WEBBIA Journal of Plant Taxonomy and Geography Firenze University Press www.fupress.com/webbia A taxonomic and phylogenetic study of some Lecidella species from Pakistan Citation: Rizwana Zulfiqar, Kamran Habib, Memoona Khan, Abdul Nasir Khalid (2020) A taxonomic and phylogenetic study of some Lecidella species from Pakistan. Webbia. Journal of Plant Taxonomy and Geography 75(2): 219-230. doi: 10.36253/jopt-9581 Received: August 8, 2020 Rizwana Zulfiqar*, Kamran Habib, Memoona Khan, Abdul Nasir Khalid Fungal Biology and Systematics Lab, Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan *Corresponding author. Email: rizwanamughal6@gmail.com Accepted: September 9, 2020 Published: November 18, 2020 Copyright: © 2020 Rizwana Zulfiqar, Kamran Habib, Memoona Khan, Abdul Nasir Khalid. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/webbia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Editor: Riccardo M. Baldini Abstract. 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. Keywords: Khyber Pakhtunkhwa, Lecanoraceae, Lecidella, phylogeny, Pakistan. 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 24–37o N latitude and 62–75o 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 Webbia. Journal of Plant Taxonomy and Geography 75(2): 219-230, 2020 ISSN 0083-7792 (print) | ISSN 2169-4060 (online) | DOI: 10.36253/jopt-9581 220 Rizwana Zulfiqar, Kamran Habib, Memoona Khan, Abdul Nasir Khalid 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. MATERIALS AND METHODS 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). 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 RESULTS Lecidella tumidula (A.Massal.) Knoph & Leuckert, Biblthca Lichenol. 68: 131. 1997. (Figure 1, A-E; Figure 5; A-D). Description Thallus crustose, continuous, up to 3 cm in diameter, up to 0.6 mm thick, granulose to rimose. Colour: light greyish green to light olive green, dull to weakly glossy. Apothecia: rounded, semi-immersed, sessile, 0.5– 1 mm, frequently present, strongly constricted at base. Margins: thin, continuous, concolorous to disc, distinct when young, indistinct when mature. Disc: black, smooth, glossy, pruinose, flat to convex. Exciple: black, 40–50 µm thick. Epihymenium: blackish, 30–36 µm tall. Hymenium: hyaline, 80–86 µm tall. Hypothecium: dark reddish brown, 30–40 µm tall. Paraphyses: aseptate, hyaline, rarely anastomosing or branched, apex swollen, 2–4 µm wide. Asci: clavate, 8-spored, 58–78 µm × 10–14 µm. Ascospores: simple, hyaline, narrowly ellipsoid to ovoid, 10–12 µm × 5–7 µm. 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 221 A taxonomic and phylogenetic study of some Lecidella species from Pakistan Table 1. Specimens used in phylogenetic analysis of Lecidella species. ITS Accession NoKT453736 KT453737 HQ650596 KT453750 KT453746 KT453748 KT453747 AF517929 KT453743 KT453742 KX132994 KX132965 JN873903 MN387031 MN387029 HQ287871 JN873884 Specimen Name Lecidella tumidula Lecidella tumidula Lecidella tumidula Lecidella elaeochromoid Lecidella elaeochromoid Lecidella effugiens Lecidella effugiens Lecidella meiococca Lecidella euphorea Lecidella euphorea Lecidella flavosorediata Lecidella flavosorediata Lecidella wulfenii Lecidella elaeochroma Lecidella elaeochroma Lecidella greenii Lecidella greenii Country China China USA China China China China Sweden China China Switzerland Switzerland Austria Poland Poland Antarctica Antarctica Voucher NoZX XL0009 (Zhao et al. 2015) ZX 20129166-2 (Zhao et al. 2015) 404720 (Schmull et al. 2011) ZX 20141142 (Zhao et al. 2015) ZX 20114966-2 (Zhao et al. 2015) ZX 20141269-2 (Zhao et al. 2015) ZX 20141148-2 (Zhao et al. 2015) Ekman 3101 (BG) (Ekman & Tonsberg, 2002) ZX XL0387 (Zhao et al. 2015) ZX 20140638 (Zhao et al. 2015) 980812 (Mark et al. 2016) 980812 (Mark