Taxonomic and phylogenetic study of the genus Diploschistes (Ostropales, Thelotremataceae) reveals one new species from PakistanABSTRACT
Diploschistes pakistanicus sp. nov. is described from the Himalayan moist temperate forest, Pakistan. ITS sequences confirm its position within the genus Diploschistes and, together with its morphology and chemistry, suggest that it is separate from other species of this genus. The taxon is characterized by grey to greyish white pruinose thallus, perithecioid-type ascomata, small apothecia 0.1‒0.4 mm wide, hypothecium 20‒25µm thick, ascus of 85‒110 × 9‒17 µm in size, 3‒5 transverse and 2‒4 longitudinal septa in large ascospores 42‒55 × 18‒30; also differing from related species in ITS region.
Keywords: Darel; Garhi Dupatta; lichenized fungi; western South Asia
Introduction
The lichen-forming genus Diploschistes Norman includes crustose species with a remarkable range of variation in morphology of the ascomata, varying from perithecioid to urceolate with a blackish pseudoparenchymatous proper exciple, lateral paraphyses and a trebouxioid photobiont (Lumbsch & Mangold 2007; Lumbsch & Huhndorf 2010). The genus is widely distributed in arid and semiarid regions worldwide, with c. 43-45 species (Kirk et al. 2008; Abbas et al. 2014).
Pakistan is located in western South Asia between 24-37 °N latitude and 62-75 °E longitudes. The country is well known for its geographical and climatic variations which is linked with rich biodiversity (IUCN 2006). Previously 5 species reported from Pakistan viz. D. candidissimus (Kremp.) Zahlbr. (Razzaq et al. 2022), D. diacapsis (Ach.) Lumbsch, D. euganeus (A. Massal.) Steiner, D. muscorum (Scop.) R.Sant. Lumbsch, and D. scruposus (Schreb.) (Aptroot & Iqbal 2012).
During our exploration of the lichen diversity of Pakistan, collections were made from Gilgit-Baltistan and various sites of Azad Jammu and Kashmir, Pakistan. Using molecular analyses as well as morphological and chemical characters, we were able to confirm the presence of one new species of the genus Diploschistes from Pakistan which are presented here.
Materials and methods
Morphological and chemical studies
Specimens were collected from the various sites of Azad Jammu and Kashmir, and Gilgit-Baltistan, Pakistan in 2020 respectively. The specimens are deposited in the herbarium of Institute of Botany, University of the Punjab, Lahore (LAH). Hand sections were prepared manually with a razor blade under a stereomicroscope, scrutinized under a compound microscope (MX4300H, Meiji Techno, Japan) at different magnifications for anatomical characterization and measurements.
Chemical characterization
The secondary chemistry was analyzed using spot tests with KOH (10%; K), and potassium hypochlorite solution (C). Thin Layer Chromatography was carried out using Solvent System G, following standard methods (Orange et al. 2001).
DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted directly from a portion of thallus with apothecia from each specimen using a modified 2% CTAB method (Gardes & Bruns 1993). The ITS-nrDNA region (Internal Transcribed Spacer of the nrDNA) was amplified using the primer pair ITS1F (forward primer) (Gardes & Bruns 1993) and ITS4 (reverse primer) (White et al. 1990) following the amplification protocol of Khan et al. (2018). PCR products were visualized on a 1% agarose gel with ethidium bromide (Sambrook & Russel 2001). PCR products were sent to Tsingke, China for sequencing.
BLAST analysis was used to retrieve highly similar sequences of ITS region. Sequence maximum query coverage and percent identity along with related taxa were noted. Sequences retrieved from GenBank and obtained from published literature (Zhao et al. 2017) were used in an initial alignment then realigned using web-PRANK with default settings (Löytynoja & Goldman 2010). Phylogenetic relationships were investigated using Maximum Likelihood bootstrapping, as implemented in RAxML-HPC2 v. 8.1.11 (Stamatakis 2014), hosted on the CIPRES Science Gateway (Miller et al. 2010). Analyses used rapid bootstrapping with 1000 iterations, and the HYK+G+I substitution model. FigTree v 1.4.3 (Rambaut et al. 2014) was used for displaying trees from the ML analysis.
