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In order to better understand Pakistan's lichen biota, lichen specimens were collected in September 2021 from Ghanche broq, Khaplu, Gilgit Baltistan. At 4,600 m above sea level (a.s.l.), near Khaplu city, the study location is located 100 km to the east of Skardu. The most notable feature of Ghanche Broq is the breathtaking view of the four 8,000-m summits in the world as well as other well-known peaks. K2, the second-highest peak in the world, stands at 8,611 m, followed by Broad Peak (8,047 m), Gashabrum-4 (7,925 m), Gashabrum-3 (7,952 m), Hrnami Ka Peak (6,325 m), Tchogho Lingsa Peak (6,325 m), Gashabrum-2 (8,035 m), K6 (7,281 m), and K7 (6,934 m). This area encompasses 8,915 square kilometres in total[11]. Khaplu is a section of the Karakoram range and is located at 35.1611° N and 76.3319° E. In this region of the Krakoram range, which is situated between 2,600 and 5,200 m above sea level, there are dry temperate deciduous forests[12]. On a typical day in January, Khaplu has temperatures ranging from a high of −11 °C to a low of −21 °C. The studied site experiences its warmest temperatures in July, with highs of 26 °C and lows of 12 °C. In these areas, the average annual precipitation is 59.3 cm, with a humidity level of 57%.[12].
Morphological and chemical characterization
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Under a stereomicroscope (Meiji Techno, EMZ-5TR, Japan) and a compound microscope (SWIFT M4000-D with a 9MP camera system), the specimens were analyzed macro- and micromorphologically. The thallus was cut out by hand in water for anatomical study. For each of the three samples, at least 20 measurements were taken. By performing spot tests with KOH (10%; K) and sodium hypochlorite solution (C), the secondary chemistry was examined. Utilizing Solvent System G and following industry standards, Thin Layer Chromatography was performed.
Molecular characterization and phylogenetic analysis
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Using the 2% CTAB procedure, genomic DNA was extracted from dried materials[1]. Polymerase Chain Reaction (PCR) was used to amplify the ITS region (Internal Transcribed Spacer) and LSU (larger sub-unit) regions ribosomal DNA of the fungal equivalent of the lichen DNA using ITS1F/ITS4 and LR0R/LR5 primer pairs respectively[13]. Following the conditions: initial denaturation at 94 °C for 1 min, final denaturation at 94 °C for 1 min followed by 35 cycles of annealing at 53 °C for 1 min, initial extension at 72 °C for 1 min and a final extension at the same temperature for 7 min[14]. In 1.2% Agarose gel, the amplified DNA fragments were visible[15]. PCR products were sequenced by BGI, Hong Kong (China). Using the BioEdit sequence alignment editor v7.0.9.0, the forward and reverse sequences were put back together[16]. To align the sequences with other sequences obtained from NCBI GenBank, Clustal W was employed[17]. On the CIPRES Portal, the HYK + G + I model was selected using jModelTest[6,18]. Using RAxMLHPC2 v8.1.11 on CIPRES, a maximum likelihood analysis was implemented using rapid bootstrapping with 1000 iterations. The resulting tree was visualised with FigTree v1.4.3[3,19]. For the phylogenetic analysis of this species based on the ITS-rDNA marker, a total of 47 sequences belonging to different genera were included in the final alignment (Table 1).
Table 1. ITS sequences included in the recent phylogenetic analysis are mentioned with GenBank accession numbers and country of the taxa.
