The Revision of Lichen Flora Around Maxwell Bay, King George Island, Maritime Antarctic

Since the floristic study of lichens at the Barton and Weaver Peninsulas of King George Island in 2006, there have been intense investigations of the lichen flora of the two peninsulas as well as that of Fildes Peninsula and Ardley Island in Maxwell Bay, King George Island, South Shetland Islands, maritime Antarctic. In this study, a total of 104 species belonging to 53 genera, are identified from investigations of lichens that were collected in austral summer seasons from 2008 to 2016. Phenotypic and molecular analyses were incorporated for taxonomic identification. In particular, 31 species are found to be endemic to the Antarctic and 22 species are newly recorded to the Maxwell Bay region. Lepra dactylina, Stereocaulon caespitosum, and Wahlenbergiella striatula are newly recorded in the Antarctic, and the previously reported taxon Cladonia furcata is excluded from the formerly recorded list due to misidentification. We also provide ecological and geographical information about lichen associations and habitat preferences. Supplementary Information The online version contains supplementary material available at 10.1007/s12275-023-00015-x.


Introduction
Lichens are symbiotic organisms that are composed of lichenized fungi (mycobiont) and microalgae and/or cyanobacteria (photobiont). They have successfully adapted to severe environmental conditions that prevail in tropical, desert, high alpine, and polar regions. Lichens usually inhabit ice-free areas (Singh et al., 2018;Smith, 1988), and they are the main vegetation in the Antarctic terrestrial ecosystems along with two vascular plants, Deschampsia antarctica Desv. and Colobanthus quitensis (Kunth) Bart, and diverse bryophytes (Ochyra et al., 2002;Øvstedal & Smith, 2001).
King George Island is one of the most appropriate places for studying the biodiversity and evolution of lichenized fungi of Antarctica because of its geographical location and the diversity of lichens (Kim et al., 2006;Lee et al., 2008;Øvstedal & Smith, 2001). Floristic notes on lichens in 1993 covering the Fildes Peninsula of King George Island and Harmony Cove of Nelson Island indicated 198 species; however, determination of many of these species is unreliable due to the unavailability of the type collections or a lack of adequate collections (Hertel, 1988;Inoue, 1991Inoue, , 1993. Information on the lichen species in Antarctica remains insufficient for comprehensive ecological studies despite its significance (Kim et al., 2006). Floristic research has been 1 3 conducted in some of the localities (Admiralty Bay, Elephant Bay, and Lions Rump area) of King George Island (Olech, 2002); however, the areas around Maxwell Bay have often been excluded. Kim et al. (2006) reported 62 lichenized fungi from Barton Peninsula and Weaver Peninsula, part of Maxwell Bay region of the island. They conducted intensive survey close to the King Sejong Korean Antarctic Research Station in Barton Peninsula to investigate the presence and distribution of vegetation. In addition, the phylogenetic analyses of 50 lichenized fungi from King George Island were analyzed using the partial large subunit ribosomal DNA sequences (Lee et al., 2008).
However, the information on lichen diversity is insufficient due to the cryptic diversity and the less visible characteristics of microlichen species in this region.
This study aims to provide complementary lichen floristic information to understand the terrestrial biodiversity of the maritime Antarctic region and to conduct comparative studies with the biodiversity of other Antarctic Conservation Biogeographical Regions (Terauds & Lee, 2016). We investigated lichen flora intensively in the Maxwell Bay region, i.e. Barton Peninsula, Fildes Peninsula, Weaver Peninsula, and Ardley Island in King George Island during an Antarctic expedition from 2008-2016. We also provide ecological and geographical information of collected specimens along with taxonomic identification. The past specimens from the region were re-examined as well.

Study Area
Maxwell Bay (62°25′S; 58°85′W) is located between King George Island and Nelson Island in the South Shetland Islands of Antarctica. It is a common fjord-like Antarctic embayment characterized by a U-shaped deep basin. Fieldwork was carried out in Barton Peninsula, Fildes Peninsula, Weaver Peninsula, and Ardley Island located around Maxwell Bay (Fig. 1). The study area includes the Antarctic Specially Protected Areas (ASPAs) that are designated to protect environmental and scientific values such as breeding bird colonies, relatively extensive flora, and geological features under the Antarctic Treaty System. Narębski Point (ASPA No. 171) encloses penguin colonies and is located on the southeast coast of Barton Peninsula, King George Island. Ardley Island is an islet that is 1.9 km long and is located off the southwest end of King George Island. Bird colonies inhabit the area, and the whole island has been designated as ASPA No. 150 (Fig. 1).

