Introduction
Horseshoe Island is located in Marguerite Bay within the West Antarctic Peninsula. According to the BAS Database (2022), 29 lichenized fungal species and 15 moss species have been reported from Horseshoe Island. Apparently, these numbers do not represent the biodiversity of Antarctic terrestrial vegetation and more detailed and professional studies should be carried out. In a project aiming to determine the lichen biodiversity of this island with the aid of DNA-barcoding, the first author collected lichens from Horseshoe Island during Turkish Antarctic Expedition VI, 2022. From these collections, Candelariella ruzgarii Halıcı, A.M. Kahraman & Güllü (Halıcı, Kahraman Yiğit, Bölükbaşı, & Güllü, Reference Halıcı, Kahraman Yiğit, Bölükbaşı and Güllü2023a) and Lendemeriella vaczii Halıcı, Kahraman Yiğit & Güllü (Halıcı, Bölükbaşı, Güllü, Kahraman Yiğit, & Barták, Reference Halıcı, Bölükbaşı, Güllü, Kahraman Yiğit and Barták2023b) have already been described and published.
Thamnolecania (Vain.) Gyeln. is a genus of lichenized fungi comprising Antarctic endemic species established by Gyelnik (Reference Gyelnik1933). This genus is a well-supported clade including T. brialmontii (Vain.) Gyeln., T. gerlachei (Vain.) Gyeln. and T. racovitzae (Vain.) S.Y. Kondr., Lőkös & Hur (Kondratyuk et al., Reference Kondratyuk, Lőkös, Kim, Kondratiuk, Jeong, Zarei-Darki and Hur2014; Reese Næsborg, Ekman & Tibell, Reference Reese Næsborg, Ekman and Tibell2007). These three species were first described under the genus Lecanora by Vainio (Reference Vainio1903) as subgenus Thamnolecania. Reese Næsborg et al. (Reference Reese Næsborg, Ekman and Tibell2007) indicated that an alternative classification would be to make a wider circumscription of the genus Bilimbia that included Thamnolecania and affiliated taxa such as Lecania croatica (Zahlbr.) Kotlov and Lecidea sphaerella Hedl.
Morphological, anatomical and phylogenetical analyses of the specimens collected on mosses from Horseshoe Island by the first author in 2022 showed that these samples belong to an undescribed species in Lecania s. lat. and very closely related to the genus Thamnolecania. Therefore, in this paper, we describe this novel taxon under the genus Thamnolecania but also suggest that it may be transferred to a novel genus when more data becomes available.
Material & methods
Morphological and anatomical studies
Samples of lichenized fungi were collected from Horseshoe Island (Antarctic Peninsula). Collected specimens are deposited in “Erciyes University Herbarium (ERCH), Kayseri, Turkey).” The specimens were identified by using standard microscope methods. The chemistry was analysed using spot tests, (i.e. 10% KOH (K) and sodium hypochlorite (C)) or thin-layer chromatography (TLC) using solvents A (toluene/1,4-dioxane/glacial acetic acid; 36:9:1) and solvent C (toluene/glacial acetic acid; 20:3; Huneck, Yoshimura, Huneck, & Yoshimura, Reference Huneck, Yoshimura, Huneck and Yoshimura1996; Orange, James & White, Reference Orange, James and White2001). Measurements were made only from sections in water. Ascospores were measured outside the asci. The average is followed by its standard deviation, and the maximum and minimum values are given in parentheses.
Molecular methods
DNA isolation, PCR and sequencing
Total genomic DNA was extracted using the commercial DNA isolation kit (DNeasy Plant Mini Kit; Qiagen). The isolation was carried out according to the instructions prepared by the manufacturer in the kit. The most universal primers were used in this study. The complete ITS plus 5.8S rDNA was amplified using primers ITS1F and ITS4 (Gardes & Bruns, Reference Gardes and Bruns1993; White, Bruns, Lee & Taylor, Reference White, Bruns, Lee and Taylor1990). The mtSSU gene region was amplified by using the primers mtSSU1F and mtSSU3R (Zoller, Scheidegger & Sperisen, Reference Zoller, Scheidegger and Sperisen1999). The RPB1 gene region was amplified using the primers RPB1-5F (Denton, McConaughy & Hall, Reference Denton, McConaughy and Hall1998) and fRPB1-11aR (Liu, Whelen & Hall, Reference Liu, Whelen and Hall1999). Each sample was prepared for a total of 50 µl of standard reaction. Optimum amplification conditions were obtained with 25 μl of 2 × Taq PCR MasterMix in each tube with 1 mM of the primers ITS1F and ITS4, ∼100 ng of DNA extracts and it was completed with 50 μl double distilled water. PCR amplifications were conducted with an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C or 56 °C and 52 °C (nrITS, mtSSU and RPB1, respectively) for 1 min, 72 °C for 90 s and a final extension at 72 °C for 10 min. Positive amplification of the gene regions was determined using agarose gel electrophoresis. Samples were visualised under UV light. Sequence analyses of lichen samples from which PCR products were obtained were performed by Epigen Biotechnology Laboratory (Ankara, Turkey).