et al. 2016) Tuerk 39666 (Ruprecht et al. 2012) 272987 (Singh et al. 2019) 272987 (Singh et al. 2019) Herbarium Tuerk 43015 (Lumbsch et al. 2011) Tuerk 33612 (Ruprecht et al. 2012) 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 oC, 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. Material examined PAKISTAN: Khyber Pakhtunkhwa Province: Kurram District, Parachinar: 33°90’ N, 70°08’ E; 1,705 m; on bark of Quercus incana W. Bartram, 18 August 2018, ITS Accession NoMK970672 HQ605934 KT453767 MK620163 MK620136 KT453764 KT453765 KT453760 KT453763 KT695322 KT695353 Specimen Name Lecidella greenii Lecidella patavina Lecidella patavina Lecidella stigmatea Lecidella stigmatea Lecidella stigmatea Lecidella stigmatea Lecidella stigmatea Lecidella stigmatea Lecidella carpathica Lecidella carpathica Country Antarctica Turkey China Argentina Argentina China China China China Canada Canada JN873899 Lecidella siplei Antarctica JN873896 Lecidella siplei Antarctica JN873898 Lecidella siplei Antarctica JN873897 Lecidella siplei Antarctica HM577376 HM577309 Rhizoplaca porterii Rhizoplaca parilis USA USA Voucher NoT48787 (Wagner et al. 2019) 574971 (Basaran et al. 2014) ZX 20140501-2 (Zhao et al. 2015) UR00128 (Ruprecht et al. 2019) UR00080 (Ruprecht et al. 2019) ZX 20140086-2 (Zhao et al. 2015) ZX 20140045-2 (Zhao et al. 2015) ZX 20140519-2 (Zhao et al. 2015) ZX 20140507-2 (Zhao et al. 2015) BIOUG24047-H06 (Telfer et al. 2015) BIOUG24047-E02 (Telfer et al. 2015) Tuerk 35895 (Ruprecht et al. 2012) Tuerk 32991 (Ruprecht et al. 2012) Tuerk 33457 (Ruprecht et al. 2012) Tuerk 33449 (Ruprecht et al. 2012) 55145 (BRY-C) (Leavitt et al. 2011) 55078 (BRY-C) (Leavitt et al. 2011) A.N. Khalid & K. Habib; LAH36399; (GenBank accession no. MT139651). Lecidella carpathica Korb., Parerga lichenol. (Breslau) 3: 212. 1861. (Figure 2, A-F; Figure 5; E-H). 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- 222 Rizwana Zulfiqar, Kamran Habib, Memoona Khan, Abdul Nasir Khalid Figure 1. Lecidella tumidula. (A & B) showing crustose thallus and abundant apothecia (C) pruinose apothecia; (D) Cross section of an apothecium (H: Hymenium; E: Epihymenium); (E) showing ascus and paraphyses (A: Ascus; P: Paraphyses). vex, pruinose. Margins: distinct, entire or flexuose, up to 0.9 mm wide. Exciple: bluish green to black, 20–30 µm thick. Epihymenium: pale brown to dark brown, 15–20 µm tall. Hymenium: hyaline, 65–90 µm tall. Hypothecium: pale brown to brown, 30–45 µm tall. Paraphyses: aseptate, hyaline, rarely anastomosing or branched, sometimes slightly swollen apically, up to 2 µm wide. Asci: clavate, 45–60 x 13–15 µm, 8-spored. Ascospores: simple, hyaline, ellipsoid to ovoid, 10–14 x 6–8 µm. Spot tests K+ yellow, C–, KC–, P– Distribution Widespread including Africa, Europe, Australia, Macaronesia, New Zealand (Smith et al. 2009), UK, USA, Ukraine (Oxner 1968; Kondratyuk et al. 1998; 2003), temperate parts of Asia (Nash et al. 2004), China (Zhao et al. 2015), India (Singh & Sinha 2010) and Pakistan (Nasim et al. 2004). Material examined PAKISTAN: Azad Jammu & Kashmir: Chikar, 34° 9’ N, 73° 41’ E, 234 m; on rock; 18 August 2018, K. Habib; LAH36400; (Genbank accession no. MT139649). Substrate and ecology L. carpathica was found on rock (saxicolous), in moist temperate forest at an altitude of 234 m, temperature ranges between -2–37 oC, average annual rainfall 1,500–1,600 mm, with dominant tree species Pinus wallichiana A.B. Jacks., Picea smithiana Boiss., Abies pindrow Royle., Quercus incana W. Bartram and Q. dilatata Lindl. ex Royle. Lecidella patavina (A.Massal.) Knoph & Leuckert, in Knoph, Bibliotheca Lichenol. 36: 116. 