Lecaimmeria pakistanica , a new lichen from Azad Jammu and Kashmir, Pakistan

. A new lichen species Lecaimmeria pakistanica K.Habib, R.Zul ﬁ qar & Khalid sp. nov. is described and illustrated from rocks in the temperate forests of the Himalaya of Azad Jammu and Kashmir, Pakistan. This species is characterized by its yellow-brown to brown thallus having areoles 0.4 to 1.5 mm across, branched and anastomosing paraphyses, a tall hymenium, large ascospores 20–32 × 10–16 μ m, and no substance detected by thin layer chromatography. All other species of the genus have ascospore dimensions in the range of 14–22 × 5–14 μ m. A phylogenetic analysis is provided based on ITS nrDNA sequences, and supports the separation of the novel species. Photographs and a comparative analysis with related species of Lecaimmeria are provided to con ﬁ rm the status of the species. tube, 20–32 × 10–16 μ m. Spot tests cortex and medulla K-, C-, KC-; medulla IKI+ blue; TLC none detected.


Introduction
The lichen genus Immersaria Rambold & Pietschm. originally included species with both lecideine and lecanorine apothecia (Calatayud & Rambold 1998). Recently, Xie et al. (2022) performed a fi ve-loci phylogenetic analysis (nrITS, nrLSU, RPB1, RPB2 and mtSSU) of the Lecideaceae Chevall. in order to test the monophyly of Immersaria. The analysis showed two clades within Immersaria: one with species with lecideine apothecia, and another with species with lecanorine apothecia. They excluded the lecanorine species from Immersaria and proposed a new genus, Lecaimmeria C.M.Xie, Lu L.Zhang & Li S.Wang to accommodate them (Xie et al. 2022).
Lecaimmeria could be distinguished from related genera by its waxy glossy, orange or red-brown thallus with an amyloid medulla, its immersed apothecia with a crypto-thalline margin, its orange epihymenium with an epinecral layer, and its Porpidia-type asci with eight halonate, non-amyloid ascospores. Species of the genus frequently grow on granite or sandstone, with the exception of one species, L. tuberculosa C.M.Xie & Xin Y.Wang, which grows on jade. The genus is represented by 10 species, distributed in alpine areas, high-latitude steppe or high-altitude dessert-steppe areas (Xie et al. 2022). Neither of the two genera had been recorded from Pakistan previously.
During our study on the lichen biota of the state of Azad Jammu and Kashmir, Pakistan, a novel species of Lecaimmeria was discovered. We present a brief diagnosis, an extensive description, illustrations, and a phylogenetic analysis based on ITS-sequence data.

Collection and preservation
Specimens were collected during surveys in the state of Azad Jammu and Kashmir, Pakistan in 2019. The specimens have been deposited in the LAH herbarium of the Institute of Botany, University of the Punjab, Lahore. Acronyms of herbaria follow Index Herbariorum (Thiers continuously updated).

Morphological and chemical characterization
Macro-and micromorphology of the specimens was examined under a stereo microscope (Meiji Techno, EMZ-5TR, Japan) and a compound microscope (SWIFT M4000-D). Thalline chemistry was analyzed by spot tests, using 10% potassium hydroxide (K) and calcium hypochlorite (C), and thin layer chromatography (TLC) (solvent C) according to the method proposed by Orange et al. (2001). Anatomical characterization and measurement of anatomical features were done by preparing and observing hand-cut apothecial sections mounted in water, K and Lugol's solution (IKI) under the compound microscope.
The amplifi ed DNA fragments (PCR products) were visualized with the help of 1% agarose gel using ethidium bromide and a gel documentation system (Sambrook & Russel 2001). The amplifi ed products were then sequenced from TsingKe BioTech Company Beijing, China, and the sequences deposited in GenBank.

Phylogenetic analysis
Bidirectional sequences (ITS1 and ITS4) were assembled by using BioEdit ver. 7.2.5 (Hall 2005). Comparative ITS sequences for the analysis were identifi ed and retrieved from GenBank using the Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990), evaluating maximum percent identifi cation and query coverage. The two newly generated sequences were aligned with 18 sequences retrieved from GenBank (Table 1) using MAFFT ver. 7 (Katoh et al. 2019). All sequences were trimmed terminally using BioEdit ver. 7.2.5.
The phylogenetic tree was constructed by MEGA X (Kumar et al. 2018) using the maximum likelihood (ML) method. The optimal model for nucleotide sequences was estimated by MEGA X (Kumar et al. 2018). The Kimura 2-parameter (K2P) was found to be the best model for a phylogenetic tree construction.

Phylogenetic analysis
The data matrix had 521 unambiguously aligned nucleotide positions of which 347 were conserved, 172 variables, 117 parsimony-informative and 54 were singletons. The new ITS nrDNA sequences nested within the phylogenetic branch of newly proposed genus Lecaimmeria (Fig. 1). The sequences of our new species formed a well-supported (BS 92) separate clade outside a group comprised of L. orbicularis

Diagnosis
Distinguished from all the known species of the genus by having large ascospores (20-32 × 10-16 μm), and relatively taller hymenium. All the other species of the genus have ascospore dimensions in the range of 14-22 × 5-14 μm. Also separated from other species of the genus by ITS nrDNA sequence data.

Etymology
The specifi c epithet ʻpakistanicaʼ refers to country in which the new species was discovered.

Discussion
During recent explorations of lichens from Azad Jammu and Kashmir, Pakistan, we observed specimens that could not be readily assigned to any known species. A phylogenetic analysis of the ITS nrDNA region confi rms their position within the genus Lecaimmeria, and morphological data showed their distinctness from other known species of the genus. We therefore describe these specimens as a new species, Lecaimmeria pakistanica sp. nov.
Lecaimmeria pakistanica sp. nov. is superfi cially similar to L. tibetica C.M.Xie & Xin Y.Wang, which was recently described from China (Xie et al. 2022). The species have a similar thallus and apothecia coloration with no substance being detected by TLC, but L. pakistanica diff ers morphologically in having areoles up to 1.5 mm across (vs 0.3-0.5 mm), and apothecia up to 0.8 mm diam. (vs 0.25-0.5 mm). The anatomical diff erences between these two species include the size of ascospores and the type of paraphyses. Ascospores are large and wider (20-32 × 10-16 μm) and paraphyses branched and anastomosing in L. pakistanica, whereas in L. tibetica, ascospores are small (12.5-15.0 × 5.0-6.0 μm) and paraphyses are unbranched and not anastomosing.
The phylogenetically close taxon L. botryoides C.M.Xie & Li S.Wang diff ers from the new taxon in having a red brown thallus, apothecia densely crowded while immature (3-6 / areolae), paraphyses only branched at the top and not anastomosing, comparatively very small ascospores 7.5-8.0 × 4.0-6.0 μm, and the presence of gyrophoric acid.
The diagnostic features distinguishing L. pakistanica sp. nov. from the related species of the genus are presented in Table 2.
Azad Jammu and Kashmir (AJK) is a state of Pakistan that exhibits a large altitudinal variation, with climatic conditions and a diverse vegetation that supports a diverse and conspicuous lichen biota. The nature reserves have abundant biological resources, it is expected that more new species of lichen may be discovered in the Azad Jammu and Kashmir in the future.