A new species of Lecidella (Lecanorales, Ascomycota) from Azad Jammu and Kashmir, Pakistan

ABSTRACT

The newly described species, Lecidella iqbalii sp. nov., is so far only known from Azad Jammu and Kashmir, Pakistan. A comparative morpho-anatomical study combined with a phylogenetic analysis based on the marker ITS placed this species together with two accessions from southern South America in a distinct and highly supported group at the base of the Lecidella stigmatea clade. The taxon is characterized by a shiny thallus, strongly convex apothecia, a thin excipulum, a dark brown epihymenium and relatively small ascospores (10-) 11-12 (-13) × (6-) 6.5-8(-9) µm.

Keywords:
crustose lichen; Ganga choti; Garhi dupatta; saxicolous; lichenized ascomycetes

Introduction

The western Himalayan region of Azad Jammu and Kashmir (AJ&K) extends from 73-75°E longitude and 33-36°N latitude. The mainly mountainous landscape is characterized by fertile, green, rocky, and undulating territory (Dar et al. 2012). The resulting diverse environmental conditions would suggest high lichen diversity. However, little is known about how diverse the lichen flora actually is in these regions, because there has been very little exploration (Ahmad et al. 1997; Aptroot & Iqbal 2012).

One of the more common and widely distributed crustose lichen genera is Lecidella (Lecanoraceae), established by Körber (1855) and comprising about 80 species (Kirk et al. 2008). This genus is mainly characterized by black lecideine apothecia with a persistent proper excipulum and Lecidella-type asci (Hafellner 1984). It is widely distributed - from the tropics to polar regions - and occurs on various substrata, including rock, detritus, bark, wood and mosses (Zhang et al. 2012). This group of lichenized ascomycetous fungi is usually regarded as taxonomically difficult due to a high degree of variation in morphological characters (Hertel 1984). In such cases, the support of molecular tools has played a significant role in systematics and species delimitation (Zhao et al. 2015).

So far, six Lecidella species have been reported from Pakistan: Lecidella carpathica, L. euphoria, L. patavina, L. pulveracea, L. stigmatea and L. tumidula (Zulfiqar et al. 2020).

As part of the study of the lichen flora of Azad Jammu and Kashmir, Pakistan, a new species of Lecidella was described using morphological and molecular methods.

Materials and methods

Collection and preservation

Study site: Two specimens of the genus Lecidella were collected from Azad Jammu and Kashmir, Pakistan in 2020 during fieldwork focused on an increasing floristic knowledge of the lichens of Pakistan. The Azad Jammu & Kashmir state falls within the Himalayan orogenic belt and the topography of northern districts (Neelum, Muzaffarabad, Hattian, Bagh, Haveli, Poonch, and Sudhnoti) is mainly hilly and mountainous with deep ravines, and rugged and undulating terrain, whereas the southern districts (Kotli, Mirpur and Bhimber) are of comparatively low relief (Abasi et al. 2019). The collected specimens were deposited in the herbarium of the Institute of Botany, University of the Punjab, Lahore (LAH).

Morphological characterization

The specimens were examined macro and micromorphologically under a stereomicroscope (Meiji Techno, EMZ-5TR, Japan) and compound microscope (SWIFT M4000-D) with a 9MP camera system, respectively. For anatomical investigation, sections of apothecia were made by hand and examined in water, KOH (10%) and Lugol’s iodine solution (50%). A minimum of twenty measurements in water were made for each diagnostic feature from the two samples.

Chemical characterization

The secondary chemistry was analyzed using spot tests, which were performed using KOH (10% K), Lugol’s iodine solution (50% I) and sodium hypochlorite solution (C). Thin Layer Chromatography was carried out using Solvent System G, following standard methods (Orange et al. 2010).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted directly from the center of the thallus with apothecia from each specimen using a modified 2% CTAB method (Gardes & Bruns 1993). The internal transcribed spacer region of the nuclear ribosomal DNA (ITS) was amplified using the primers ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. 1990) following the amplification protocol of Khan et al. (2018). PCR products were sent to Tsingke, China for sequencing.

The sequences of the marker ITS were assembled and edited using Geneious Pro 6.1.8 (www.geneious.com), aligned with MAFFT v7.017 (Katoh et al. 2002) and placed in context of the most recently published phylogeny of Ruprecht et al. (2020) based on the concept of Zhao et al. (2015). On the CIPRES Portal (Miller et al. 2010), the HYK+G+I model was selected using jModelTest (Posada 2008). Using RAxML-HPC2 v. 8.1.11 on CIPRES, a maximum likelihood analysis was implemented (Stamatakis 2014), using rapid bootstrapping with 1000 iterations. The resulting tree was visualised with FigTree v 1.4.3 (Rambaut et al. 2014).