Results
Phylogenetic analysis
ITS sequences of the holotype of the new species and of the collections CKT-04, DR-40 and BLP-15 were identical. The final dataset of ITS consisted of 45 sequences (See Table 1 for voucher details). The aligned ITS1-5.8S-ITS2 region comprised 657 sites, of which 358 were conserved and 278 variable; 162 sites were parsimony-informative. In our phylogram (Fig. 1) the sequences of Diploschistes pakistanicus is sister to a clade of two sequences of D. diploschistoides (Vain.) G. Salisb., and together these are sister to a clade comprising D. euganeus (A. Massal.) Steiner, (KF688485, KC166986), D. candidissimus (Kremp.) Zahlbr. (KC166977, KC166976, MN103134), D. caesioplumbeus (Nyl.) Vain. (KC166974, KC166973, KC166975) and D. actinostoma (Pers. Ex Ach.) Zahlbr. (MN586952, MN586953, AF229194, MN586951) demonstrating their status as independent species.
Specimens used in the phylogenetic analyses of Diploschistes species. New sequences are in bold.
Taxonomic treatment
Diploschistes pakistanicus Fayyaz, M. S. Iqbal & Afshan sp. nov. (Figure 1 & 2).
Phylogenetic relationships of Diploschistes pakistanicus based on a Maximum Likelihood analysis of the ITS region. Sequences from Pakistan are in bold.
Diploschistes pakistanicus (LAH37419-holotype) A-C: A & B: Showing crustose thallus B: Cross section of apothecium C: Ascospore. Scale bar = A: 3cm, B: 125 µm, C: 20 µm.
MycoBank No: MB844722
Etymology: The specific epithet “pakistanicus” (Latin) refers to the type locality Pakistan.
Diagnosis: The taxon is characterized by grey to greyish white pruinose thallus, perithecioid-type ascomata, small apothecia 0.1‒0.4 mm wide, hypothecium 20‒25µm thick, ascus of 85‒110 × 9‒17 µm in size, 3‒5 transverse 2‒4 longitudinal septa in large ascospores 42‒55 × 18‒30 µm; also differing from related species in ITS region.
Holotype: Pakistan: Azad Jammu and Kashmir, Garhi Dupatta (34°36′ N, 73°35′ E) 817 m alt., on rock, September 23 2020, I. Fayyaz, N. S. Afshan & A. R. Niazi (CKT-03) (LAH37419-holotype), ITS GenBank accession number ON891114
Thallus: crustose, epilithic, rimose-areolate, regular, up to 3 cm across, pruinose. Color: grey to greyish white, unchanged when wet. Areoles: plane to strongly convex, large and strongly convex at margin, 0.2‒0.5 mm in diam., regular, contiguous, weakly and thinly wrinkled. Upper cortex: greyish black, 20‒25 µm thick, composed of dead cells. Algal layer: continuous, even, 70‒115 µm thick. Photobiont: trebouxioid with cells globose, 15‒20 µm in diam. Medulla: hyphae hyaline to light brown, 3‒4.5 µm wide. Apothecia: common, immersed, perithecioid, 4‒6 per areole, 0.1‒0.4 mm in diam. Disk: grayish black, concealed by margin. Proper exciple: well developed, pseudoparenchymatous, black, carbonized, 75‒90 µm thick. Epihymenium: weakly developed, 20‒30 µm thick. Hymenium: hyaline, 110‒150 µm thick. Hypothecium: hyaline, 20‒25 µm thick. Paraphyses: hyaline, anastomosing, simple, flexuose, not swollen at apex 1.5‒2 µm thick. Asci: clavate, 6‒8 spored, 85‒110 × 9‒17 µm. Ascospores: hyaline when young, becoming dark brown when mature, muriform, 3‒5 transverse septa and 2‒4 longitudinal septa, 42‒55 × 18‒30 µm.
Chemistry: K‒, C+ red, KC+ red. Major substance: Lecanoric acid and minor substance: Gyrophoric acid.