1990. (Figure 3, A-G; Figure 6; A-D). Description Thallus indistinctly crustose, inconspicuous, intermingled with rock particles, up to 4 cm in diameter. A taxonomic and phylogenetic study of some Lecidella species from Pakistan 223 Figure 2. Lecidella carpathica. (A & B) Crustose thallus showing abundant apothecia; (C) black, pruinose apothecia; (D) Cross section of an apothecium (H: Hymenium; E: Epihymenium); (E) Ascus; (F) Paraphyses. 224 Rizwana Zulfiqar, Kamran Habib, Memoona Khan, Abdul Nasir Khalid Figure 3. Lecidella patavina. (A & B) Crustose thallus showing abundant apothecia; (C) black, pruinose fascicle of apothecia; (D) Cross section of an apothecium (H: Hymenium; E: Epihymenium); (E) Ascus; (F) Ascospores; (G) Paraphyses. Colour: light green to dark green. Apothecia: rounded to irregular, 0.2–1.5 mm in diameter, sessile, constricted at base, frequent, separate, rarely fascicle. Margins: thin, continuous, concolorous to disc, distinct when young, indistinct when mature. Disc: black, flat to strongly convex, smooth and slightly glossy, slightly pruinose. Exci- A taxonomic and phylogenetic study of some Lecidella species from Pakistan 225 ple: bluish green, 40–50 µm thick. Epihymenium: dark blue with blackish tint, 20–30 µm tall. Hymenium: hyaline to grey, 110–120 µm tall, inspersed. Hypothecium: hyaline to brown, 90–110 µm tall. Paraphyses: hyaline, aseptate, apically branched, not anastomosing, apex slightly swollen, 1–2 µm wide. Asci: clavate, 8-spored, 30–67 µm × 12–20 µm. Ascospores: hyaline, simple, narrowly ellipsoid to ovoid, 9–13 µm × 5–6 µm. 78–88 µm thick, bluish green to brown. Epihymenium: dark brown, 12–16 µm high. Hymenium: hyaline, 75–80 µm tall, not inspersed. Hypothecium: hyaline to brown, 65–75 µm tall. Paraphyses: aseptate, hyaline, rarely anastomosing or branched, slightly swollen apically, up to 1.2 µm wide. Asci: lecanoral-type, clavate, 8-spored, 40–57 x 14–18 µm; Ascospores: simple, hyaline, thick and smooth wall, ellipsoid to ovoid, 9–14 x 5–8 µm. Spot Test Spot tests K+ (Slight yellow), C–, KC–, P– (atranonin or lichexanthone might be predicted). K+ yellow, C–, KC–, P– (atranonin, zeorin, lichexanthone or norlichexanthone might be predicted). Substrate and ecology Substrate and ecology L. patavina was found on nutrient enriched siliceous rocks near water falls, in moist temperate forest at an altitude of 1,705 m, temperature ranges between -2–37 oC, average annual rainfall 1,500– 1,650 mm, with dominant tree species Cedrus deodara (Roxb. ex Lambert) G.Don, Pinus wallichiana A.B. Jacks., Picea smithiana Boiss., Abies pindrow Royle., Quercus incana W.Bartram, Q. dilatata Lindl. ex Royle. and Q. semecarpifolia Sm. L. stigmatea was found on bark of Pinus wallichiana A.B. Jacks., in moist temperate forest, with dominant tree species Cedrus deodara (Roxb. ex Lambert) G.Don, Pinus wallichiana A.B. Jacks., Picea smithiana Boiss., Abies pindrow Royle., Quercus incana W. Bartram, Q. dilatata Lindl. ex Royle. and Q. semecarpifolia Sm. at an altitude of 3,300 m, temperature ranges between -2–37 oC, average annual rainfall 1650 mm. 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 oC, average annual rainfall 1,500– 1,650 mm. Distribution Mainly arctic-alpine, UK, N.W. Scotland (Smith et al. 2009), Africa, Antarctica, Europe, USA, (Knoph and Leuckert 2004; Knudsen and Kocourková 2012), Ukraine (Vondrak et al. 2010), temperate parts of Asia (Nash et al. 2004), China (Zhao et al. 2015) and Pakistan (Aptroot and Iqbal 2012). Material examined PAKISTAN: Khyber Pakhtunkhwa Province: Kurram District, Parachinar: 33°90’ N, 70°08’ E; 1705 m; on rock; 18 August 2018, A.N. Khalid & K. Habib; LAH3640; (GenBank accession no. MT139652). Lecidella stigmatea (Ach.) Hertel & Leuckert, Willdenowia 5: 375. 