Research Article |
Muhammad Usman‡§, Paul S. Dyer§, Matthias Brock§, Christopher M. Wade§, Abdul Nasir Khalid‡
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Corresponding author: Muhammad Usman ( musmanmughal52@yahoo.com ) Academic editor: Gerhard Rambold
© 2024 Muhammad Usman, Paul S. Dyer, Matthias Brock, Christopher M. Wade, Abdul Nasir Khalid.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Usman M, Dyer PS, Brock M, Wade CM, Khalid AN (2024) Two novel species of arctic-alpine lichen-forming fungi (Ascomycota, Megasporaceae) from the Deosai Plains, Pakistan. MycoKeys 102: 285-299. https://doi.org/10.3897/mycokeys.102.113310
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Abstract
Members of the lichen-forming fungal genus Oxneriaria are known to occur in cold polar and high altitudinal environments. Two new species, Oxneriaria crittendenii and O. deosaiensis, are now described from the high altitude Deosai Plains, Pakistan, based on phenotypic, multigene phylogenetic and chemical evidence. Phenotypically, O. crittendenii is characterised by orbicular light-brown thalli 1.5–5 cm across, spot tests (K, C, KC) negative, apothecia pruinose, hymenium initially blue then dark orange in response to Lugol’s solution. Oxneriaria deosaiensis is characterised by irregular areolate grey thalli 1.5–2 cm across, K test (light brown), KC test (dark brown), apothecia epruinose, hymenium initially blue then dark blue in response to Lugol’s solution. Both species share the same characters of thalli with black margins and polarilocular ascospores. The closest previously reported species, O. pruinosa, differs from O. crittendenii and O. deosaiensis in having non-lobate margins, thin thalline exciple (45–80 μm thick), short asci (55–80 × 25–42 μm) and K positive (yellow) and KC negative tests and divergent DNA sequence in the ITS, LSU and mt SSU regions. The newly-described Oxneriaria species add to growing evidence of the Deosai Plains as a region of important arctic-alpine biodiversity.
Key words
Aspicilia, Gilgit-Baltistan, Himalaya, Karakorum, Maximum Likelihood, Pertusariales, Skardu
Introduction
The Deosai Plains are located between the Himalaya and Karakorum, two of the world’s most famous mountain ranges, with an average elevation of over 4,000 m (
Members of the lichen-forming genus Oxneriaria S.Y. Kondr. & Lőkös are distributed in cold polar and high-altitude localities of Eurasia and the Northern Hemisphere (
Four species of the genus Oxneriaria have, so far, been described from Pakistan with a distance of 300 to 650 km from Deosai Plains, namely O. iqbalii R. Zulfiqar, H. S. Asghar, K. Habib & Khalid from Kohistan (350 km) and Swat (500 km), O. kohistaniensis R. Zulfiqar, K. Habib & Khalid from Kohistan (350 km), O. pakistanica M. S. Iqbal, Usman, K. Habib & Khalid from Darel (300 km) and O. pruinosa H. S. Asghar., Usman, K. Habib & Khalid from Chitral (650 km). These were all found at relatively high altitudes up to ca. 2,500 m (
Materials and methods
Sample collection
More than half of the Deosai Plains are situated between an elevation (elev.) of 4,000 and 4,500 m with an average daily temperature ranging from -20 °C (January-February) to 12 °C (July-August). Annual precipitation varies from 350 to 550 mm, mostly received during winter as snow (
Morpho-anatomical and chemical studies
Methods for the examination of external morphology, macroscopic and microscopic characters and their measurements were followed and recorded according to the terminology of
Molecular and phylogenetic analyses
Nuclear DNA was extracted from apothecia present on thalli using a GF1 Plant DNA extraction kit according to the manufacturer’s instruction (Vivantis, Selangor Darul Ehsan, Malaysia). Primers used for amplifications were ITS1F 5'-CCT GGT CAT TTA GAG GAA GT A A-3 ' and ITS4 5'-TCC TCC GCT CTA TTG ATA TGC-3' for the internal transcribed spacer (ITS1-5.8S-ITS2) region, while LROR 5'-ACC CGC TGA ACT TAA GC-3' and LR5 5'-TCC TGA GGG AAA CTT CG-3' were used for the nuclear large subunit (LSU) ribosomal RNA region (
Forward and reverse sequences of the ITS, LSU and mt SSU regions were obtained in FASTA format and sequences were assembled using BIOEDIT v. 7.2.5 (
Sequences used in the phylogenetic analyses. Novel sequences generated during this study are shown in bold. Note that sequences were not available for all regions for certain taxa.
