A new species of the genus Anamylopsora (Baeomycetaceae; Ascomycota) from Deosai National Park, Gilgit-Baltistan, Pakistan

Abstract A novel lichen species occurring on rocks was collected from three different localities within Deosai National Park, Gilgit-Baltistan, Pakistan. Phylogenetic analyses of the nrDNA ITS and nuLSU regions revealed that it clustered within the genus Anamylopsora. Further chemical and morpho-anatomical analyses confirmed its uniqueness, and it is described here as a new species under the name A. pakistanica. The distinguishing characters are: an irregularly squamulose appressed thallus on rocks without rhizines; an epinecral layer up to 25 μm thick; ascospores that are hyaline, simple, thick-walled with a smooth surface; septate paraphyses with a pigmented apical cell in a gel-like matrix; globose to subglobose pycnidia with hyaline and bacilliform pycnidiospores. In particular, the species is distinguished from other members of the genus by morpho-anatomical features including the coloration of the thalli, the presence of a thick lower cortex (up to 100 μm), and the presence of simple, thick-walled ascospores. Specimens were found at altitudes up to 4587 m, the highest elevation yet reported for Anamylopsora. A key and comparison to all existing species of the genus Anamylopsora is also given.


Introduction
The genus Anamylopsora Timdal was established in 1991 based on the type specimen of A. pulcherrima (Vain.) Timdal (Timdal 1991). Initially, it was described as Lecidea pulcherrima Vain. in 1888 (Vainio 1888) 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 1904). 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 1991). Timdal (1984) 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 1991). 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. 1995). 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. 2015).
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 1991;Zuo et al. 2018;Ahat et al. 2019;Esslinger 2021).
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 2002). Deosai National Park is the second highest plateau in the world, covering an area of 2240 km 2 with an altitudinal range between 3500-5200 m a.s.l. and located between the Himalaya and Karakorum ranges in Pakistan (Usman et al. 2021). 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 2015;Frolov et al. 2016;Hafellner & Türk 2016;Timdal et al. 2016). A further species, Placidium deosaiense Usman & Khalid, was also recently described from this locality (Usman et al. 2021). 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.

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. (2021). 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. (2021). For detection of lichen secondary metabolites, thin-layer chromatography (TLC) with solvents A and G were used, as described by Orange et al. (2010). Measurements are given as (min-) x ± SD (-max), where 'min' and 'max' are the extreme values observed, x 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. 1990;Gardes & Bruns 1993). Polymerase chain reaction (PCR) conditions adapted from those of Gardes & Bruns (1993) were followed according to Usman & Khalid (2020). 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 1999) 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 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.  (Hall 1999). 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 1974;Darriba et al. 2012). Phylogenetic trees were visualized using FigTree v. 1.4.2 (Rambaut 2012). 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.
The final ITS phylogram ( Fig. 1) consisted of 25 sequences; 24 of these formed an ingroup clade B distinct from Baeomyces rufus, 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.
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.

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).
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.

Discussion
High altitudinal regions such as Deosai National Park offer specialized habitats for the evolution and growth of lichen species (Khan & Jan 2018;Usman et al. 2021). 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 2012). 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. 2019). 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 1991;Zuo et al. 2018).
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 1991;Zuo et al. 2018;Ahat et al. 2019).
It is also noted that A. pakistanica is saxicolous in nature and rhizines are absent, whereas A. pruinosa is terricolous (Zuo et al. 2018), 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 1991;Zuo et al. 2018;Ahat et al. 2019). 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 1991;Zuo et al. 2018;Ahat et al. 2019). Based upon this combination of characters, the new species A. pakistanica is clearly distinct.
A key to species of Anamylopsora