Results

Phylogenetic analyses

The final alignment contained 46 sequences of the marker ITS with a length of 536 characters. The tree (Fig. 1) was rooted with species of the genera Carbonea, and Lecanora (see Tab. 1 for voucher details). According to Zhao et al. (2015) and Ruprecht et al. (2020), the tree shows four main clades: Lecidella stigmatea, L. enteroleucella, L. elaeochroma and Lecidella ssp. nov. The two specimens from Pakistan, described below as L. iqbalii, are sister to two specimens from southern South America (sSA; MK620141, MK620152), which form together a well supported group basal to the L. stigmatea clade.

Figure 1
Phylogeny (ML) of the genus Lecidella with two accessions of the newly described species Lecidella iqbalii, based on single OTUs of the species concepts of Ruprecht et al. (2020) and Zhao et al. (2015).

Table 1
Specimens used in the ITS phylogenetic analyses of Lecidella species. New sequences are in bold.

Name of species	Country/Origin	Voucher specimen	Genbank no.
Carbonea vorticosa	Antarctica: Ross Dependency, Brown Hills	R. Tuerk 35647 (SZU)	JN873866
Carbonea vorticosa	Norway	R. Tuerk 43429 (SZU)	JN873870
Lecanora fuscobrunnea	Antarctica: Ross Dependency, Diamond Hills	R. Tuerk 44628 (SZU)	JN873875
Lecanora polytropa	USA: California, Tranverse Range	U.C. Riverside 47815UCR1	JN873881
Lecidella carpathica	China: Inner Mongolia	ZX 20141477	KT453739
Lecidella carpathica	China: Xinjiang	ZX 20140367-2	KT453741
Lecidella carpathica	China: Xinjiang	ZX 20129069	KT453740
Lecidella effugiens	China: Yunnan	ZX YN0203	KT453754
Lecidella effugiens	China: Jilin	ZX 20141269-2	KT453748
Lecidella enteroleucella	China: Yunnan	ZX YN0201	KT453757
Lecidella aff. Elaeochroma	China: Jilin	ZX 20141284	KT453751
Lecidella elaeochroma	China: Xinjiang	ZX XL0395-2	KT453749
Lecidella elaeochroma	United Kingdom	_	FR799214
Lecidella elaeochroma	Turkey: Kayseri	_	HQ605938
Lecidella elaeochroma	United Kingdom	_	FR799215
Lecidella elaeochroma	_	U. Arup L98156 (Arup priv.herb.)	AY541275
Lecidella elaeochroma	United Kingdom	_	FR799217
Lecidella elaeochromoides	China: Xinjiang	ZX 20141142	KT453750
Lecidella euphoria	China: Xinjiang	ZX 20140638	KT453742
Lecidella euphoria	China: Xinjiang	ZX XL0351-2	KT453745
Lecidella aff. Euphoria	China: Yunnan	ZX YN0186-2	KT453755
Lecidella aff. Euphoria	China: Shaanxi	ZX 20114605	KT453756
Lecidella flavosorediata	United Kingdom	_	FR799213
Lecidella greenii	Antarctica: Ross Dependency, Granite Harbour	R. Tuerk 33586 (SZU)	HQ287871
Lecidella greenii	Antarctica: Victoria Land, Taylor Valley	R. Tuerk 33586 (SZU)	JN873883
Lecidella iqbalii	Pakistan	LAH37006	OL843364
Lecidella iqbalii	Pakistan	LAH37007	OL843364
Lecidella meiococca	Sweden	Ekman 3101 (BG)	AF517929
Lecidella patavina	China: Xinjiang	ZX 20140501-2	KT453767
Lecidella siplei	Antarctica: Victoria Land, Botany Bay	R. Tuerk 32991(SZU)	JN873896
Lecidella siplei	Antarctica: Quenn Maud Mountains, Mt Kyffin	R. Tuerk 33457(SZU)	JN873898
Lecidella sp. UR00090	Chile: Region de Magallanes y de la Antartica Chilena	UR00090 (SZU)	MK620141
Lecidella sp. UR00107	Chile: Region de Magallanes y de la Antartica Chilena	UR00107 (SZU)	MK620152
Lecidella stigmatea	China: Xinjiang	ZX 20141254	KT453758
Lecidella stigmatea	Argentina: Provinzia de Santa Cruz	UR00162(SZU)	MK620188
Lecidella stigmatea	Argentina: Provincia de Chubhut	UR00208 (SZU)	MK620216
Lecidella stigmatea	Argentina: Provinzia de Santa Cruz	UR00133 (SZU)	MK620166
Lecidella stigmatea	Austria: Kärnten, Gailtaler Alpen	R. Tuerk 38608 (SZU)	JN873901
Lecidella stigmatea	Turkey: Kayseri	_	HQ605937
Lecidella stigmatea	Chile: Region de Magallanes y de la Antartica	UR00095 (SZU)	MK620144
Lecidella tumidula	China: Xinjiang	ZX XL0009	KT453736
Lecidella tumidula	_	AFTOL-ID 1374	HQ650596
Lecidella wulfenii	Austria: Salzburg, Hochkönigmassiv	R. Tuerk 39666 (SZU)	JN873903
Lecidella sp. 3	Chile: Region de Magallanes y de la Antartica Chilena	UR00120 (SZU)	MK620158
Lecidella sp. 1	Argentina: Provincia de Chubhut	UR00213 (SZU)	MK620221
Lecidella sp. 2	Antarctica: Ross Dependency	MAF-GS1-64 (MAF)	MK208746