| Taxon name | Voucher number | GenBank accession | Country | ||
|---|---|---|---|---|---|
| ITS | LSU | mt SSU | |||
| Oxneriaria crittendenii | LAH37193 | OR037223 | OR037219 | OR037259 | Pakistan |
| Oxneriaria crittendenii | LAH37194 | OR037224 | OR037220 | OR037260 | Pakistan |
| Oxneriaria dendroplaca | UPS:Nordin 5952 | HQ259259 | HM060744 | HM060706 | Sweden |
| Oxneriaria dendroplaca | UPS:Nordin 6366 | HQ259260 | HM060758 | – | Finland |
| Oxneriaria deosaiensis | LAH37200 | OR037225 | OR037221 | OR037261 | Pakistan |
| Oxneriaria deosaiensis | LAH37416 | OR037226 | OR037222 | OR037262 | Pakistan |
| Oxneriaria iqbalii | LAH37155 | ON392710 | – | Pakistan | |
| Oxneriaria iqbalii | LAH37156 | ON392709 | ON392708 | Pakistan | |
| Oxneriaria kohistaniensis | LAH37152 | ON392707 | ON392711 | – | Pakistan |
| Oxneriaria kohistaniensis | LAH37151 | ON454505 | – | – | Pakistan |
| Oxneriaria mashiginensis | Nordin 5790 (UPS) | EU057912 | HM060732 | HM060694 | Sweden |
| Oxneriaria mashiginensis | UPS:Tibell 23557 | HQ259266 | – | – | Sweden |
| Oxneriaria pakistanica | LAH37495 | OP114649 | – | – | Pakistan |
| Oxneriaria pakistanica | LAH37501 | OP627196 | – | – | Pakistan |
| Oxneriaria permutata | Nordin 6027 (UPS) | EU057918 | HM060747 | HM060709 | Sweden |
| Oxneriaria permutata | Nordin 6029 (UPS) | EU057919 | – | – | Sweden |
| Oxneriaria permutata | Nordin 6039 (UPS) | EU057921 | – | – | Sweden |
| Oxneriaria permutata | Nordin 5980 (UPS) | EU057930 | – | – | Sweden |
| Oxneriaria permutata | Wheeler 4463 | – | – | MW424810 | Alaska, USA |
| Oxneriaria pruinosa | LAH37556 | OP352770 | – | – | Pakistan |
| Oxneriaria pruinosa | LAH37555 | OP352771 | – | – | Pakistan |
| Oxneriaria rivulicola | Nordin 5957 (UPS) | EU057922 | HM060753 | – | Sweden |
| Oxneriaria rivulicola | Nordin 5960 (UPS) | EU057923 | – | – | Sweden |
| Oxneriaria sp | Nordin 6003 (UPS) | EU057931 | – | – | Sweden |
| Oxneriaria sp | Nordin 6004 (UPS) | EU057932 | – | – | Sweden |
| Oxneriaria supertegens | Owe-Larsson H-168a (UPS) | EU057935 | – | – | Sweden |
| Oxneriaria supertegens | Owe-Larsson 9011 (UPS) | EU057937 | – | – | Norway |
| Oxneriaria supertegens | Nordin 6023 (UPS) | EU057938 | HM060751 | – | Sweden |
| Oxneriaria supertegens | Owe-Larsson 9002 (UPS) | – | HM060742 | HM060704 | Norway |
| Oxneriaria verruculosa | Owe-Larsson 9007 (UPS) | EU057940 | HM060741 | HM060703 | Norway |
| Oxneriaria verruculosa | Owe-Larsson 9003 (UPS) | EU057941 | – | – | Norway |
| Oxneriaria verruculosa | Nordin 5942 (UPS) | EU057942 | – | – | Sweden |
| Oxneriaria virginea | UPS:Nordin 6017a | HQ259270 | – | – | Sweden |
| Oxneriaria virginea | UPS:Ebbestad SVL1-1 | HQ259271 | – | – | Svalbard |
| Oxneriaria virginea | Wheeler 7153 (hb. Wheeler) | – | – | MW424818 | Montana, USA |
| Outgroup | |||||
| Megaspora cretacea | B 600200932 | KX253974 | – | – | Armenia |
| Megaspora cretacea | B 600199170 | KX253975 | – | – | Armenia |
| Megaspora verrucosa | St. Clair C54042 (BRY) | – | KC667062 | – | Colorado, USA |
| Megaspora verrucosa | UPS:Nordin 6495 | – | – | HM060687 | Sweden |
