Introduction
The genus Anamylopsora Timdal was established in 1991 based on the type specimen of A. pulcherrima (Vain.) Timdal (Timdal Reference Timdal1991). Initially, it was described as Lecidea pulcherrima Vain. in 1888 (Vainio Reference Vainio1888) but then Elenkin transferred it to the genus Psora due to the saxicolous and squamulose characters of the thallus, renaming it Psora pulcherrima (Vain.) Elenkin (Elenkin Reference Elenkin1904). Since the species also deviated from the genus Psora in a number of characteristics (e.g. having a non-amyloid tholus and hymenial gelatine, lacking anthraquinones in the hymenium, and having a different type of upper cortex and pycnidium), Timdal therefore established the monotypic new genus Anamylopsora in the family Lecideaceae (Timdal Reference Timdal1991). Timdal (Reference Timdal1984) had previously synonymized Lecidea hedinii Magnusson with L. pulcherrima and when proposing the genus Anamylopsora he also synonymized L. undulata H. Magn. with A. pulcherrima (Timdal Reference Timdal1991). In 1995, Lumbsch then established a new family Anamylopsoraceae, which differed from the Lecideaceae and Psoraceae due to the presence of gymnocarpous ascoma development and stipitate apothecia (Lumbsch et al. Reference Lumbsch, Lunke, Feige and Huneck1995). Later, the family Anamylopsoraceae was synonymized with Baeomycetaceae based on multigene phylogenetic analyses and currently Anamylopsora is included in the family Baeomycetaceae (Baeomycetales) (Resl et al. Reference Resl, Schneider, Westberg, Printzen, Palice, Thor, Fryday, Mayrhofer and Spribille2015).
To date, three species of Anamylopsora are recognized: A. altaica Ahat et al. from China, A. pruinosa D. L. Liu & X. L. Wei from China, and A. pulcherrima (Vain.) Timdal from Russia and North America. All of these species have been reported from high-altitudinal regions at elevations up to 3900 m (Timdal Reference Timdal1991; Zuo et al. Reference Zuo, Liu, Li, Chen and Wei2018; Ahat et al. Reference Ahat, Tumur and Guo2019; Esslinger Reference Esslinger2021).
The Gilgit-Baltistan region of Pakistan, formerly known as the Northern Areas, is a highly mountainous region which includes parts of four great mountain ranges, namely the Himalaya, Hindukush, Karakoram and Pamir ranges. Besides mountains, this region is also famous for the spectacular Deosai Plateau, an almost isolated tract of land located north-west of Skardu and the neighbouring Kargil sector of Indian-administered Kashmir (Mock & O'Neil Reference Mock and O'Neil2002). Deosai National Park is the second highest plateau in the world, covering an area of 2240 km2 with an altitudinal range between 3500–5200 m a.s.l. and located between the Himalaya and Karakorum ranges in Pakistan (Usman et al. Reference Usman, Dyer and Khalid2021). Previously, only a small number of lichens have been documented from the Deosai Plains including Acarospora anatolica H. Magn., Psora himalayana (C. Bab.) Timdal, Psora vallesiaca (Schaer.) Timdal and Pyrenodesmia micromontana (Frolov et al.) Hafellner & Türk (Knudsen & Kocourková Reference Knudsen and Kocourková2015; Frolov et al. Reference Frolov, Vondrák, Fernández-Mendoza, Wilk, Khodosovtsev and Halıcı2016; Hafellner & Türk Reference Hafellner and Türk2016; Timdal et al. Reference Timdal, Obermayer and Bendiksby2016). A further species, Placidium deosaiense Usman & Khalid, was also recently described from this locality (Usman et al. Reference Usman, Dyer and Khalid2021). Here we describe another new species from Deosai National Park, based on phylogenetic analyses and the presence of unique morpho-anatomical and chemical characteristics. This study is a continuation of efforts to unveil the lichen flora of high-altitude areas of Pakistan to provide information about biodiversity and support conservation efforts.
Material and Methods
Sampling site
Surveys were conducted in Deosai National Park and its adjacent areas during May and September 2019 as part of the Ph.D. research work of the corresponding author. For a more detailed description of the sampling site, see Usman et al. (Reference Usman, Dyer and Khalid2021). Vouchered specimens are deposited in the Institute of Botany, University of the Punjab, Lahore, Pakistan (LAH).