Lecidella iqbalii Fayyaz, Afshan, Niazi & Khalid

Mycobank number MB842789

Thallus crustose, areolate, whitish to grey, apothecia lecideine, disc strongly convex; asci Lecidella-type, ascospores hyaline, non-septate, ellipsoid, ascospores relatively small, (10-) 11-12 (-13) × (6-) 6.5-8(-9) µm.

Type:-Pakistan: Azad Jammu and Kashmir, Garhi Dupatta (34-36° °N, 73-35°E), 817 m a.s.l., on rock, 22 September 2020, N. S. Afshan and A. R. Niazi (JV-08H) (LAH37006-holotype), ITS GenBank accession number OL843364

Thallus: Crustose, well developed, shiny, granulose-rimose, areolate or rimose-areolate to verruculose-areolate, 7-8 cm in diameter, up to 0.1 mm thick, prothallus and pycnidia not found. Areoles: Distinct, plane, smooth, angular to irregular in outline, 0.01-0.1 mm in diameter. Surface colour: greyish brown to grey. Cortex: dark brown, 15-10 µm. Medulla: white, 25-30 µm. Photobiont cells: cells globose, 10-15 µm. Apothecia: frequent, scattered, shiny, lecideine, black, but sometimes becoming reddish when mature, sessile, 0.1-1.2 mm in diameter. Disc: black, strongly convex. Margins: distinct, concolorous with disc, round to irregular, thin, becoming excluded. Exciple: 40-60 µm thick, bluish green to dark brown in section. Epihymenium: dark brown, 10-15 µm. Hymenium: hyaline to brown, 80-95 µm, not inspersed. Hypothecium: hyaline, 75-150 µm K+, N+. Paraphyses: aseptate, hyaline, apices usually expanded to 3.0 µm, dark greenish to brown, separate in KOH. Asci: Lecidella-type, clavate, 8-spored, (40-) 45-50-53 (-55) × (18-) 20-21-22(-24) µm. Ascospores: simple, hyaline, thick and smooth walled, ellipsoid to ovoid, (10-) 11-12 (-13) × (6-) 6.5-8(-9) µm.(Fig. 2)

Secondary chemistry: Atranorin; spot test: thallus K+, C-, KC-

Habitat and distribution: The only known specimens were collected in the sub-tropical forest of Azad Jammu and Kashmir, Pakistan. The forest is dominated by Pinus roxburghii, Quercus oblongata, Quercus glauca and Pyrus pashia and others. The maximum daily temperature of the region is 20 to 30º C during the summer and the average temperature is 4° C during the winter, and there is moderate rainfall. Additionally, the area is a beautiful wonderland in view of its transcendent Himalayan peaks, peaceful lakes, majestic glaciers and waterfalls.

Etymology: The epithet “iqbalii” honours the late Pakistani mycologist S.H. Iqbal for his significant contribution to the knowledge of lichen diversity in Pakistan.