Lichen Location Accession number ITS Protoparmeliopsis achariana Austria AF070019 Protoparmeliopsis bipruinosa Austria AF159932 Protoparmeliopsis bolcana Ukraine MK672838 Protoparmeliopsis chejuensis South Korea MK672839
MK672840Protoparmeliopsis zareii Pakistan KHP05 ITS Lecanora garovaglii Austria
USA
USA
Poland
Poland
IranAF189718
KT453728
KU934537
MK084624
MK084626
MK672841Protoparmeliopsis kopachevskae Korea
South Korea
South KoreaMK672845
MK672846
MK672847Protoparmeliopsis laatokkensis China MN912366 Protoparmeliopsis muralis Austria
Romania
Germany
USA
Russia
PolandKC791770
KP059048
KT818623
KU934555
KU934560
KY379232Protoparmeliopsis nashii Austria AF159931 Protoparmeliopsis peltata USA
Iran
Kazakhstan
RussiaKT453722
KT453723
KU934746
KU934751Protoparmeliopsis pseudogyrophorica China MK672851 Protoparmeliopsis zareii Iran KP059049 Protoparmeliopsis sp. Russia KU934865 Protoparmelia badia Germany KF562191 Protoparmelia badia Austria AF070023 Lecanora sp China MW590816 Lecanora muralis Austria AF070015 Lecanora muralis Austria AF159922 Protoparmeliopsis laatokkensis China MT875031 Protoparmeliopsis laatokkensis China MN912366 Protoparmeliopsis nashii Czech Republic ON447553 Lecanora garovaglii Austria AF189718 Rhizoplaca peltata China AY530887 Rhizoplaca peltata China AY509802 Rhizoplaca melanophthalma USA JX948289 Rhizoplaca melanophthalma USA JX948294 Rhizoplaca subdiscrepans China AY509789 Rhizoplaca chrysoleuca USA HM577252 Lecanora perpruinosa Austria AF070025 Lecanora contractula Austria AF070032 Myriolecis contractula USA HQ650604 Lecanora intricata Austria AY398703 Lecanora intricata Austria AF070022 Lecanora polytropa USA HQ650643 Lecanora polytropa Korea DQ534470 Rhizocarpon geminatum China KP314320 Protoparmelia picea Germany KF562194 Protoparmelia montagnei Spain AF101277 Protoparmelia montagnei Spain AF101275 Lecanora achroa USA JN943715 Lecanora achroa USA JN943719 Lecanora tropica USA JN943718 Lecanora tropica USA JN943720 Lecanora caesiorubella USA JN943722 Lecanora caesiorubella USA JN943727 Lecanora farinacea Austria AY541261 Lecanora farinacea Austria AY541262 Lecanora flavopallida USA JN943724 Lecanora carpinea Austria AY541249 Lecanora carpinea Austria AY541248 Lecanora allophana Austria AF070031 Lecanora allophana Austria AF159939 Xanthoria alfredii Sweden EU681345 Xanthoria ulophyllodes Sweden EU681341 Xanthomendoza fallax Sweden EU681346 Oxneria fallax USA MZ922253 -
Protoparmeliopsis zareii S.Y. Kondr.S. J. (2012) Fig. 1
Figure 1.
Protoparmeliopsis zareii. (a) Thallus. (b) Ascus with ascospores. (c) Algal cells. (d) Ascospores. Scale bars: (a) = 1 cm, (b) = 10 μm, (c) = 100 μm, (d) = 5 μm.
Thallus Placodioid, 1.5–3.5 cm or wider, 0.5–2 mm or thicker in the center, forming compact rosettes, but frequently either confluent or dispersed and irregular, tightly to loosely linked; areolate to squamulose; central areoles (0.5–)0.7–1.5 mm wide/across; upper surface greenish-grayish often with whitish edges of thalline lobes and areoles; whitish reticulum on the upper surface of the central areoles; numerous apothecia immersed into thalline areoles; dull brown to greenish brown apothecium discs; prosoplectecnchymatous true exciple. The outermost edges, especially the extreme lobe ends, frequently have darker blue-green to black areoles. The lobes (peripheral) are approximately 4–5 mm long, 1–2 mm wide, and somewhat enlarged to 2.0–2.5 mm wide at the tips. The thallus is 1 mm thick in section; the cortical layer is 50 μm thick and uniformly split from the algal zone; the algal zone is 50 μm thick (possibly 200 μm thick) and the algal cluster is occasionally vertically extended to 200–250 μm thick.
Apothecia 0.5–1.5 mm diam., typically strongly elevated with relatively narrowing stipa to 1 mm long, ranging from buried into areoles to moderately verrucose before rising slightly but without distinguishable stipa (as in P. muralis); the disc is dull brown or greenish brown, and the thalline margin is greenish-grayish, rounded by whitish edges that are the same as the edges of the thalline lobes and areoles (frequently, young apothecia appear to be identical to thalline areoles); thalline exciple with cortical layer to 30–40 μm thick; true exciple relatively thin to 50(–100) μm broad in the lateral portion and quite thick (75–)100–200 μm thick in basal portion, thalline edge to 0.3–0.35 mm wide. lax; algal zone below true exciple in vertically elongated clusters to 50–200(–250) μm thick; epihymenium dull greyish or dull brownish with pigment granules, in K becoming hyaline; Hymenium 70–80 μm high; subhymenium 20–40 μm thick with multiple oil cells to 4.8–7.2 μm in diameter; asci 8–spored, 48–60 × 22–24 μm; ascospores hyaline, generally ovoid to broadly ellipsoid, slightly smaller (7.2–)10.8–13.2 × 7.2–9.6 μm in K while larger with huge oil droplet, (8.4)10.8–13.2(–14.4) × 8.4–9.6(–10.8) μm in water.
Spot tests: Thallus usually K-, C-, KC-, P-; cortex usually KC+ yellow; medulla usually KC- .