Morpho-anatomical and chemical studies
Methods for the examination of external morphology, macroscopic and microscopic characters and their measurements, and colour reactions of the thallus using potassium (K), sodium hypochlorite (C), sodium hypochlorite following potassium (KC) and Lugol's solution (I) follow Usman et al. (Reference Usman, Dyer and Khalid2021). For detection of lichen secondary metabolites, thin-layer chromatography (TLC) with solvents A and G were used, as described by Orange et al. (Reference Orange, James and White2010). Measurements are given as (min‒) x̄ ± SD (‒max), where ‘min’ and ‘max’ are the extreme values observed, x̄ the arithmetic mean and SD the standard deviation.
Molecular and phylogenetic studies
Nuclear DNA was extracted using a GF1 Plant DNA extraction kit according to the manufacturer's instructions (Vivantis, Selangor Darul Ehsan, Malaysia). Primers used during amplifications were ITS1F and ITS4 for the ITS region, with LR0R and LR5 for the nuLSU region (White et al. Reference White, Bruns, Lee, Taylor, Innis, Gelfand, Sninsky and White1990; Gardes & Bruns Reference Gardes and Bruns1993). Polymerase chain reaction (PCR) conditions adapted from those of Gardes & Bruns (Reference Gardes and Bruns1993) were followed according to Usman & Khalid (Reference Usman and Khalid2020). The PCR amplicons were purified using a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and then sent for sequencing at TsingKe, China, using the aforementioned primers.
Forward and reverse sequences of ITS and nuLSU regions were assembled using BioEdit v. 7.2.5 (Hall Reference Hall1999) and compared with sequences on GenBank (https://www.ncbi.nlm.nih.gov/). A comprehensive representation of currently available sequences used for the phylogenetic analyses are presented in Table 1, together with GenBank Accession numbers, country distribution and reference. The sequences used in the ITS and LSU dataset were retrieved from GenBank based on inclusion of all published sequences from the genus Anamylopsora (Zuo et al. Reference Zuo, Liu, Li, Chen and Wei2018; Ahat et al. Reference Ahat, Tumur and Guo2019) together with unpublished Anamylopsora sequence data showing 79% or greater nucleotide identity. Sequences of Baeomyces rufus (Huds.) Rebent. (AF448457 from China) were used as outgroup given that this is phylogenetically the closest genus to Anamylopsora and has been used as outgroup previously in Anamylopsora publications (Zuo et al. Reference Zuo, Liu, Li, Chen and Wei2018; Ahat et al. Reference Ahat, Tumur and Guo2019).
Table 1. Sequences of lichen taxa used in the ITS phylogenetic analyses of the genus Anamylopsora, with voucher information, GenBank Accession numbers and associated references. Newly generated sequences are shown in bold. * = outgroup.
The final alignments were carried out in Clustal W implemented in BioEdit (Hall Reference Hall1999). The maximum likelihood phylogram was inferred in RAxML-HPC2 using XSEDE (v. 8.2.10) with 1000 bootstrap replicates. The GTR + GAMMMA nucleotide substitution model was used on the CIPRES Web Portal, following verification using jModelTest v. 2.1.6 and the Akaike information criterion (Akaike Reference Akaike1974; Darriba et al. Reference Darriba, Taboada, Doallo and Posada2012). Phylogenetic trees were visualized using FigTree v. 1.4.2 (Rambaut Reference Rambaut2012). Newly generated sequences were deposited in GenBank and the sequence alignment files for the phylogenetic trees are available in the Supplementary Material (available online).
Results
During field sampling within Deosai National Park, an apparently novel lichen was identified on stones. Three thalli were collected from different locations for morphological and phylogenetic analyses (see below for precise locations). Sectioning revealed further details of the anatomy, as described below.
Phylogenetic analyses
DNA sequences from three different thalli (LAH37090, LAH37091, LAH37092) were successfully obtained after PCR amplification for the ITS (c. 625 bp) and nuLSU (c. 908 bp) regions. Distinct, well-supported clades were recovered for both the ITS (Fig. 1) and nuLSU regions (Supplementary Material Fig. S1, available online). There was no conflict in the unique position of our taxon in both trees, which was distinct from all previously submitted sequences in GenBank. Note that LSU sequences were not available for any of the previously published species of the genus Anamylopsora. Therefore, only unpublished sequences (which may be described in the future) obtained directly from GenBank on the basis of sequence similarity close to our taxon were included in the LSU phylogenetic tree (Supplementary Material Fig. S1). Clade names were provisionally assigned as described below.