Additional specimens examined: PAKISTAN. Azad Jammu and Kashmir, Ganga choti (34-36° °N, 73-35°E), 3, 675 m a.s.l on rock, 25 September 2020, N. S. Afshan and A. R. Niazi (JV-08) (LAH37007) (ITS GenBank accession number OL843365).

Discussion

The species Lecidella iqbalii can be distinguished from the two formerly L. stigmatea to assigned specimens from southern South America (sSA, UR00090, UR00107; Ruprecht et al. 2020) by several morphological characters. The thallus is more areolate, the apothecia are more frequent with a strongly convex disc, a small exciple and a dark brown epihymenium in contrast to the granulose to rimose thallus, dull black and partially flat apothecia, a dark green epihymenium and in one case much smaller ascospores (UR00090). The phylogenetically distinct group formed by the new species and the two specimen from sSA can be distinguished to L. stigmatea by smaller ascospores (Wirth et al. 2013), to L. greenii by a different chemistry (stictic acid chemosyndrome) and by a pale thallus in contrast to the mainly darker pigmented thallus of L. siplei (Ruprecht et al. 2012) (compare Figs. S1, S2; Text S1, S2; Tab. 2).

Figure 2
Diagnostic characters of Lecidella iqbalii (LAH37006). A. Typical thallus of the type specimen. B. Close up of Apothecia. C. Hand-cut apothecial section in KOH. D. Cross section of apothecium. Bars: A = 2 cm, B = 1 mm, C = 60 µm, D = 80 µm.

Table 2
Comparison of the non-uniform characteristics.

Characters	Lecidella iqbalii	Lecidella sp. UR00090	Lecidella sp. UR00107	Lecidelaa stigmatea (Kantvilas & Elix, 2013)	Lecidella greenii (Ruprecht et al., 2012)	Lecidella siplei (Ruprecht et al., 2012)
Thallus colour	Whitish to grey	Greyish to beige	Greyish to beige	Pale cream to grey-brown, sometimes discoloured brownish	Whitish, grey to beige.	Whitish, beige, pale gray to dark gray
Apothecia	Strongly convex	Plane, pruinose, black	Plane to strongly convex	Plane to convex	Flat to convex	Flat to convex
Exciple	40-60 µm thick, bluish green to dark brown	Up to 120 µm thick, hyaline, J-, prominent, dark-green margin.	Up to 100 µm thick, hyaline, J-, dark green margin	35-50 (-80) µm thick	Up to 200 µm thick, hyaline, dark green margin	Up to 200 µm thick, hyaline, dark green to brown margin
Ascus	40-55 x 18-24 µm	Up to 45 µm	Up to 55µm	45-60 × 13-20 µm	Lecidella-type, 40-50 µm	Lecidella-type, 40-50 µm
Ascospores	10-13 x 6-9 µm.	8-11 x 6-8 µm	10-12 x 6-8 µm	(10-) 11-13.4-15(-16) × (6-) 6.5-7.8-9(-10) µm	10-11 (-12) x 6-7 (-8) µm	(10-) 12-14 (-15) x (5-) 6-7 µm
Apical cell of paraphyses	Up to 3.0 µm	Up to 3.0 µm	Up to 4.0 µm	2.5-5 µm wide	Up to 4.0 µm	Up to 4.0 µm
Epihymenium	Dark brown	Dark-green	Dark-green		Olive to dark green	Olive to dark green
Hypothecium	Hyaline to light brown at the base,	Hyaline, inspersed with dark crystals.	Hyaline , inspersed with crystals	60-150 µm thick, colourless or occasionally pale yellow-brown at the base.	Hyaline, inspersed with brown crystals.	Hyaline, inspersed with brown crystals

Lecidella iqbalii was so far only found in Azad Jammu and Kashmir, Pakistan. It is phylogenetically clearly separated from the two closely related sSA specimens (UR00090, UR00107) which have a distinct morphology to L. iqbalii and to each other. However, these two accessions from sSA can be treated at least on subspecies level (Figs. S1, S2; Tab. 2). Unfortunately, there is only one specimen each available and therefore we refrain from describing these accessions as new species.

At this stage of knowledge, it is not viable to make a definite assignment regarding the state of endemism of the new species. Further investigations are required to clarify this unresolved aspect.

Acknowledgements

We are highly grateful to University of the Punjab, Lahore, Pakistan for providing research project (Rs.250,000/-) No.D/72/EST-I Dated 14/01/2022 to conduct this work. We are also highly thankful to Dr. Kamran Habib (Institute of Botany, University of the Punjab, Lahore) for assistance in anatomical characterization.