Results
Out of almost 300 samples collected from the Deosai plains and its adjacent areas during the 2019 and 2020 surveys, four lichen thalli were putatively assigned to the genus Oxneriaria on the basis of gross morphological features (Figs
Multigene phylogenetic analyses
DNA was extracted from the four different collections and used successfully in PCR to generate amplicons for the ITS, LSU and mt SSU regions, which ranged in size from 500–800, 900–950 and 900–960 base pairs, respectively. Sequence data of amplicons were aligned and used to construct separate ITS, LSU and mt SSU trees via Maximum Likelihood analyses to examine phylogenetic relationships. Distinct, well-supported clades were recovered from all datasets with minimal conflict, each taxon showing a unique position in all phylogenetic analyses with sequence divergence from other taxa. Clade names were provisionally assigned.
The ITS phylogenetic tree (Fig.
The LSU phylogenetic tree (Fig.
The mt SSU phylogenetic tree (Fig.
Taxonomy
Oxneriaria crittendenii
Etymology
The specific epithet “crittendenii” refers to the British lichenologist Prof. Peter D Crittenden in recognition for his outstanding contributions to lichenology.
Holotype
Diagnosis
It differs from its closest species O. pruinosa by having lobate black margins (vs. non-lobate), orbicular thallus 1.5–5 cm (vs. irregular 3–8 cm), K test negative (vs. K positive yellow), distinct proper-exciple 17–40 µm wide (vs. indistinct) and polarilocular ellipsoid ascospores (vs. simple ellipsoid).
Description
Thallus crustose, epilithic, orbicular, 1.5–5 cm across, zonate, fine bullate to areolate in the centre to poorly areolate towards margin, in the centre areoles 0.5–1 mm diam. and a few areoles changing to squamules up to 1.8 mm in length, lobate at margins, determinate and radiate. Hypothallus distinct, shiny light brown. Upper surface grey with white powdery texture and black at margins. Thallus heteromerous, upper cortex 20–60 µm thick, globose to sub-globose hyaline paraplectenchymatous cells, 6–11 µm in diam. Algal layer discontinuous, 90–140 µm thick, photobiont Trebouxia sp, coccoid cells, globose to sub-globose 6–14 µm in diam. Medulla and lower cortex not differentiated and consisting of paraplectenchymatous, globose to sub-globose hyaline cells 25–45 µm in diam.
Apothecia
without stipe, aspicilioid, one apothecium per areole, rounded, 600–950 µm in diam., pruinose with black disc 450–700 µm, dull and concave. Proper exciple 17–40 µm thick. Thalline exciple 140–190 µm thick. Epihymenium brown, 10–20 µm thick. Hymenium hyaline, 85–110 µm thick. Hypothecium hyaline, 35–55 µm thick. Asci clavate, 8–spored, 60–100 × 22–30 µm. Ascospores hyaline, ellipsoid, polarilocular, 13–18 × 7–11 µm. Paraphyses moniliform, septate, cylindrical cells 3–10 × 1–2.5 µm, with internally brown terminal cells. Pycnidia roccella type (
Ecology
Saxicolous, calcareous, known only from Deosai Plains, Gilgit-Baltistan, occurring at elevations between 4,117 m and 4,651 m in extremely cold conditions.
