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Taxonomic and phylogenetic study of the genus Diploschistes (Ostropales, Thelotremataceae) reveals one new species from Pakistan

ABSTRACT

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 2007Lumbsch HT, Mangold A. 2007. Diploschistes elixii (Ostropales: Thelotremataceae), an overlooked terricolous species from Western Australia. The Lichenologist 39: 459-462. doi: 10.1017/s0024282907007049
https://doi.org/10.1017/s002428290700704...
; Lumbsch & Huhndorf 2010Lumbsch HT, Huhndorf SM. 2010. Myconet volume 14. Part one. Outline of Ascomycota - 2009; Part Two. Notes on Ascomycete Systematics, Nos 4751-5113. Fieldiana (Life and Earth Sciences) 1: 1-42.). The genus is widely distributed in arid and semiarid regions worldwide, with c. 43-45 species (Kirk et al. 2008Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi. 10th. edn. Wallingford, CAB International. ; Abbas et al. 2014Abbas A, Guo SY, Ababaikeli G, Abdulla A, Xahidin H. 2014. Diploschistes xinjiangensis, a new saxicolous lichen from northwest China. Mycotaxon 129: 465-471. doi: 10.5248/129.465.
https://doi.org/10.5248/129.465...
).

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 2006IUCN - International Union of Conservation for Nature. 2006. News release: Pakistan’s rich biodiversity faces serious threats. https://www.iucn.org/
https://www.iucn.org/...
). Previously 5 species reported from Pakistan viz. D. candidissimus (Kremp.) Zahlbr. (Razzaq et al. 2022Razzaq F, Habib K, Aslam S, Khalid AN. 2022. Additions to the List of Graphidaceae (Lichenized Ascomycetes) in Pakistan. Biology Bulletin 49: 14-20.), D. diacapsis (Ach.) Lumbsch, D. euganeus (A. Massal.) Steiner, D. muscorum (Scop.) R.Sant. Lumbsch, and D. scruposus (Schreb.) (Aptroot & Iqbal 2012Aptroot A, Iqbal S. H. 2012. Annotated checklist of the Lichens of Pakistan, with reports of new records. Herzogia 25: 211-229. doi: 10.13158/heia.25.2.2010.211
https://doi.org/10.13158/heia.25.2.2010....
).

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. 2001Orange A, James PW, White FJ. 2001. Microchemical Methods for the Identification of Lichens. London, British Lichen Society.).

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 1993Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes‐application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118. doi: 10.1111/j.1365-294X.1993.tb00005.x
https://doi.org/10.1111/j.1365-294X.1993...
). The ITS-nrDNA region (Internal Transcribed Spacer of the nrDNA) was amplified using the primer pair ITS1F (forward primer) (Gardes & Bruns 1993Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes‐application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118. doi: 10.1111/j.1365-294X.1993.tb00005.x
https://doi.org/10.1111/j.1365-294X.1993...
) and ITS4 (reverse primer) (White et al. 1990White TJ, Bruns T, Lee SJWT, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds.). PCR protocols: a guide to methods and applications. Cambridge, Academic Press. p. 315-322.) following the amplification protocol of Khan et al. (2018Khan M, Khalid AN, Lumbsch HT. 2018. A new species of Lecidea (Lecanorales, Ascomycota) from Pakistan. MycoKeys 38: 25-34. doi: 10.3897/mycokeys.38.26960
https://doi.org/10.3897/mycokeys.38.2696...
). PCR products were visualized on a 1% agarose gel with ethidium bromide (Sambrook & Russel 2001Sambrook J, Russel DW. 2001. Rapid isolation of yeast DNA. In: Sambrook J, Russel DW (eds). Molecular cloning: A laboratory manual (S). New York, Cold Spring Harbor Laboratory Press. p. 631-632.). 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. 2017Zhao X, Fernández-Brime S, Wedin M, Locke M, Leavitt SD, Lumbsch HT. 2017. Using multi-locus sequence data for addressing species boundaries in commonly accepted lichen-forming fungal species. Organisms Diversity & Evolution 17: 351-363.) were used in an initial alignment then realigned using web-PRANK with default settings (Löytynoja & Goldman 2010Löytynoja A, Goldman N. 2010. webPRANK: a phylogeny-aware multiple sequence aligner with interactive alignment browser. BMC Bioinformatics 11: 579. doi: 10.1186/1471-2105-11-579
https://doi.org/10.1186/1471-2105-11-579...
). Phylogenetic relationships were investigated using Maximum Likelihood bootstrapping, as implemented in RAxML-HPC2 v. 8.1.11 (Stamatakis 2014Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313. doi: 10.1093/bioinformatics/btu033
https://doi.org/10.1093/bioinformatics/b...
), hosted on the CIPRES Science Gateway (Miller et al. 2010Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), 14 November 2010, New Orleans. p. 1-8.). Analyses used rapid bootstrapping with 1000 iterations, and the HYK+G+I substitution model. FigTree v 1.4.3 (Rambaut et al. 2014Rambaut A, Suchard MA, Xie D, Drummond AJ. 2014. FigTree 1.4.2 Software. Institute of Evolutionary Biology, University of Edinburgh.) 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.

Table 1
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).

Figure 1
Phylogenetic relationships of Diploschistes pakistanicus based on a Maximum Likelihood analysis of the ITS region. Sequences from Pakistan are in bold.