References

• Abasi F, Ahmad I, Ahmad KS, Ulfat A, Khurshid R. 2019. Estimation of Genetic Diversity in Genus Mentha Collected from Azad Jammu and Kashmir, Pakistan. BioRxiv 866111. https://doi.org/10.1101/866111.
  » https://doi.org/10.1101/866111
• Ahmad S, Iqbal SH, Khalid AN. 1997. Fungi of Pakistan. Lahore, Sultan Ahmad Mycological Society of Pakistan.
• Aptroot A, Iqbal SH. 2012. Annotated checklist of the lichens of Pakistan, with reports of new records. Herzogia 25(2): 211-229.
• Dar MEUI, Cochard R, Shreshta RP, Ahmad S. 2012. Plant resource utilization by local inhabitants around Machiara National Park, Azad Kashmir, Pakistan. Journal of Food, Agriculture & Environment 10(3&4): 1139-1148.
• Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes‐application to the identification of mycorrhizae and rusts. Molecular Ecology 2(2): 113-118.
• Hafellner J. 1984. Studien in Richtung einer natürlicheren Gliederung der Sammelfamilien Lecanoraceae und Lecideaceae. Beiheft zur. Nova Hedwigia 79: 241-371
• Hertel H. 1984. Über saxicole, lecideoide Flechten der Subantarktis. Beihefte zur Nova Hedwigia (79): 399-499.
• Katoh K, Misawa K, Kuma KI, Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic acids research 30(14): 3059-3066.
• Khan M, Khalid AN, Lumbsch HT. 2018. A new species of Lecidea (Lecanorales, Ascomycota) from Pakistan. MycoKeys 38: 25-34.
• Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Ainsworth & Bisby's Dictionary of the Fungi. 10th ed. Wallingford, CAB International.
• Körber GW. 1855. Systema Lichenum Germaniae (I-XXXIV). Die Flechten Deutschlands mikroscopisch gepruft, kritisch gesichtet, charakteristisch beschrieben und systematisch geordnet. Breslau, Verlag Trewendt & Granier.
• Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Gateway Computing Environments Workshop: 1-8.
• Orange A, James PW, White FJ. 2010. Microchemical methods for the identification of lichens. British Lichen Society. London.
• Posada D. 2008. jModelTest: phylogenetic model averaging. Molecular Biology and Evolution 25(7): 1253-1256.
• Rambaut A, Suchard MA, Xie D, Drummond AJ. 2014. FigTree 1.4. 2 Software. Edinburgh, Institute of Evolutionary Biology, University of Edinburgh.
• Ruprecht U, Fernández-Mendoza F, Türk R, Fryday AM. 2020. High levels of endemism and local differentiation in the fungal and algal symbionts of saxicolous lecideoid lichens along a latitudinal gradient in southern South America. The Lichenologist 52(4): 287-303.
• Ruprecht U, Lumbsch HT, Brunauer G, Green TGA, Türk R. 2012. Insights into the diversity of Lecanoraceae (Lecanorales, Ascomycota) in continental Antarctica (Ross Sea region). Nova Hedwigia 94: 287-306.
• Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9): 1312-1313.
• White TJ, Bruns T, Lee SJWT, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 18(1): 315-322.
• Wirth V, Hauck M, Schultz M. 2013. Die Flechten Deutschlands. 2nd. edn. Stuttgart (Hohenheim): Eugen Ulmer GmbH & Co.
• Zhang LL, Wang LS, Wang HY, Zhao ZT. 2012. Four new records of lecideoid lichens from China. Mycotaxon 119(1): 445-51.
• Zhao X, Zhang LL, Zhao ZT, Wang WC, Leavitt SD, Lumbsch HT. 2015. A molecular phylogeny of the lichen genus Lecidella focusing on species from mainland China. Plos One 10(9): e0139405. https://doi.org/10.1371/journal.pone.0139405.
  » https://doi.org/10.1371/journal.pone.0139405
• Zulfiqar R, Habib K, Khan M, Khalid AN. 2020. A taxonomic and phyloge-netic study of some Lecidella species from Pakistan. WEBBIA Journal of Plant Taxonomy and Geography 83(2): 219-230.

Publication Dates

• Publication in this collection
  12 Sept 2022
• Date of issue
  2022

History

• Received
  29 Oct 2021
• Accepted
  12 May 2022