Additional sequences
The final dataset consisted of newly generated sequences (1 sequence for ITS, 1 sequence for mtSSU and 1 sequence for RPB1) from this study and 39 nrITS, 45 mtSSU and 11 RPB1 sequences obtained from GenBank (Table 1). Sphaerophorus globosus was used as the outgroup in nrITS, mtSSU and RPB1 phylogenetic trees.
Table 1. nrITS, mtSSU and RPB1sequences used in the analyses. The new sequences are in bold.
Sequence alignment and phylogenetic analysis
nrITS, mtSSU and RPB1 sequences of all species were aligned and optimised manually using ClustalW in BioEdit V7.2.6.1 for preparing the phylogenetic trees. In MEGA XI, only parsimony-informative regions were used for analysis. Indeterminate regions were excluded from the alignment (Hall, Reference Hall1999; Tamura, Stecher & Kumar, Reference Tamura, Stecher and Kumar2021). The final alignment comprised 579 (nrITS), 785 (mtSSU) and 619 (RPB1) columns.
One-thousand bootstrap replications were performed by bootstrap analysis for the estimation of confidence levels of the clades. Phylogenetic relationships and support values were investigated using maximum likelihood (ML) bootstrapping, as implemented in MEGA XI. Kimura's two-parameter model was used for the analysis of the ML method. Genbank numbers of used sequences in phylogenetic trees within this study are given in Table 1. Sequence data obtained from ITS and mtSSU gene regions were combined using the Geneious v2023.2.1.
Results
Evaluating phylogenetic analysis results
Three independent phylogenetic trees for Thamnolecania and related genera were produced from 68 sequences (39 for nrITS, 15 for mtSSU) from GenBank and three new sequences (1 sequence for nrITS, 1 sequence for mtSSU and 1 sequence for RPB1) from the new species. We obtained the RPB1 gene of the species, but because of the insufficient RPB1 data of Thamnolecania genus in GenBank, we did not construct the RBP1 gene phylogenetic tree. All species names are followed by the GenBank accession numbers or voucher information (Table 1). When concatenated maximum likelihood (ITS + mtSSU) tree was examined, Thamnolecania yunusii, which we described as a new species, clearly showed branching with other species belonging to the genus Thamnolecania with a high bootstrap value (Fig. 1). Within the genus Thamnolecania, the new species clearly occupied a separate branch from the rest of the genus. The phylogenetic analysis did not designate any species identical to the new species in the genus Thamnolecania. Polymorphism statistics and Blast comparisons of the new species and related species are shown in Table 2.
Figure 1. Concatenated maximum likelihood phylogenetic tree (ITS + mtSSU) of Thamnolecania and related genera. Posterior probabilities are shown above branches. Numbers at nodes represent the ML bootstrap support (values ≥ 50%). The new species T. yunusii is highlighted. Sphaerophorus globosus was used as outgroup.
Table 2. Polymorphism statistics for each marker (nrITS and mtSSU) from datasets corresponding to the genus Thamnolecania and Blast comparison of the new species with related species in the GenBank.
Taxonomy
Thamnolecania yunusii Halıcı, Güllü, Bölükbaşı & Kahraman sp. nov. (Fig. 2)
Figure 2. Thamnolecania yunusii. (a) Habitus. (b) Apothecial section in water. (c) Asci and paraphyses in methylene blue. (d) 3– septate hyaline ascospores.
MycoBank No.: MB 848346
Type: Antarctic Peninsula, Horseshoe Island: Sally Cove, Bourgeois Fjord, Marguerite Port, 67°48′30″S 67°17′39″W, alt. 10 m, on mosses, 17 February 2022, leg. M. G. Halıcı ERCH HS 0.044 (ERCH—holotype).
Diagnosis: Characterised by cream to greyish brown granulose, crustose thallus without vegetative propagules growing on mosses with shorter hymenium (30–50 μm high) compared with the other known members of the genus.