Figure 1. Molecular phylogenetic analyses of Anamylopsora species by maximum likelihood (ML) based on ITS sequence data. Bootstrap values >70% are shown at the branches and novel sequences generated during this study are in bold. Clades are indicated by letters.
The final ITS phylogram (Fig. 1) consisted of 25 sequences; 24 of these formed an ingroup clade B distinct from Baeomyces rufus, which formed the outgroup clade A. Within clade B, clade C consisted of seven sequences belonging to A. pruinosa and four sequences (MN545147, MN545148, MN545149 and MN545150) named here as Anamylopsora sp. which are available from GenBank but so far unpublished in a formal publication. Clade E comprised two sequences of A. pulcherrima. Our novel taxon, named A. pakistanica here, formed a separate clade G (containing all three thalli) alongside clade H. Clade H contained three sequences of Anamylopsora altaica and four unpublished sequences of Anamylopsora. It is noted that all previously reported sequences of species described from the genus Anamylopsora are different from our novel taxon, including A. altaica, A. pruinosa and A. pulcherrima, with differences of 44, 31 and 22 base pairs, respectively.
A separate phylogenetic tree was constructed based on LSU sequence data from available sequences in GenBank (Supplementary Material Table S1, available online). The LSU phylogram (Supplementary Material Fig. S1) consisted of nine sequences, eight of which formed an ingroup clade distinct from Baeomyces rufus forming the outgroup clade. The analyses showed the separate position of our taxon in both phylogenetic trees, a position further supported by morpho-anatomical and chemical evidence as described below.
Taxonomy
Anamylopsora pakistanica Usman & Khalid sp. nov.
MycoBank No.: MB 843629
Differing from A. altaica by having larger squamules, up to 3 mm diam. (vs normally ≤ 1 mm diam. for the latter), a light brown to dark brown upper surface (vs white to whitish grey), the presence of an epinecral layer up to 25 μm thick (vs absent), thick-walled ascospores with smooth surfaces (vs thin-walled with warty surfaces) and immersed, non-marginal pycnidia (vs marginal).
Type: Pakistan, Gilgit Baltistan, Deosai National Park, saxicolous, on calciferous rock with other lichen species, c. 4216 m a.s.l., 35°1ʹ1.12ʺN, 75°12ʹ9.09ʺE, 13 May 2019, M. Usman DEO-01 (LAH37090—holotype). GenBank Accession nos.: ON175977 (ITS) and ON175979 (nuLSU).
Figure 2. Anamylopsora pakistanica sp. nov. holotype (LAH37090). A, dry form on rock (arrows). B–D, cross-section of thallus viewed under stereomicroscope. E, thallus with apothecium and pycnidia (arrows). Scales: A = 10 mm; B, D & E = 500 μm; C = 1 mm. In colour online.
Figure 3. Anatomical structures of Anamylopsora pakistanica sp. nov. holotype (LAH37090). A, upper cortex with algal layer. B, asci in Lugol's solution (I). C, algal cells in I. D, apothecia in I (initially hymenium turns green then brown). E, ascus with ascospores and paraphyses in I. F, epihymenium and hymenium. Scales: A = 50 μm; B = 20 μm; C = 5 μm; D = 200 μm; E = 15 μm; F = 10 μm. In colour online.
Thallus with irregular squamules, (290–)347 ± 54(–400) μm thick at margins, appressed on rocks with soil present between thalli; squamules 0.7–3 mm diam., slightly overlapping. Soredia and isidia absent, pruinose upper surface bright brown to dark brown; margin whitish, entire to subentire, usually upturned; lower surface white to dirty white near margins, without rhizines, lacking well-developed lower cortex. The thallus is heteromerous, epinecral layer hyaline, up to 25 μm thick; upper cortex paraplectenchymatous (25–)67 ± 17(–100) μm thick, brown to light brown; algal layer (211–)289 ± 119(–427) μm thick, continuous, unicellular, globose to subglobose (3–)4.3 ± 0.5(–6) μm diam., medulla (246–)346 ± 21(–395) μm thick; lower cortex hyaline, up to 100 μm thick near margins, while 5 mm thick towards the centre near the rock surface.