1969. (Figure 4, A-G; Figure 6; E-H). Description Thallus crustose, areolate or rimose-areolate to verruculose-areolate, 7–8 cm in diameter, up to 0.2 mm thick, rarely thick up to 0.6 mm. Areoles: indistinct to distinct, flat to slightly convex, angular to irregular in outline, 0.2–0.6 mm in diameter. Surface: finely granulose, rough, dull. Colour: greyish to grey to brownish grey. Apothecia: lecideine, black, sessile, 0.4–0.9 mm in diameter. Disc: black, flat to somewhat convex, pruinose. Margins: distinct, thin, becoming excluded. Exciple: Distribution Probably cosmopolitan, including UK, USA, Antarctica, Australia (Smith et al. 2009), Ukraine (Oxner 1968, Kondratyuk et al. 1998, 2003), temperate parts of Asia (Nash et al. 2004), China (Zhao et al. 2015), India (Singh & Sinha 2010) and Pakistan (Ahmad 1965). Material examined PAKISTAN: Gilgit Baltistan, Fairy Meadows: 35°23’ N, 74°35’ E; 3300 m; on bark of Pinus wallichiana A.B. Jacks., 18 July 2017, A.N. Khalid & M. Khan; LAH36402; (GenBank accession no. MT139653); Azad Jammu & Kashmir: District Muzaffarbad, Peer Chanasi: 34°2’ N, 73°33’ E; 2900 m; on rock; 18 August 2018, K. Habib; LAH36403; (Genbank accession no. MT139650). Key to Genus Lecidella in Pakistan 1a. On Bark ................................................................. L. tumidula 1b. On rock, Cortex K-, C ............................................................ 2 1c. On rock, or on bark, Cortex K+, C ...................................... 3 226 Rizwana Zulfiqar, Kamran Habib, Memoona Khan, Abdul Nasir Khalid Figure 4. Lecidella stigmatea. (A & B) Crustose thallus showing abundant apothecia; (C) black, pruinose disc of apothecia; (D) Cross section of an apothecium (H: Hymenium; E: Epihymenium); (E) Ascus; (F) Ascospores; (G) Paraphyses. 1d. On rock, Cortex K+, C ........................................................... 4 2a. Thallus crustose, areolate, epihymenium 15–20 µm tall, hypothecium brownish grey, on rock ...............L. carpathica 2b. Thallus crustose, granulose, epihymenium 30–36 µm tall, hypothecium dark reddish brown, on bark ....... L. tumidula 3a. Thallus rimose-areolate, paraphyses rarely branched, exciple greenish black, hymenium not inspersed ... L. stigmatea A taxonomic and phylogenetic study of some Lecidella species from Pakistan 227 Figure 5. (A-D): Line drawings of Lecidella tumidula A: Granulose thallus; B: Ascus; C: Ascospores; D: Paraphyses; (E-H): Line drawings of Lecidella carpathica E: Crustose-areolate thallus; F: Ascus; G: Paraphyses; H: Ascospores. Figure 6. (A-D): Line drawings of Lecidella patavina A: crustose thallus B: Ascus; C: Ascospores; D: Paraphyses; (E-H): Line drawings of Lecidella stigmatea E: Rimose-areolate thallus; F: Ascus; G: Paraphyses; H: Ascospores. 3b. Thallus inconspicuous, paraphyses mostly branched, exciple bluish green, hymenium inspersed ................L. patavina 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). 4a. Thallus crustose, granulose to rimose-areolate, hymenium 55–100 µm tall ....................................................... L. euphorea 4b. Thallus sorediate, farinose, hymenium 60–70 µm tall, ......... ........................................................................ L. pulveraceae 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. 228 Rizwana Zulfiqar, Kamran Habib, Memoona Khan, Abdul Nasir Khalid Figure 7. Phylogenetic analysis of species of Lecidella, comprised of 39 sequences. This tree has been inferred using maximum likelihood method. The bootstrap values based on 1000 replicates are shown below the branches. Sequences generated from local collection are marked with . 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. A taxonomic and phylogenetic study of some Lecidella species from Pakistan 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). 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