Additional specimen examined Azad Jammu and Kashmir, Ganga Choti (34°15′ N, 73°25′ E), 2960 m alt., on rock, September 25 2020, I. Fayyaz, N. S. Afshan & A. R. Niazi (CKT-04, BLP-15) Gilgit Baltistan, Darel Valley (35°37' N, 73°27'E), 3843 m alt., on rock, October 21, 2020, A. N. Khalid, K. Habib & M. S. Iqbal (DR-40).
Habitat and distribution
The known collections of the new species are from moist temperate forest of the Himalaya in Pakistan. The specimens were found on siliceous rocks. The coniferous forest is dominated by species of Pinus roxburghii Sarg., Quercus oblongata D. Don, Q. glauca Thumb and Pyrus pashia L. etc. The maximum daily temperature of the region varies from 20 to 30 ºC during the summer and averages 4 °C during the winter, and there is moderate rainfall.
Discussion
Diploschistes pakistanicus morphologically and phylogenetically closely resembles to D. euganeus (A. Massal.) J. Steiner. Our species can be distinguished from the D. euganeus in having larger ascospores (42‒55 × 18‒30 µm vs. 24‒36 × 15‒18 µm and presence of Lecanoric acid vs. absence of secondary metabolites (Pant & Upreti 1993). Our species can be distinguished from the D. candidissimus in having smaller apothecia (0.1‒0.4 mm vs. 1.5 mm), thicker excipulum (75‒90 µm vs. 70 µm), smaller ascus (85‒110 × 9‒17 µm vs. 90‒125 × 12‒22 µm) and larger ascospores (42‒55 × 18‒30 µm vs. 24‒34 × 14‒20 µm (Lumbsch 1989; Razzaq et al. 2022). Similarly our new taxon is clearly differentiated from the D. actinostomaitalic> in having a pruinose thallus, smaller apothecia (0.1‒0.4 mm vs. 3 mm), thicker excipulum (75‒90 µm vs. 70 µm), smaller ascus (85‒110 × 9‒17 µm vs. 110‒150 × 15‒40 µm) and larger ascospores (42‒55 × 18‒30 µm vs. 16‒32 × 10‒20 µm) (Lumbsch 1989).
The new species also differs from D. caesioplumbeus in having grey to greyish white pruinose thallus, smaller apothecia (0.1‒0.4 mm vs. 1.6 mm), thicker excipulum (75‒90 µm vs. 70 µm), smaller ascus (85‒110 × 9‒17 µm vs. 120‒160 × 15‒35 µm) and larger ascospores (42‒55 × 18‒30 µm vs. 28‒45 × 12‒25 µm) (Lumbsch 1989).
The new taxon differs from D. diploschistoides in having grey to greyish white pruinose thallus, smaller apothecia (0.1‒0.4 mm vs. 0.5 mm), thicker hypothecium (20‒25 µm vs. 15‒20 µm), smaller ascus (85‒110 × 9‒17 µm vs. 100‒160× 15‒45 µm) and larger ascospores (42‒55 × 18‒30 µm vs. 30‒52 × 15‒26 µm) (Guderley & Lumbsch 1996).
The new taxon differs from the non-sequenced species- D. austroafricanus in having grey to greyish white pruinose thallus, smaller apothecia (0.1‒0.4 mm vs. 2.5 mm), thicker hypothecium (20‒25 µm vs. 10‒15 µm), smaller ascus (85‒110 × 9‒17 µm vs. 90‒140× 20‒40 µm) and larger ascospores (42‒55 × 18‒30 µm vs. 30‒40 × 19‒22 µm) (Guderley & Lumbsch 1996) (Table 2).
Acknowledgements
We are highly thankful to the pre-submission reviewer Dr. Thorsten Lumbsch (The Field Museum) for his valuable comments and reviewing article that greatly improved the manuscript. We are also highly grateful to University of the Punjab, Lahore, Pakistan for providing project No.D/72/EST-I Dated 14/01/2022 to conduct this research work.
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Publication Dates
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Publication in this collection
31 Mar 2023 -
Date of issue
2023
History
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Received
13 May 2022 -
Accepted
03 Mar 2023