Apothecia marginal, lecideine, up to 0.5 mm diam. when single, up to 1.7 mm diam. in cluster form, often globose; disc dark brown to black, shiny, epruinose, sometimes cracked; epihymenium brown, (8–)12 ± 3(–15) μm thick; hymenium (102–)121 ± 37(–170) μm high. Subhymenium hyaline, up to 155 μm thick; hypothecium black, up to 458 μm high. Paraphyses (47–)10.1 ± 1.3(–63) × (1.9–)1.6 ± 0.7(–2.9) μm, septate, with pigmented apical cells in a gel-like matrix. Asci narrowly clavate to subcylindrical, tholus amyloid, 8-spored, (67–)75.5 ± 6(–86) × (8.5–)11.8 ± 2.8(–13.5) μm. Ascospores hyaline, simple, ellipsoid, thick-walled, (7.9–)10.1 ± 1.3(–11.9) × (5.3–)6.6 ± 0.7(–7.9) μm, l/w ratio (1.3–)1.53 ± 0.17(–1.8) μm, simple with smooth surface.
Pycnidia immersed in the medulla and in the upper cortex forming light brown outgrowths on the squamules, globose to subglobose, (300–)324 ± 35(–352) μm diam. Conidia hyaline and bacilliform (3.5–)4 ± 0.5(–4.9) × (0.7–)0.97 ± 0.15(–1.16) μm, l/w ratio (3.3–)4.25 ± 0.75(–5.6) μm.
Chemistry
Thallus upper surface K+ red, KC+ black, C−; upper cortex K+ red, KC+ black, C−; medulla K+ yellowish brown, KC+ brown, C−; algal layer K+ black, KC+ dark black, C−; apothecial disc I+ blue, K−, KC−, C−, UV−. Secondary metabolites detected were atranorin, norstictic acid, salazinic acid whilst stictic acid was absent.
Etymology
The specific epithet pakistanica (Latin) refers to Pakistan, the country of the type locality.
Distribution
The species has so far been found only infrequently on stones between 4008–4587 m a.s.l. in well-drained locations in Deosai National Park, Gilgit Baltistan, Pakistan.
Additional specimens examined
Pakistan: Gilgit Baltistan: Deosai National Park, saxicolous, on calciferous rock, c. 4587 m a.s.l., 35°0ʹ47.36ʺN, 75°13ʹ16.31ʺE, 2019, M. Usman DEO-57 (LAH37091—paratype; GenBank Accession nos.: ON1759778 (ITS) and ON175980 (nuLSU)); ibid., saxicolous, on calciferous rock, 4008 m a.s.l., 35°5ʹ49.48ʺN, 75°32ʹ37.03ʺE, 2019, M. Usman & K. Habib GPS-2 (LAH37092—paratype; GenBank Accession no. (ITS): MW418153).
Discussion
High altitudinal regions such as Deosai National Park offer specialized habitats for the evolution and growth of lichen species (Khan & Jan Reference Khan and Jan2018; Usman et al. Reference Usman, Dyer and Khalid2021). The lichen flora has previously been partially investigated using classical morphology for identification, with a variety of lichens described including catapyrenoid genera found commonly as part of biological soil crusts (Aptroot & Iqbal Reference Aptroot and Iqbal2012). By contrast, we now describe a new saxicolous species. Superfically, the new species resembles Anamylopsora altaica, due to the thallus shape and presence of black apothecia (Ahat et al. Reference Ahat, Tumur and Guo2019). It also shares some common characteristics with the two other remaining species of the genus, A. pulcherrima and A. pruinosa, including dark brown to black marginal apothecia, a pruinose thallus, globose to subglobose and unicellular algal cells, clavate to subcylindrical asci and hyaline bacilliform pycnidiospores (Timdal Reference Timdal1991; Zuo et al. Reference Zuo, Liu, Li, Chen and Wei2018).
However, our novel taxon Anamylopsora pakistanica is clearly different from these taxa since it forms a separate clade based on ITS and nuLSU DNA sequence divergence, and phylogenetically is a sister group to A. altaica with strong bootstrap support. This proposal is supported by morpho-anatomical characters which distinguish the species, including coloration of the thalli, the presence of a thick lower cortex up to 100 μm, up to 5 mm thick towards the centre near the rock surface and the presence of simple thick-walled ascospores as discussed below.
Anamylopsora pakistanica has a light to dark brown-coloured thallus upper surface and the apothecia have a thin epihymenium up to 15 μm thick, whereas A. altaica has a white to whitish grey thallus upper surface and an epihymenium up to 30 μm thick. Further morpho-anatomical details of A. pakistanica include a continuous thick medulla, 325–367 μm in depth, which contrasts that in A. altaica (only 190–280 μm deep), A. pruniosa (112–250 μm deep), and A. pulcherrima which has a discontinuous medulla. Anamylopsora pakistanica also has a thicker algal layer, 229–360 μm in depth, compared to that in A. altaica (135–195 μm), A. pruniosa (50–150 μm) and A. pulcherrima (120–220 μm). In addition, A. pakistanica has a thick hymenium, 90–151 μm, in contrast to that present in A. altaica (95–115 μm), A. pruniosa (75–100 μm) and A. pulcherrima (60–100 μm) (Timdal Reference Timdal1991; Zuo et al. Reference Zuo, Liu, Li, Chen and Wei2018; Ahat et al. Reference Ahat, Tumur and Guo2019).