Specimen Collection
A total of 982 specimens were collected from the study area and were examined during Antarctic expeditions from 2008 to 2016. Ecological information such as habitats, substrata, and other ambient traits at nearby locations were recorded for all specimens. Geographic coordinates of collection sites were recorded with GPSMAP 64 s (Garmin) or Trimble R8 GNSS /Trimble HPB450 /Geoexplorer 6000 XH Handheld, (Geosystems Inc.). The specimens were then deposited at the Korea Polar Research Institute (KOPRI).

Taxonomic Nomenclature
All the species listed in Tables 1 and 2 followed the current name on MycoBank (www. mycob ank. org/) and were confirmed with Index Fungorum (www. index fungo rum. org/), and GBIF (www. gbif. org/). If the taxonomic nomenclature was controversial in those references, the synonymy and/ or taxonomy opinions of MycoBank were prioritized. The broad determination of the species was primarily conducted following Øvstedal and Smith (2001) and other relevant literature (mentioned below).

Phenotypic Analysis
Morphological characteristics were examined under a microscope (Zeiss Boom Stand Stemi 2000 Stereo Microscope) and the components were analyzed by Thin Layer Chromatography (TLC). TLC analyses were performed according to standardized methods using solvent systems A and C, and Merck TLC silica gel with Lethariella cladonioides (Nyl.) Krog. as the control (Culberson, 1972;Orange et al., 2001).

Molecular Analysis
Molecular phylogenetic analyses were applied to 49 lichen species that were not clearly identified by morphological and chemical characteristics. Seventy-three specimens were identified using molecular analyses and among them, we obtained nucleotide sequences from 61 specimens through this study (Table S1). The samples of thalli were preserved at -80 °C in 100% ethanol for molecular analyses. For DNA extraction, samples were washed three times with 0.85% NaCl by vigorous mixing, spin down, and discarding of the supernatant. Samples were then freeze-dried and ground into a fine powder using a Tissue lyser (QIAGEN). DNA extraction was performed according to the DNA extraction protocol of the Exgene soil DNA mini kit (GeneAll,. ITS1-5.8S-ITS2 (ITS) were amplified using ITS1F and LR5 (Gardes & Bruns, 1993;Vilgalys & Hester, 1990) and sequenced using the ITS1F and LR5 primers by the procedures described in a previous study (Park et al., 2012).
Sequence alignments of ITS2-nucLSU rDNA were conducted using the program jPHYDIT (Jeon et al., 2005) and were manually adjusted. Ambiguously aligned sites were excluded for phylogenetic analyses. Phylogenetic trees (Fig. S1) were inferred for ITS by maximum parsimony (MP), neighbor joining (NJ) and maximum likelihood (ML). The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) (Nei & Kumar, 2000), the NJ tree was obtained using the p-distance method and the ML tree was obtained using the GTR + I + G evolutionary model of MEGA X (Lanave et al., 1984;Nei & Kumar, 2000) with search level 5, in which the initial trees were obtained by bootstrap method (1000 replicates). All positions containing gaps were treated as missing data. Genbank accession numbers of the sequences are included in Table S1. Obtained sequences were handled for the determination of sequence similarity with reference sequences from the NCBI database (https:// blast. ncbi. nlm. nih. gov/).

