Lecanora chlarotera Nyl.

Lecanora chlarotera Nyl., Flora, Regensburg 55 (35): 550 (1872) Fig. 5 Description. Thallus crustose, verrucose-areolate to rimose-areolate, up to 1 cm across, indeterminate, continuous, irregular to orbicular, esorediate. Areoles: verruculose, smooth to rough, warty. Upper surface: greenish-white to yellowish-grey, smooth to rough. Prothallus: absent. Algal layer: 100–130 µm thick, even, continuous, cells chlorococcoid, spherical to sub-spherical, 6–15 µm in diameter. Apothecia: lecanorine, frequent, scattered to aggregated, sessile, epruinose. Disc: orange to reddish-brown, smooth, concave to slightly convex, rounded, 0.3–0.8 mm in diameter, epruinose to slightly pruinose. Margin: distinct, entire, smooth, thick, concolorous to thallus, persistent, continuous. Thalline exciple: well-developed. Paraphyses: hyaline, unbranched, 1.5–2 µm wide, simple, apically swollen, apex 3–4 µm wide. Epihymenium: reddish-brown to brownish-orange, 6–18 µm tall. Hymenium: hyaline, 70–100 µm tall. Hypothecium: hyaline, 25–42 µm tall. Asci: Lecanora - type, hyaline, thin-walled, sub-cylindrical to clavate, 8 - spored, 60–78 × 10–15 µm. Ascospores: ovoid to broadly ellipsoidal, hyaline, simple, aseptate, 12–15 × 6–9 µm. Chemical study. K + ve (yellow), C - ve, KC - ve. Atranorin was detected through TLC. Phylogenetic analysis. For phylogenetic analysis, sequences of the genus Lecanora were retrieved from GenBank based on their similarity to our sequences. A total of 48 ITS nrDNA sequences were analyzed during this study, including four newly generated sequences. The final aligned file was comprised of 559 nucleotides, among which 285 were conserved, 268 were variable, 251 were parsimony-informative, and 17 were singleton variants. Lecanora saxigena Lendemer & R. C. Harris (KP 224466 and KP 224467) was chosen as an outgroup. In our analysis, distinct clades were formed with high bootstrap values (Fig. 6). The newly generated sequences of Lecanora chlarotera (KUM- 18 and SN- 07) and Lecanora hybocarpa (FM- 22) form a well-supported clade (99 % ML) alongside Lecanora paramerae I. Martínez, Aragón & Lumbsch. The Pakistani sequences of L. chlarotera (KUM- 18 and SN- 07) cluster with sequences of the same taxon from the United Kingdom (FR 799201, FR 799204, FR 799203) and Sweden (MW 375013, MW 375016, MW 375017, MW 375018, and MW 374998), indicating they represent the same species. Additionally, the sequence of L. hybocarpa (FM- 22) groups with a sequence of the same taxon from Spain (EF 105412) with strong bootstrap support (89 % ML). The sequence of the Pakistani collection (K- 165) forms a clade with Lecanora horiza sequences reported from North Macedonia (OQ 717441), Switzerland (KX 132998), and the USA (MG 554692), indicating they represent the same species. L. horiza is closely related to L. campestris (Schaer.) Hue, L. allophana (Ach.) Nyl., and L. glabrata (Ach.) Nyl., forming a clade with high bootstrap support (82 % ML). Notes. Lecanora chlarotera is a common epiphytic species within the L. subfusca complex, widespread in Europe and North America, growing on nutrient-rich bark from lowlands to mountainous regions (Bolognini 1992). The morpho-anatomical characteristics of the Pakistani specimen agree with the published description of L. chlarotera from the Czech Republic, except slightly smaller apothecia 0.3–0.8 mm (vs. 0.5–1 mm) and smoother apothecial margins (vs. smooth to crenate) (Malicek 2014). L. chlarotera is both morphologically and phylogenetically related to L. hybocarpa (Tuck.) Brodo, but the latter one has larger apothecial discs and yellow-white to grey thallus coloration (Cannon et al. 2022). Phylogenetically, L. chlarotera is also closely related to L. paramerae I. Martínez, Aragón & Lumbsch but differs in having smaller apothecia 0.3–0.8 mm (vs. 1–3 mm), pruinose apothecial disc (vs. epruinose) and taller hymenium 70–100 µm (vs. 60–70 µm) (Martínez et al. 1999). Habitat and ecology. The sample (KUM- 18) was found at an elevation of 2,500 m, growing on a bark in a cold, temperate mountainous region. The coldest month in this region is December, while June is the hottest month of the year, with average monthly temperatures of 3 ° C and 25 ° C, respectively, and rainfall varying between 1000 and 1200 mm. The second collection (SN- 07) was made from Kaghan Valley which is drained by the River Kunhar originating from Lulusar Lake. The average rainfall for spring, summer and winter is 363, 655, and 190 mm, respectively. The maximum daily temperature of the region varies from 20 ° C to 30 ° C during the summer and averages 4 ° C during the winter with moderate rainfall. Distribution. This species is widespread in the Czech Republic, Europe, Japan and Turkey (Miyawaki 1988; Öztürk et al. 2010; Malicek 2014). From Pakistan, it is reported for the first time. Materials examined. Pakistan ∙ Khyber Pakhtunkhwa Province, District Upper Dir, Kumrat Valley, 35°33.6'N, 72°12.4'E; alt. 2,500 m a. s. l.; 23 Sep. 2023; M. Usman & A. N. Khalid leg.; on bark; KUM- 18, ITS GenBank accession number PQ 113695. Pakistan ∙ Khyber Pakhtunkhwa Province, District Manshera, Kaghan Valley; 34°42.2'N, 73°26.2'E; alt. 2,454 m a. s. l.; 24 Aug. 2022; A. R. Niazi & M. Nadeem leg.; on bark; SN- 07, ITS GenBank accession number PQ 106505.