Furthermore, A. pakistanica has squamules between 0.7–3 mm diam., compared to A. altaica which forms squamules ≤ 1(–2.5) mm diam. and A. pruniosa which has squamules of 2–3 mm diam. Anamylopsora pakistanica forms asci up to 82 μm in length while A. pulcherrima has larger asci up to 125 μm in length. Anamylopsora pakistanica also has ellipsoid ascospores with a thick-walled and smooth surface whereas A. altaica and A. pruinosa have thin-walled ascospores with a warty surface (Timdal Reference Timdal1991; Zuo et al. Reference Zuo, Liu, Li, Chen and Wei2018; Ahat et al. Reference Ahat, Tumur and Guo2019). Other differences of A. pakistanica are the thick epinecral layer up to 25 μm, whereas in A. pulcherrima the layer is only 5–10 μm thick and is absent in A. altaica. Anamylopsora pakistanica has a thinner upper cortex, 50–84 μm thick, in contrast to A. pruniosa and A pulcherrima where the upper cortex is 125–150 μm and 35–180 μm deep, respectively (Timdal Reference Timdal1991; Zuo et al. Reference Zuo, Liu, Li, Chen and Wei2018; Ahat et al. Reference Ahat, Tumur and Guo2019).
It is also noted that A. pakistanica is saxicolous in nature and rhizines are absent, whereas A. pruinosa is terricolous (Zuo et al. Reference Zuo, Liu, Li, Chen and Wei2018), providing a key differentiating character separating these species. A further difference is that A. pulcherrima produces alectorialic acid, A. pruinosa produces alectorialic and barbatolic acids and A. altaica produces psoromic acid, whereas A. pakistanica produces atranorin, norstictic acid and salazinic acid (Timdal Reference Timdal1991; Zuo et al. Reference Zuo, Liu, Li, Chen and Wei2018; Ahat et al. Reference Ahat, Tumur and Guo2019). A final significant difference among Anamylopsora species lies in their altitudinal locations. The new species A. pakistanica was found on rocks at a high altitude between 4008 and 4587 m a.s.l., compared to A. altaica found at 960–1087 m, A. pruinosa at 1577 m and A. pulcherrima from 550 to 3900 m (Timdal Reference Timdal1991; Zuo et al. Reference Zuo, Liu, Li, Chen and Wei2018; Ahat et al. Reference Ahat, Tumur and Guo2019). Based upon this combination of characters, the new species A. pakistanica is clearly distinct.
A key to species of Anamylopsora
1 On soil, ascospores subglobose, rhizines abundant ……… A. pruinosa
On rock, ascospores ellipsoid, rhizines absent ……… 2
2(1) Epinecral layer present, ascospore surface smooth ……… 3
Epinecral layer absent, ascospore surface warty ……… A. altaica
3(2) Upper surface ochraceous brown, algal layer discontinuous, epinecral layer up to 15 μm thick, upper cortex up to 180 μm thick ……… A. pulcherrima
Upper surface light brown to dark brown, algal layer continuous, epinecral layer up to 25 μm thick, upper cortex up to 84 μm thick ……… A. pakistanica
Acknowledgements
The authors are very grateful to Prof. Dr Pradeep Kumar Divakar (Departamento de Farmacología, Universidad Complutense de Madrid, Spain), Dr José Pizarro (Curator, Herbario MAF, Universidad Complutense de Madrid, Spain), and Prof. Peter Crittenden and Dr Chris Wade (School of Life Sciences, University of Nottingham, UK) for sending herbarium specimens of lichens for thin-layer chromatography and phylogenetic suggestions, respectively.
Author ORCIDs
Muhammad Usman, 0000-0002-3490-058X; Qudsia Firdous, 0000-0002-3096-0198; Paul S. Dyer, 0000-0003-0237-026X; Abdul Nasir Khalid, 0000-0002-5635-8031.
Supplementary Material
The Supplementary Material for this article can be found at https://doi.org/10.1017/S002428292300018X.
Open access