Lichen Flora in Maxwell Bay Region
All lichen species surveyed in this study are listed under the taxonomic hierarchy in Table 1. Identification of several species was supported by molecular phylogeny in addition to morphology and chemistry. A total of one hundred-four species, belonging to 53 genera, were identified in the Maxwell Bay region (Table 1). Among them 22 species marked in bold letters are newly recorded around the bay and 31 species are endemic to Antarctica. Photographs of several Antarctic endemic species are provided in Fig. 2. Lecanorales showed the highest species diversity and followed by Caliciales and Teloschistales (Table 1).
The observations of ecological traits were made at the time of specimen collection (Table 2). Among the sampling regions, Barton Peninsula showed the highest lichen diversity. Some species occurred on multiple substrates and habitats. Rock is the most general substrate for the lichen species in Maxwell Bay region (Fig. 3A). Seven epilichenic species were found and three crustose lichens were observed on a weathered whalebone. A few species favored several habitats: seashore, coastal cliff, and intertidal zones (Table 2). Some species showed habitat preference. Seven species were closely associated with the intertidal zone, especially the taxa of Verrucariaceae. Fifteen species were observed on perch boulders nearby nests of penguins and flying birds (Fig. 3B).
Lichen thallus crustose and subgelatinous, dark green, irregular shape of perithecia are common on rocks, Cladonia furcata (Huds.) Schrad. has been previously reported once in Antarctica, based on the specimens from Barton Peninsula (Kim et al., 2006). The specimens were reexamined in this study (no. HUR ANT050856 and no. HUR ANT050857; Fig. 4F). They showed a morphology that was typical of Cladonia gracilis subsp. elongata (Wulfen) Vain. An unbranched to somewhat branched podetia and closed axil were observed (Fig. 4F). Tips were pointed and seldom narrowly scyphose. Typically, open axil and numerous squamules were observed in C. furcata. Cracks on the podetia and damaged tips were frequently observed and these may have been misconceived as the 'open axil' trait.
In these specimens, only fumarprotocetraric acid was found by TLC. The acid is found in both C. gracilis (L.) Willd. and C. furcata (Ahti & Stenroos, 2013). It was also proven to be closely related to C. gracilis based on the molecular phylogeny (Fig. 5). We suggest the delisting of C. furcata from the lichen list provided in Kim et al. (2006).

