Additions to the lichen genus Rhizocarpon in Pakistan and their comparative analysis

As part of a comprehensive study of lichen diversity of northern areas of Pakistan using molecular and morphological approaches, we found four species of the genus Rhizocarpon. The analysis revealed two new species in the lichen biota of Pakistan namely, Rhizocarpon lavatum and Rhizocarpon petraeum, while the other two, Rhizocarpon disporum and Rhizocarpon geminatum are reported for the second time, from new localities. In comparison to literature information and GenBank sequences of the same taxa, variation in the morpho-anatomical features and nucleotide differences in the ITS marker of nrDNA have been documented. Descriptions and phylogenetic analysis of the taxa are given, along with information on ecology and distribution.


INTRODUCTION
Rhizocarpon Ramond ex DC. (Rhizocarpaceae, lichenized Ascomycota) is a large genus of lichenized or lichenicolous fungi, comprising approximately 200 currently accepted species (Davydov and Yakovchenko 2017). The members of the genus are usually recognized by a crustose thallus, a distinct prothallus, black lecideine apothecia, Rhizocarpon-type asci, and hyaline to brown, halonate ascospores that can be transversely septate or submuriform to muriform (Fletcher et al.2009;McCarthy and Elix 2014). The genus has a wide distribution throughout temperate to polar and alpine regions (Feuerer and Timdal 2004;Ihlen 2004;Kirk et al. 2008), occurring on siliceous or calcareous rocks (Moniri et al. 2009). Ihlen and Ekman (2002) proposed a variety of infrageneric arrangement in Rhizocarpon. Previously suggested infrageneric arrangements based on presence or absence of the yellow substance rhizocarpic acid in the thallus or the septation of the ascospores are unnatural. Some species with grey or brown thallus may have evolved from a yellow ancestor. They have also justified that during the course of evolution, spore septation and colour, amyloidity of the thalline medulla, and the presence of stictic acid complex and rhizocarpic acid are changed multiple times (Ihlen and Ekman, 2002). The most widely-used classification is the one proposed by Thomson (1967), who divided the genus into taxa with a yellow thallus containing the pigment rhizocarpic acid (subgenus Rhizocarpon), and taxa with white, grey or brown thalli lacking rhizocarpic acid (subgenus Phaeothallus) (Ihlen 2004).

Morphological and chemical studies
The collections were made during a lichen survey of different sites of Azad Jammu and Kashmir, Pakistan, in 2019. Morphological characters were observed under a stereomicroscope (Meiji Techno, EMZ-5TR, Japan). Standard microscopy and spot tests (Hale 1979) were used for further identification. Measurements were made from free hand sections of apothecia mounted in water on glass slides. The sections were observed with a compound microscope (MX4300H, Meiji Techno, Japan).

DNA extraction and PCR amplification
We extracted fungal DNA from the thallus using a 2% CTAB protocol (Gardes and Bruns 1993). Molecular data were generated for the internal transcribed spacer (ITS) region. The primer pair ITS1F (Gardes and Bruns 1993) and ITS4 (White et al.1990) was used to amplify the ITS region under the PCR conditions used by Khan et al. (2018). PCR products were visualized in a 1 % agarose gel (Sambrook and Russell 2001) and sent to BGI Hong Kong, for sequencing.

Phylogenetic analysis
The ITS locus was amplified and sequenced for all specimens yielded fragments of about 700-800 bp each. The BioEdit sequence alignment editor was used to reassemble forward and reverse sequences (Hall 2005). The nucleotide sequence comparison was performed using the Basic Local Alignment Search Tool (BLAST) of the National Centre for Biotechnology Information (NCBI) (Altschul et al. 1990). The multiple sequence alignment was performed using MAFFT v.7 with all parameters set to default values (Katoh and Standley 2013). The phylogenetic tree was executed using software MEGA 10.0 (Kumar et al. 2018) with ML method based on Tamura 3-parameter model. Nodal support was evaluated using the "rapid bootstrapping" option with 1,000 replicates. For rooting purpose of the tree, Catolechia wahlenbergii (Ach.) Körb. (HQ650649) was selected as an out-group.

