A taxonomic and phylogenetic study of some Lecidella species from Pakistan

In this study, a number of species of the genus Lecidella were collected from different areas of Pakistan and characterized using morpho-anatomical and molecular techniques. The present work revealed that collected specimens belong to four species of Lecidella. Among which L. tumidula is a new record for Pakistan while L. carpathica, L. patavina and L. stigmatea are being reported here from new localities, from Pakistan, representing their wider distribution. Complete morpho-anatomical descriptions, ecology and distribution, along with ITS-based molecular analysis is provided.


INTRODUCTION
The genus Lecidella Korb. (Lecanoraceae), established by Korber in 1855, is comprised of 80 species (Zhao et al. 2015). This genus of crustose lichens is mainly characterized by black lecidiene apothecia with persistent proper excipulum and Lecidella-type asci (Zhao et al. 2015). It is a cosmopolitan genus, frequently found in temperate latitudes on wood, bark or rock (Kantvilas and Elix 2013). This group of lichenized ascomycetous fungi is usually regarded as taxonomically difficult due to a high degree of variation in morphological characters. In such cases, molecular study has played a significant role in systematics and species delimitation (Zhao et al. 2015).
Pakistan is located in western South Asia between 24-37 o N latitude and 62-75 o E longitudes. The country is well known for its geographical and climatic variations which is linked with rich biodiversity (IUCN 2006). The lichen diversity in this region is probably very high but little known due to the lack of surveys in many areas (Ahmad et al. 1997;Aptroot and Iqbal 2012). So far, 375 lichen species have been reported from Pakistan (Ahmad 1965;Aptroot and Iqbal 2012;Habib et al. 2017;Khan et al. 2018;Habib and Khalid 2019). In the past, attempts were made to describe the lichen diversity of the country using morpho-anatomical techniques (Ahmad 1965;Iqbal et al. 1978;Aptroot and Iqbal 2012). Recently, molecular techniques have also been used to study lichen flora of Pakistan (Habib et al. 2017;Khan et al. 2018;Habib and Khalid 2019).
From Pakistan, five species of Lecidella have been reported so far, viz; L. carpathica Korb., L. euphorea (Florke) Hertel, L. pulveraceae (Florke) Th.Fr., L. patavina (A.Massal.) Knoph & Leuckert, and L. stigmatea (Ach.) Hertel & Leuckert (Aptroot and Iqbal 2012). In the present study, different species of Lecidella have been collected from different areas of Pakistan. Use of morpho-anatomical techniques along with phylogenetic analysis led to identification of four different species. L. tumidula (A.Massal.) Knoph & Leuckert has been collected and described for the first time from Pakistan which made an addition to the lichen flora of this country. Now, the number of Lecidella species reported from Pakistan has been raised from five to six.

Morphological and chemical studies
Collections were made during a lichen survey of Chikar, Muzaffarabad (Azad Jammu and Kashmir), Parachinar, and Fairy Meadows (Gilgit Baltistan) in 2017 and 2018. Morphological characters were observed under a stereomicroscope (Meiji Techno, EMZ-5TR, Japan). Standard microscopy and spot tests (Hale 1979) were used for identification. Measurements were made from free hand section of apothecia mounted in water on a glass slide. The sections were observed using a compound microscope (MX4300H, Meiji Techno Co., Ltd., Japan). Minimum twenty measurements in water were made for each diagnostic feature.

DNA extraction and PCR amplification
We used thallus material along with apothecial material to extract fungal DNA using a 2% CTAB protocol (Gardes and Bruns 1993). The primer pair ITS1F (Grades and Bruns 1993) and ITS4 (White et al. 1990) was used to amplify the internal transcribed spacer (ITS) region under PCR conditions used by Khan et al. (2018). PCR products were visualized in a 1 % agarose gel using ethidium bromide (Sambrook and Russell 2001). PCR products were sequenced from BGI, China.

Phylogenetic analysis
The ITS regions of all specimens were amplified and sequenced. Bio-edit sequence alignment editor was used to reassemble forward and reverse sequences (Hall 2005). Sequences of other Lecidella species based on initial BLAST searches and those used in a study on phylogeny of Lecidella by Zhao et al. (2015) were used in phylogenetic analysis (Table 1). The multiple sequence alignment was performed using MAFFT v7 with all parameters set to default values (Katoh and Standley 2013). The ends of the alignment were trimmed to nearly an equal number of sites for all sequences. All gaps were treated as missing data. Maximum Likelihood analysis was performed with MEGA6 using a GTR model for bootstrapping (Tamura et al. 2013). One thousand rapid bootstrap replicates were run to infer the evolutionary history of each species.
The length of the final aligned file was 541 nucleotides, of which 355 sites were conserved, 181 were variable, 156 were parsimony informative and 25 were singleton, Rhizoplaca porterii and R. parilis (HM577376, HM577309) were chosen as an outgroup (Zhao et al. 2015).

