics: very small apothecia in contrast to L. elaeochroma (Wirth et al. 2013); lacking soralia and a different chemistry to L. flavosorediata (Wirth et al. 2013); more oblong spores and a different chemistry to L. wulfenii (Wirth et al. 2013); a dark green instead of violet-brown epihymenium and a different chemistry to L. euphorea (Zhao et al. 2015); a thinner thallus and also a different chemistry to L. meiococca (Knoph & Leuckert 1994).Neither species described here is related to the Southern Hemispheric, not yet molecularly confirmed species L. sublapicida (C.Knight) Hertel.This species is distinguished by its different chemistry (arthothelin, isoarthothelin) and reddish brown hymenium (Knoph & Leuckert 1994).Kappen (1985)  Lecidella ayazii, which was found by the last author only in the maritime Antarctic, was also found to cluster with a collection from alpine areas of the La Sal Mountains, Utah, USA (Leavitt The Lichenologist et al. 2021) and the Kivalliq Region, Nunavut, in the north of Canada (this study).The specimen from the La Sal Mountains was determined as Lecidella wulfenii because of its growth on moss, which is one of the most important traits for assigning this species.Unfortunately, there is another published sequence of L. wulfenii determined by Roman Türk (Ruprecht et al. 2012) which is not related and which is located in another clade (Fig. 2).This specimen was found on moss over calcareous rock, which is another important distinguishing characteristic (Wirth et al. 2013), in contrast to the specimens from the La Sal Mountains (Utah, USA) and the Kivalliq Region (Nunavut, northern Canada), where it can be assumed that the substratum is siliceous.The specimen of L. ayazii from the maritime Antarctic was also growing only over siliceous substrata.Lecidella wulfenii was described as Lichen muscorum by Wulfen (Jacquin 1790), but because that name is illegitimate, Hepp (1853) introduced the replacement name Biatora wulfenii Hepp, which was transferred to Lecidella by Körber (1861).Since Wulfen's species was described from Austria and the Türk collection mentioned above is also from Austria, we have no doubt that the epithet 'wulfenii' should be applied to the European species.
Thus far, Lecidella drakensis shows a similar pattern to that of Usnea aurantiacoatra (Lagostina et al. 2021).It occurs in southern South America as well as in maritime Antarctica.However, five other accessions were also recorded from the maritime Antarctic belonging to the cosmopolitan L. stigmatea clade, including one assigned to the bipolar species L. siplei.
distinct clades.The terricolous species Lecidella ayazii sp.nov., which occurs in maritime Antarctica, as well as in the alpine areas of the La Sal Mountains, Utah, USA and the Kivalliq Region, Nunavut in the north of Canada, is sister to accessions of L. elaeochroma, L. effugiens (Nilson) Knoph & Hertel and L. elaeochromoides (Nyl.)Knoph & Hertel.Lecidella drakensis sp.nov., restricted to southern Patagonia (sSA) and maritime Antarctica, is sister to a heterogeneous group consisting of accessions assigned to L. wulfenii (Hepp) Körb, L. elaeochroma, L. euphorea (Flörke) Hertel and L. flavosorediata (Vĕdza) Hertel & Leuckert.Lecidella meiococca (Nyl.)Leuckert & Hertel is basal to this highly supported group.Taxonomy Lecidella ayazii Halıcı & U. Rupr.sp.nov.MycoBank No.: MB 851244 Differing from L. wulfenii by having a golden brown instead of a reddish or orange-brown hypothecium, growing on mosses over a siliceous instead of calcareous substratum and by a different

Figure 1 .
Figure 1.Collection sites of Lecidella species in maritime Antarctica, the subantarctic areas of southern South America (Chile), and North America.Turquoise triangles = Lecidella ayazii; pink circles = Lecidella drakensis.In colour online.

Figure 2 .
Figure 2. Phylogenetic analysis of ITS sequences of the genus Lecidella with the newly described species L. ayazii (turquoise) and L. drakensis (pink), and five other accessions (bold) integrated in the species concepts of Zhao et al. (2015) and Ruprecht et al. (2020).Maximum likelihood (ML) bootstrap values ≤ 95 were directly mapped on the Bayesian tree with posterior probability values ≤ 0.95 (branches in bold).Branch tips are labelled with voucher ID/Accession number_species_OTU.In colour online.

Table 1 .
Voucher information and GenBank Accession numbers of the investigated specimens of the genus Lecidella collected in the subantarctic areas of southern South America (Chile), maritime Antarctica and northern Canada.
(Garrido-Benavent & Pérez-Ortega 2017;Hale et al. 2019y described, that forms a greyish pulvinate crust on rock, from northern Victoria Land (H.Hertel, personal communication) and is clearly morphologically different from L. ayazii and L. drakensis.Two prominent examples are the common species Lecidea cancriformis C. W. Dodge & G. E. Baker and Usnea aurantiacoatra (Jacq.)Bory(Ruprechtetal. 2020;Lagostina et al. 2021).Both species are distributed not only in the subantarctic areas of southern South America but also in the maritime and/ or continental Antarctic.Other species such as Usnea antarctcia Du Rietz are even more restricted to southern polar areas and occur only in the maritime Antarctic(Lagostina et al. 2021).Conversely, species such as Lecidea polypycnidophora(Hale et al. 2019)and Lecidea andersonii(Hertel 2007;Ruprecht et al. 2010;Hale et al. 2019) have an alpine and/or bipolar distribution which suggests that there are migration routes especially along the American continent(Garrido-Benavent & Pérez-Ortega 2017;Hale et al. 2019).The two newly described species, L. ayazii and L. drakensis, are part of the cosmopolitan genus Lecidella.They are both located in the L. elaeochroma clade, to which mostly Northern Hemisphere species have been assigned until now, and form clearly distinguished groups.