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

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.


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 Lecidellatype 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).

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.

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.

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).

Iram Fayyaz, Najam-ul-Sehar Afshan, Abdul Rehman Niazi, Abdul Nasir Khalid and Ulrike Ruprecht
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.