Material examined Pakistan: Gilgit-Baltistan: Ghanche, Khaplu, 35.1611° N, 76.3319° E; 3,900 m a.s.l., dry temperate area, cold desert, on rock; coll.: A. Bano (KHP-05), 20 September 2021.
Ecology: On silicate rocks beside Bromus tectorum L., Valeriana cymbicarpa C.A. Mey., and Malva sylvestris L. var. silvestris.
Distribution: Known so far from Ghanche Broq Khaplu, Gilgit Baltistan, Pakistan.
Phylogenetic analyses
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The final dataset contained 605 locations. Rhizocarpon geminatum (L.) DC. was used to root the tree (Fig. 2). Protoparmelia complex, Lecanora complex, Lecanora complex, Rhizoplaca complex, Protoparmeliopsis complex, and Lecanora complex are the six clades that are represented in the tree. The specimen from Pakistan, designated Protoparmeliopsis zareii below, is grouped with the species from Iran (KP059049), both of which belong to clade V.
Figure 2.
The phylogenetic tree of the representatives of the family Lecanoraceae including genera Protoparmeliopsis, Lecanora, Rhizoplaca and Protoparmelia based ITS-rDNA marker sequences. All the genera are separated in the form of distinct clades in the tree. The maximum likelihood method is used in this phylogenetic analysis showing Protoparmeliopsis zareii is the same as the Iranian collection. Pakistani collection is labeled with a black circle in the figure.
For LSU marker analysis, 26 sequences have been extracted from the GenBank database and included in the final alignment (Table 2). The final dataset contained 2,317 locations and these sequences were recovered in major clades in the phylogenetic analysis (Fig. 3).
Table 2. LSU sequences used in the phylogentic analysis mentioned with acession numbers and type locality.
Lichen Location Accession number LSU Lecanora achroa Thailand
AustraliaJN939502
JN939527
JN939504Lecanora tropica Thailand
Fiji
KenyaJN939518
JN939533
JN939537Lecanora caesiorubella USA
Australia
AustraliaJN939509
JN939506
JN939508Lecanora farinacea Australia
AustraliaJN939511
JN939513Lecanora flavopallida Australia JN939514
JN939516Lecanora intricata Sweden DQ787345 Lecanora polytropa USA DQ986792 Lecanora sulphurea Sweden DQ787355 Lecanora carpinea Sweden DQ787363 Lecanora aff. Sweden AY853376 Lecanora campestris Sweden DQ787361 Lecnora contractula USA DQ986746 Lecanora perpruinosa Sweden DQ787343 Protoparmeliopsis achariana Sweden
USADQ787341
DQ973027Lecanora muralis Germany HQ660533 Lecanora muralis Sweden DQ787339 Figure 3.
Phylogenetic analysis of Protoparmeliopsis zareii (KHP05 LOR) based on LSU sequences. The evolutionary history was inferred by using the Maximum Likelihood method and Jukes-Cantor model . The tree with the highest log likelihood (–7,214.55) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 26 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. There were a total of 2317 positions in the final dataset. Evolutionary analyses were conducted in MEGAX.
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The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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About this article
Cite this article
Bano A, Razaq A, Ishaq A, Ilyas S, Liaqat H. 2024. Molecular identification and phylogenetic analysis of a lichen, Protoparmeliopsis (lecanoraceae, ascomycota), a new lichen genus for Khaplu valley, Gilgit-Baltistan, Pakistan. Studies in Fungi 9: e005 doi: 10.48130/sif-0024-0002
Molecular identification and phylogenetic analysis of a lichen, Protoparmeliopsis (lecanoraceae, ascomycota), a new lichen genus for Khaplu valley, Gilgit-Baltistan, Pakistan
- Received: 19 May 2023
- Accepted: 02 February 2024
- Published online: 08 May 2024
Abstract: Protoparmeliopsis is reported for the first time from Pakistan and is represented here by Protoparmeliopsis zareii as a species record of this genus. This species has been identified using macro- and micromorphological descriptions, spot tests, ITS (Internal Transcribed Spacers), and LSU (Larger Sub Unit) of rDNA molecular markers analyses. Molecularly, in the phylogenetic analysis based on the ITS sequence of Pakistani collection of Protoparmeliopsis zareii (KHP 05) clustered with DNA sequences of Protoparmeliopsis zareii reported from Iran (KP059049) while in LSU analysis, it assembled with Lecanora muralis as the LSU sequences of P. zareii are not available in the DNA database. Morphological data was also found and the studied specimen is found to be conspecific with Protoparmeliopsis zareii.
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Key words:
- Fungal taxonomy /
- Lichenology /
- Mycology /
- ITS /
- rDNA