Chemical study
K -ve, C -ve, KC -ve, UV +ve (light green), hymenium initially blue then turning dark orange after Lugol’s solution. Substictic acid detected through TLC.
Oxneriaria deosaiensis
Etymology
The specific epithet “deosaiensis” refers to the Deosai Plains, the type locality.
Holotype
Diagnosis
It differs from its closest species O. pruinosa by having lobate black margins (vs. non-lobate), K test positive light brown (vs. K positive yellow), KC test positive dark brown (vs. KC negative), apothecia epruinose (vs. densely pruinose), distinct proper-exciple 30–50 µm wide (vs. indistinct) and polarilocular ellipsoid ascospores (vs. simple ellipsoid).
Description
Thallus crustose, epilithic, irregular, 1.5–2 cm across, zonate, areolate to poorly bullate up to 0.8 mm in diam. to lobate up to 1.5 mm at margins, determinate and radiate. Hypothallus light grey. Upper surface dull grey, black at margins. Thallus heteromerous, upper cortex 20–55 µm thick, paraplectenchymatous hyaline cells 6–15 µm in diam. Algal layer discontinuous, 50–90 µm thick, photobiont Trebouxia sp, coccoid cells, globose to sub-globose, 13–21 µm in diam. Medulla and lower cortex not differentiated and consisting of paraplectenchymatous, globose to sub-globose hyaline cells, 5–12 µm diam.
Apothecia
without stipe, aspicilioid, epruinose, one apothecium per areole, rounded, 520–700 µm in diam., with black disc 350–550 µm in diam., dull, concave. Proper exciple, 30–50 µm thick. Thalline exciple 90–145 µm thick. Epihymenium brown, 10–24 µm thick. Hymenium hyaline, 90–160 µm thick. Hypothecium hyaline, 50–90 µm thick. Asci clavate, 8–spored, 75–110 × 16–27 µm. Ascospores hyaline, ellipsoid, polarilocular 11–18 × 7–10 µm. Paraphyses moniliform, septate, cylindrical cells 4–10 × 1–2 µm, with internally brown terminal cells. Pycnidia roccella type (
Ecology
Saxicolous, Quartz, known only from Deosai Plains, Gilgit-Baltistan, occurring at elevations between 4,364 m and 4,689 m in extreme cold conditions.
Chemical study
K +ve (light brown), C -ve, KC +ve (dark brown), UV +ve (light green), hymenium initially blue then turning dark blue after Lugol’s solution. Substictic acid and two unknown substances detected through TLC.
Discussion
The genus Oxneriaria was introduced by
The two new proposed species share some morphological similarities to each other such as dull coloured grey to brown, areolate to bullate, heteromerous thalli with black lobate margins, a discontinuous algal layer, medulla consisting of paraplectenchymatous cells and concave black apothecia showing as light green in response to UV. However, the two species, O. crittendenii and O. deosaiensis, also exhibit differences from each other in thallus growth- pattern (orbicular vs. irregular), hypothallus appearance (shiny brown vs. light grey), algal layer (90–140 µm vs. 50–90 µm), size of algal cells (6–14 µm vs. 13–21 µm), size of lower cortex cells (25–45 µm vs. 5–12 µm), apothecia (pruinose 0.6–0.95 mm diam. vs. epruinose 0.52–0.7 mm diam.) and thalline exciple (140–190 µm vs. 90–145 μm thick) and hypothecium (35–55 µm vs. 50–90 µm thick), respectively. Additionally, in response to Lugol’s solution, the hymenium of O. crittendenii turned dark orange, whilst that of O. deosaiensis turned dark blue.