Discussion
Around the Maxwell Bay, twenty-two new lichen species have been identified (Table 1). Above all, Lepra dactylina, Stereocaulon caespitosum, and Wahlenbergiella striatula were previously unrecorded around the Maxwell Bay area, besides being foreign to Antarctica. L. dactylina was previously classified as Pertusaria dactylina (Ach.) Nyl. and is known as circumpolar species in the Arctic (Brodo et al., 2001), as reported by Olech (2002Olech ( , 2004 from Lion Rump, King George Island; however, the records were subsequently revised to exclude this species (Olech and Slaby, 2016). Our specimen (no. 2017-Ant-053) exhibited a whitish thallus with numerous tall isidia ( Fig. 4A and B), not just a thick crust, compared to the other Pertusaria spp. such as P. excludens Nyl. and P. signyae Øvstedal. (Brodo et al., 2001;Thomson, 1984). Stereocaulon caespitosum observed in New Zealand and Tasmania were flattened and had cracked pseudopodetia without enlarged tips (Smith & Øvstedal, 1991), and those collected from South Georgia had the same features. This species had been found in the subantarctic region but not in the Antarctic (Øvstedal & Smith, 2001). In this study, the specimen (no. 2017-Ant-061) collected from the Barton Peninsula was comparable to and corresponded with the descriptions of S. caespitosum (Fig. 4C). It should be noted that the Antarctic specimen showed large pale orange apothecia which has not been observed previously (Fig. 4D). Gueidan et al. (2009)  to the molecular and morphological evidence. This species is a common lichen on the littoral zone in the Northern Hemisphere but has seldom been recorded in the Southern Hemisphere. The specimen collected from New Zealand was described by Santesson (1939) and named V. striatula subsp. australis. Currently, two of the subspecies are in the New Zealand lichen flora list together as Wahlengergiella (de Lange et al., 2018). Verrucaria striatula subsp. australis showed a more effuse thallus margin (McCarthy, 1991) than that in the Northern Hemisphere, and our specimen shared characteristics with the former. The prominent perithecia (a small flask-shaped fruiting body that contains the ascospores) are often irregular in shape (Fig. 4E), and ostioles are comparatively large. The thallus is distinctive in its cover of black dots and ridges which gives the surface a rough texture. The combination of dark ridges and tiny black dots on a green-black thallus is a typical feature of this species. Verrucaria amphibia (Clemente) may be confused with W. striatula; however, it is darker with ridges but lacks the dot matrix that gives the thallus surface texture (Brodo & Santesson, 1997). TLC was negative in the examined specimen. Wahlenbergiella striatula is a pyrenolichen occurring on siliceous maritime rocks in the mid-to high-tide mark on rocky coasts. Wahlenbergiella striatula and Verrucaria spp. is usually found on the boulders, particularly near the Chinstrap penguin colonies (Table 2).
We identified 104 species of 53 genera and added 22 unrecorded species to the previous lichen floristic studies around the Maxwell Bay region (Guzmán & Redón, 1981;Hu, 1998;Inoue, 1991Inoue, , 1993Jianbin, 1996;Kim et al., 2006;Olech, 1996Olech, , 2004Øvstedal & Smith, 2001). The Barton Peninsula showed the highest lichen diversity with 101 species among the sampling regions for the number of lichen taxa recorded. A Polish lichenologist, Olech conducted a comprehensive lichen floristic study of King George Island and 153 sampling points were visited for the study (Olech, 2004). In the study, 253 lichen species were reported, of which 74 taxa were newly recorded. Only 16% of the newly recorded taxa were found around Maxwell Bay, as most of the sampling points (101 points) were concentrated at the Admiralty Bay where the Polish Antarctic research station is located. From Barton Peninsula, Olech (2004) reported just 16 species from three points. Because of the harsh environment and remoteness of Antarctica, detailed Antarctic lichen floristic studies have been limited to areas that are near the Antarctic research stations, so that our investigation was also carried out around the Korean Antarctic Research Station. However, the topography of the Barton Peninsula is much dynamic, glacio-marine origin, and is marked by fjords, cirque, flatforms, and hanging valleys covered with bedrocks, rock fragments, moraines, raised beaches, patterned grounds, and patterned beaches (Chang et al., 2003). The complex topography of the peninsula provides a variety of habitats for diverse lichens despite the relatively narrow exposed area. This intensive survey noted that the Barton Peninsula exhibited noticeable lichen diversity and variety and may be referred to as 'The diversity hotspot' (Green et al., 2011;Ji et al., 2016). Given the geographical and ecological significance of the Antarctic research, the comprehensive study of the peninsula was comparatively lacking prior to this survey.
Our result shows that Lecanorales had the highest species diversity, followed by Caliciales and Teloschistales (Table 3 and Fig. 6). This trend was seen in other studies in the regions of King Gorge Island and South Shetlands Islands. Lecanorales is also the most diverse taxa of lichens worldwide. Ostropales and Arthoniales are highly diverse worldwide (Table 3), few have been reported from the Antarctic region because of their micro size or inconspicuous parasitic (lichenicolous) taxa and a lack of specialists; Olech (2004) only reported six taxa of Arthoniales and one of Ostropales from King George Island. On the other hand, while most diverse orders were the same, the proportions of Lecanorales and Caliciales were similar in Cape Hallett in the Eastern Antarctic region (i.e., a continental Antarctic climate region), one of the richest sites for terrestrial biodiversity in the Ross Sea region where fiftynine lichen species were reported for eight sites (Green et al., 2015). Most species were crustose and a few were foliose near an Adelie penguin colony, which acted as a major source of ammonia (Theobald et al., 2013). Other eastern Antarctic Lichen flora reported from Schirmacher Oasis and Larsemann Hills, Prydz Bay included up to 69 and 25 lichen species respectively (Singh & Nayaka,  . The major lichen taxa recorded in the region consisted of Buellia with 10 species, followed by 9 species of Lecanora, and 5 species each of Caloplaca and Umbilicaria. However, owing to the diverse habitats of lichen in the Antarctic terrestrial ecosystem, we expect that more cryptic taxa will be reported in future studies, particularly, in the eastern Antarctic region where fewer studies have been conducted than the western Antarctic region. Furthermore, a significant number of unrecorded species were discovered and most of them were microlichens, which suggests that the biodiversity of the Antarctic lichens may be not to be fully revealed. Lichen flora in the Antarctic are surprisingly rich, and are equally at risk from environmental change as lichens in other regions (Chown et al., 2015). In particular, glacial outposts are worthy to be investigated for further understanding of the Antarctic floristic composition and their roles in the ecosystem in the context of global warming, and more information on the lichen diversity (as the most dominant component of Antarctic terrestrial ecosystems) is required.