Etymology: Named in honour of Yunus Emre, also known as Derviş Yunus (Yunus the Dervish) (1238–1328), who was a Turkish folk poet and Sufi mystic who greatly influenced Turkish culture. Yunus Emre, who passed away 700 years ago, especially examined the love of humanity and nature in his poems. In order to keep the name of this important heartfelt person alive in nature, we decided to name the new species after him.
Description: Thallus effuse, cream to greyish brown sometimes with a greenish tinge, granulose, up to 0.3 mm thick; granules up to 0.5 mm diam; medulla I-. Photobiont trebouxioid, cells subglobose to globose, 9–13 × 8–12 µm diam.
Apothecia common, mostly aggregated, biatorine, dark brown to almost black, epruinose, mostly flat, sometimes slightly concave, 0.3–0.4 mm diam., with a distinct thalline margin concolourous with the thallus. The thalline margin is frequently apparent. In section: proper exciple c. 25 μm thick, thalline margin c. 40 μm thick, inner section hyaline to becoming brown towards the cortex (K+ purple-brown; Lecania-brown pigment); composed of narrow irregularly radiating hyphae c. 1 μm wide; epihymenium 15–25 μm, brown. Hymenium hyaline, 30–50 μm high, upper parts greenish brown, K+ purple-brown (Lecania-brown pigment); paraphyses simple or sometimes branched at the tips, septate, capitate to moniliform mostly with a brown cap. Hypothecium hyaline, 60–80 μm high, is composed of randomly orientated hyphae. Asci Biatora-type, cylindrical or sometimes clavate, 40–45 × 8–10 μm, ascospores hyaline, 3– septate, randomly arranged in ascus, mostly multiseriate towards the apices, vermiform with rounded or sharp ends, (13.5–)15.5–17.5–19.5(–22.5) × (3–)3.5–4.5–5.5(–6.5) µm (n = 30) and l/w ratio: (2.46–)3.43–4.27–5.1–(6.17) µm (n = 30). Conidiomata is not observed.
Chemistry: No secondary components were detected in TLC. All spot tests are negative.
Ecology and distribution: The species is so far only known from Horseshoe Island, Antarctica. On Horseshoe Island (Antarctic Peninsula, Antarctica), it grows on mosses with Parvoplaca athallina (Darb.) Arup, Søchting & Frödén.
Discussion
Our phylogenetic analyses show that Thamnolecania yunusii clearly differs from other species of the genus. The phylogenetic trees obtained as a result of using nrITS and mtSSU gene regions showed that the new species grouped together with T. gerlachei, T. brialmontii and T. racovitzae species. Although the new species forms a clade with other Thamnolecania species, it occurred on a different branch (with higher bootstrap support) from other species. However, since we do not currently have sufficient sequence data to justify erecting a new genus, we include it within the genus Thamnolecania. With more detailed studies in the future, it is possible that this new species can be transferred to a novel genus separated from Thamnolecania s. str. in Lecania s. lat.
The genus Thamnolecania is characterised by most species having a fruticose habit, but sometimes also foliose, squamulose or crustose (Øvstedal & Smith, Reference Øvstedal and Smith2001), having three-septate relatively large ascospores up to 22 µm long, a high hymenium (60–100 µm) and relatively large apothecia (up to 3 mm diam.) (Reese Næsborg et al., Reference Reese Næsborg, Ekman and Tibell2007). Interestingly, all the members of the genus are apparently Antarctic endemics. The new species, T. yunusii, is morphologically characterised by its granulose, crustose thallus without vegetative propagules. Although all the other known species of the genus have a higher hymenium (60–100 µm) as indicated above, T. yunusii has a lower hymenium (30–50 μm high) differing from the other species. The only other Thamnolecania species with a crustose thallus is T. racovitzae, but this species differs from T. yunusii by growing on rocks, having an effuse to subeffigurate thallus that is sometimes isidiate, and with shorter and narrower ascospores (c. 15 × 3.5 µm vs. 15.5–19.5 × 3.5–5.5 µm) (Øvstedal & Smith, Reference Øvstedal and Smith2001).
Acknowledgements
This study was carried out under the auspices of the Presidency of the Republic of Turkey, supported by the Ministry of Industry and Technology, coordinated by TUBITAK MAM Polar Research Institute.
Financial support
This work was supported by TUBITAK 121Z771 coded project and Turkish Academy of Science (TÜBA).
Competing interests
The authors declare none.
Authorship
Mehmet Gökhan Halıcı collected the specimen and write the manuscript. Mithat Güllü made the phylogenetic analyses. Ekrem Bölükbaşı made the molecular studies. Merve Kahraman Yiğit made the morphological and anatomical analyses.
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