Phylogeny
Closest matching sequences were downloaded from GenBank for subsequent phylogenetic analysis. A total of 47 ITS rDNA sequences were analysed. The data matrix had 523 unambiguously aligned nucleotide positions among which 246 were constant, 271 variable, 213 parsimony-informative and 57 were singleton variants.

Substrate and Ecology
It was found in temperate climate at an altitude of 4,173 m.a.s.l., growing on rocks exposed to sun and rain. The region has hilly topography having maximum and minimum temperature of 35 o C and -8C, respectively, and receive heavy snowfall in winter.

Substrate and Ecology
It was found at an altitude of 2,509 m.a.s.l., growing on sun and rain exposed rocks in partially forested area. The region has hilly topography with mean maximum and minimum temperature: 21.5°C and -2°C respectively, and receive heavy snowfall in winter.

Substrate and Ecology
It was found in Himalayan temperate forest, growing on sun and rain exposed rocks at an altitude of 2,097 m.a.s.l. The mean maximum and minimum temperature of the area is 28 o C and -2 o C, respectively, and receive an average annual rainfall of 749 mm. The dominant tree species around were Cedrus deodara (Roxb. ex Lambert) G.Don, Pinus wallichiana A.B. Jacks., Picea smithiana Boiss., Abies pindrow Royle.
Chemistry: K+ (yellowish), C-; Secondary metabolite Stictic acid is reported according to literature (Ihlen 2004), not examined in the present material.

Substrate and Ecology
It was found in temperate climate at an altitude of 2,097 m.a.s.l., growing on base-rich rocks. The mean maximum and minimum temperature of the area is 28 o C and -2 o C, respectively, and receive an average annual rainfall of 749 mm. The dominant tree species around were Cedrus deodara (Roxb. ex Lambert) G.Don, Pinus wallichiana A.B. Jacks., Picea smithiana Boiss., Abies pindrow Royle.

Comments
In our phylogenetic analysis, R. petraeum and R. reductum were found to be strongly supported sister taxa ( Figure 5). Morphologically, R. petraeum is also very close to R. reductum, as both contain stictic acid and usually have the same insoluble lichen pigments in the ascomata, but R. petraeum is distinct in having broader apothecia, and larger ascospores containing more cells.
There were two nucleotide differences found in comparison to the sequence of R. petraeum (HQ605942) reported from Turkey. This study reports it as a new record to the lichen biota of Pakistan.

COMPARATIVE ANALYSIS AND CONCLUSIONS
Our study on four Rhizocarpon species (Table 1) revealed some deviations in the areole size, presence or absence of pruina, height and pigmentation of hymenium and number of cells per ascospore) with in the same taxa published descriptions (Ihlen 2004;Wang et al. 2015).
Rhizocarpon lavatum shows great morphological variation having a wide ecological amplitude (Ihlen 2004) The areole size, pruinose disc of apothecia and lesser number of cells per ascospore of the Pakistani material were found to be in disagreement with the published descriptions (Ihlen 2004;Wang et al. 2015). The areole size was a big difference found between Chinese and Pakistani R. lavatum i.e., 0.4-1.2 mm (vs. 0.2-0.4 mm). The height and pigmentation of hymenium in Pakistani material was almost identical to Chinese specimen as compared to Nordic collection in which the height of hymenium was larger (Table 1).
In Pakistani R. petraeum we found thickness of areoles, presence and absence of pruina, height and pigmentation of hymenium and number of cells per ascospore in disagreement with the published descrip-  tions of Chinese and Nordic (Ihlen 2004;Zhao et al. 2013). The number of cells per ascospore were less in Pakistani specimen than the Nordic countries and Chinese taxa i.e., 8-15 (vs. 13-21) and (vs. 12-21), respectively. The areole thickness of Pakistani specimen was greater as compared to the Chinese taxon i.e., 0.2-0.6 mm (vs. 0.14-0.2 mm), while the hymenium pigmentation was largely in agreement with the Chinese description (Zhao et al. 2013), whereas the height and pigmentation of hymenium is not similar to the description of Ihlen (2004).
The description of Pakistani Rhizocarpon disporum is very close to North American samples, which however differs by its colour of thallus, blackish brown to black (vs. grey to grey brown), taller epinecral layer 10-20 µm (vs. 10 µm) and slightly curved ascospores (vs. not curved) (Nash et al. 2004).