Figure 2
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 1993Pant G, Upreti DK. 1993. The lichen genus Diploschistes in India and Nepal. The Lichenologist 25: 33-50.). 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 1989Lumbsch HT. 1989. Die holarktischen Vertreter der Flechtengattung Diploschistes (Thelotremataceae). Journal of the Hattori Botanical Laboratory 66: 133-196.; Razzaq et al. 2022Razzaq F, Habib K, Aslam S, Khalid AN. 2022. Additions to the List of Graphidaceae (Lichenized Ascomycetes) in Pakistan. Biology Bulletin 49: 14-20.). 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 1989Lumbsch HT. 1989. Die holarktischen Vertreter der Flechtengattung Diploschistes (Thelotremataceae). Journal of the Hattori Botanical Laboratory 66: 133-196.).

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 1989Lumbsch HT. 1989. Die holarktischen Vertreter der Flechtengattung Diploschistes (Thelotremataceae). Journal of the Hattori Botanical Laboratory 66: 133-196.).

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 1996Guderley R, Lumbsch HT. 1996. The lichen genus Diploschistes in South Africa (Thelotremataceae). Mycotaxon (USA) 58: 268-292.).

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 1996Guderley R, Lumbsch HT. 1996. The lichen genus Diploschistes in South Africa (Thelotremataceae). Mycotaxon (USA) 58: 268-292.) (Table 2).

Table 2
Comparison of some non-uniform characters of Diploschistes species.

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.

References

  • Abbas A, Guo SY, Ababaikeli G, Abdulla A, Xahidin H. 2014. Diploschistes xinjiangensis, a new saxicolous lichen from northwest China. Mycotaxon 129: 465-471. doi: 10.5248/129.465.
    » https://doi.org/10.5248/129.465
  • Aptroot A, Iqbal S. H. 2012. Annotated checklist of the Lichens of Pakistan, with reports of new records. Herzogia 25: 211-229. doi: 10.13158/heia.25.2.2010.211
    » https://doi.org/10.13158/heia.25.2.2010.211
  • Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes‐application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118. doi: 10.1111/j.1365-294X.1993.tb00005.x
    » https://doi.org/10.1111/j.1365-294X.1993.tb00005.x
  • Guderley R, Lumbsch HT. 1996. The lichen genus Diploschistes in South Africa (Thelotremataceae). Mycotaxon (USA) 58: 268-292.
  • IUCN - International Union of Conservation for Nature. 2006. News release: Pakistan’s rich biodiversity faces serious threats. https://www.iucn.org/
    » https://www.iucn.org/
  • Khan M, Khalid AN, Lumbsch HT. 2018. A new species of Lecidea (Lecanorales, Ascomycota) from Pakistan. MycoKeys 38: 25-34. doi: 10.3897/mycokeys.38.26960
    » https://doi.org/10.3897/mycokeys.38.26960
  • Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi. 10th. edn. Wallingford, CAB International.
  • Löytynoja A, Goldman N. 2010. webPRANK: a phylogeny-aware multiple sequence aligner with interactive alignment browser. BMC Bioinformatics 11: 579. doi: 10.1186/1471-2105-11-579
    » https://doi.org/10.1186/1471-2105-11-579
  • Lumbsch HT, Mangold A. 2007. Diploschistes elixii (Ostropales: Thelotremataceae), an overlooked terricolous species from Western Australia. The Lichenologist 39: 459-462. doi: 10.1017/s0024282907007049
    » https://doi.org/10.1017/s0024282907007049
  • Lumbsch HT, Huhndorf SM. 2010. Myconet volume 14. Part one. Outline of Ascomycota - 2009; Part Two. Notes on Ascomycete Systematics, Nos 4751-5113. Fieldiana (Life and Earth Sciences) 1: 1-42.
  • Lumbsch HT. 1989. Die holarktischen Vertreter der Flechtengattung Diploschistes (Thelotremataceae). Journal of the Hattori Botanical Laboratory 66: 133-196.
  • Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), 14 November 2010, New Orleans. p. 1-8.
  • Orange A, James PW, White FJ. 2001. Microchemical Methods for the Identification of Lichens. London, British Lichen Society.
  • Pant G, Upreti DK. 1993. The lichen genus Diploschistes in India and Nepal. The Lichenologist 25: 33-50.
  • Rambaut A, Suchard MA, Xie D, Drummond AJ. 2014. FigTree 1.4.2 Software. Institute of Evolutionary Biology, University of Edinburgh.
  • Razzaq F, Habib K, Aslam S, Khalid AN. 2022. Additions to the List of Graphidaceae (Lichenized Ascomycetes) in Pakistan. Biology Bulletin 49: 14-20.
  • Sambrook J, Russel DW. 2001. Rapid isolation of yeast DNA. In: Sambrook J, Russel DW (eds). Molecular cloning: A laboratory manual (S). New York, Cold Spring Harbor Laboratory Press. p. 631-632.
  • Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313. doi: 10.1093/bioinformatics/btu033
    » https://doi.org/10.1093/bioinformatics/btu033
  • White TJ, Bruns T, Lee SJWT, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds.). PCR protocols: a guide to methods and applications. Cambridge, Academic Press. p. 315-322.
  • Zhao X, Fernández-Brime S, Wedin M, Locke M, Leavitt SD, Lumbsch HT. 2017. Using multi-locus sequence data for addressing species boundaries in commonly accepted lichen-forming fungal species. Organisms Diversity & Evolution 17: 351-363.

Publication Dates

  • Publication in this collection
    31 Mar 2023
  • Date of issue
    2023

History

  • Received
    13 May 2022
  • Accepted
    03 Mar 2023
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