Spot Tests
All negative (diploicin and lichexanthone reported according to literature but not examined in our specimen).
Substrate and ecology L. tumidula was found on bark of Quercus incana W. Bartram in dry temperate forest, at an altitude of 1,705 m, dominant vegetation including Pinus gerardiana Wall. ex D. Don, Quercus ilex L., Juniperus macropoda Boiss. and Picea smithiana Boiss, temperature ranges between 6-16 o C, average annual rainfall 300-500 mm.

Distribution
Temperate areas of Asia including China (Zhao et al. 2015), have also been reported from Europe and North America (Nash et al. 2004). Here it is been reported for the first time from Pakistan.

Description
Thallus crustose, effuse, rimose areolate to subsquamulose, dull, up to 5 cm wide, up to 300 µm thick. Areoles: scattered to crowded, smooth or rough, up to 0.5 mm in diameter, irregular in outline. Colour: pale green to grey green. Apothecia: sessile, semi-immersed, constricted at base, scattered to crowded, up to 1 mm in diameter. Disc: black, plane, flat to moderately con-
The second collection of L. stigmatea was found on a rock (saxicolous), close to water falls, in moist temperate forest at an altitude of 2,900 m, temperature ranges between -2-37 o C, average annual rainfall 1,500-1,650 mm.

Discussion
The present study of the genus Lecidella from northern Pakistan revealed one new record for the lichen flora of Pakistan while other Lecidella species have been collected from new localities thus indicating their wide distributional range in Pakistan.
In the ITS-based phylogenetic analysis, the Pakistani collection of L. tumidula (PR-112) clustered with specimens of the same taxon reported from China (Accession no. KT453736, KT453737) with strong support (99%, Fig.  7) and formed a sister group relationship with L. tumidula collected from USA (Accession no. HQ650596) that was wrongly reported in GenBank (Schmull et al. 2011), but after re-examination it was L. tumidula (Zhao et al. 2015).
Morphological comparison also confirms its identity as L. tumidula (Nash et al. 2004) except the presence of a pruinose disc in the specimen collected from Pakistan. There is only one nucleotide difference between the Pakistani collection and the Chinese L. tumidula specimen. It is also the second report of this taxon from Asia after China (Zhao et al. 2015).  In the phylogenetic analysis, the ITS sequence of Lecidella carpathica (CHK-04) clustered with ITS sequences of L. carpathica collected from Canada (KT695322, KT695353). Morphological comparison also confirms its identity as L. carpathica (Nash et al. 2004). There is only one nucleotide difference between Pakistani and Canadian L. carpathica (KT695322, KT695353). Previously, it was reported from dry temperate forest of Kalam (Swat), Pakistan at an elavation of about 2,001 m. a.s.l. The new collection is from moist temperate forest of AJK state, found at an elevation of 2,900 m. a.s.l.
The ITS sequence of Lecidella patavina (PR-11) clustered with a Chinese collection of L. patavina (Accession no. KT453767) in the phylogenetic analysis and made a sister branch with L. stigmatea (Ach.) Hertel & Leuckert. Morphologically, both Lecidella species are similar in having the same chemistry, a crustose epilithic thallus with a black disc of apothecia (Basaran et al. 2014) but differ due to the presence of the conspicuous and thicker thallus in the latter.
There are also two nucleotide difference between Pakistani and Chinese collection of L. patavina (KT453767). From Pakistan, it was previously reported but locality was not recorded. Here it is described from high elevation in Parachinar i.e. 1,705 m. a.s.l., where it is widespread in warm and temperate climates. The ITS sequence of Lecidella stigmatea (MKF-7 and PC-34), clustered with L. stigmatea reported from China (KT453763, KT453764, KT453765). The L. stigmatea (MK620163, MK620136) in our tree formed separate subclade. Results from phylogenetic analysis of L. stigmatea requires revision between specimens identified as L. stigmatea from Argentina, are distantly related to samples of our clade. Morphologically our specimen is similar to the Sonoran L. stigmatea, in having rimose-areolate thallus and lecideine apothecia (Nash et al. 2004). From Pakistan, this taxon has previously been reported from dry temperate forest of Kalam (Swat), Pakistan, at an elavation of about 2,001 m. a.s.l. but recent collections are from moist temperate forests of Azad Jammu & Kashmir and Gilgit Baltistan, at high elevations 2,900 m and 3,300 m. a.s.l. respectively.