The two new proposed species O. crittendenii and O. deosaiensis were found to be phylogenetically closely related to certain other Oxneriaria species, in particular O. pakistanica, O. pruinosa and O. rivulicola, although clear molecular differences were apparent in the ITS, LSU and mt SSU sequences. There were, in addition, some striking phenotypic characters showing the distinctive characteristics of the novel taxa along with the closest species and these are shown in Table
| Characters | O. crittendenii | O. deosaiensis | O. pakistanica | O. pruinosa | O. rivulicola |
|---|---|---|---|---|---|
| Margins | lobate, determinate, black | lobate, determinate, black | areolate, indeterminate, whitish-grey | lobate, determinate, whitish-grey | Non-elongate areoles |
| Hypothallus | shiny light brown | light grey | light brown | light grey | light grey |
| Upper Cortex | 20–60 µm thick | 20–55 µm thick | 10–25 μm thick | 30–50 μm thick | 25–40 μm thick |
| Algal Layer (thick) | 90–140 µm | 50–90 µm | 30–50 μm | 70–140 μm | 30–50 μm |
| Algal Cells (in diam.) | 6–14 µm | 13–21 µm | 10–15 μm | 10–17 μm | 7–15 μm |
| Apothecia (in diam.) | pruinose, 450–700 µm | epruinose, 520–700 µm | epruinose, up to 2 mm | densely pruinose, up to 1 mm | up to 2 mm |
| Hypothecium (thick) | 35–55 µm | 50–90 µm | 90–170 μm | 50–120 μm | 80–100 μm |
| Asci | 60–100 × 22–30 µm | 75–110 × 16–27 µm. | 60–80 × 30–40 μm | 55–80 × 25–42 μm | 70–85 × 20–24 μm |
| Pycnidia | roccella type | roccella type | absent | globose | globose |
| Conidia | 17–24 × 1 µm | 19–35 × 1 µm | absent | 14–18 × 1 µm | 30–37 × 1 µm |
| References | This Study | This Study |
|
Asgar et al. (2023) |
|
In addition to the differences of Table
Conclusions
In summary, as a result of all the distinct phenotypic and phylogenetic characters, we here propose the addition of two new species in the genus Oxneriaria from high altitudinal environments in Pakistan. Whilst these were found infrequently, the detection of the two new species O. crittendenii and O. deosaiensis add to reports of the discovery of other new species of lichen-forming fungi from the Deosai Plains in Pakistan (
Acknowledgements
The corresponding author (MU) thanks the Higher Education Commission of Pakistan for funding research work under the International Research Support Initiative Program (IRSIP) scheme. All the authors are grateful to Prof. Dr. Peter D Crittenden (School of Life Sciences, University of Nottingham, UK) and Prof. Dr. Pradeep Kumar Divakar (Departamento de Farmacología, Farmacognosia y Botánica Facultad de Farmacia, Universidad Complutense de Madrid Plaza de Ramón y Cajal, Madrid, Spain) for providing lichen herbarium specimens for thin layer chromatography.
Additional information
Conflict of interest
The authors have declared that no competing interests exist.
Ethical statement
No ethical statement was reported.
Funding
This research was supported by Higher Education Commission of Pakistan for funding research work under the International Research Support Initiative Program (IRSIP) scheme.
Author contributions
Conceptualization: MU. Data curation: MU. Formal analysis: MU. Funding acquisition: ANK. Investigation: MU. Methodology: MU. Software: MU. Supervision: CMW, ANK, PSD, MB. Validation: ANK, MB, CMW, PSD. Visualization: CMW, ANK, MB, PSD. Writing - original draft: MU. Writing - review and editing: CMW, ANK, PSD, MB.
Author ORCIDs
Muhammad Usman https://orcid.org/0000-0002-3490-058X
Paul S. Dyer https://orcid.org/0000-0003-0237-026X
Matthias Brock https://orcid.org/0000-0003-2774-1856
Christopher M. Wade https://orcid.org/0000-0002-1269-9694
Abdul Nasir Khalid https://orcid.org/0000-0002-5635-8031
Data availability
All of the data that support the findings of this study are available in the main text.
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