New records of the genus Ramalina (Lecanorales, Ascomycota) in Dagestan with a key to species

Summary . Based on morphological, chemical and molecular evidences, Ramalina europaea , R. lacera , and R. obtusata are reported for the first time for Dagestan. Ramalina lacera is the first confirmed record for the Russian part of the Caucasus and the second one for Russia. ITS of nuclear ribosomal DNA barcodes for Caucasian specimens of R. lacera and R. obtusata are generated for the first time. Specimen descriptions for firstly revealed species are presented. A key for identification of the 13 known taxa of the genus Ramalina in Dagestan is provided.


Introduction
The genus Ramalina Ach. is a globally distributed genus with large ecological amplitude (found also in Antarctica) and currently combined up to 230 taxa.It is included in 50 largest genera of lichenized fungi being the 18th most diverse (Lücking et al., 2016).In general, the genus is characterized by fruticose Ismailov A. B., Volobuev S. V., Kataeva O. A. New records of the genus Ramalina (Lecanorales, Ascomycota) in Dagestan with a key to species thallus with flat to cylindrical, solid to hollow branches, often with pseudocyphellae, hyaline 1-septate ascospores and producing usnic acid (Smith et al., 2009).
In the work "A checklist of the lichen flora of Russia" (Urbanavichus, 2010) this genus is represented by 39 species, of them 18 species are recorded for the Russian Caucasus.Among them, R. elegans (Bagl.et Carestia) Jatta, R. montana Barkh., and R. panizzei De Not. are known within Russia only from the Caucasus.Later, in virgin mixed coniferous-broad-leaved forest in the Western Caucasus R. subgeniculata Nyl. was found for the first time for Russia (Urbanavichus, Urbanavichene, 2014).Further studies on lichens in different Russian regions and taxonomic studies have expanded the list of Ramalina to 44 species (Kataeva, 2014;Gasparyan et al., 2017;Davydov, Yakovchenko, 2023).
During the fieldwork as continuing studies of lichens in broad-leaved forests (Ismailov et al., 2017;Ismailov, 2020) at lowland and in the foothills of Dagestan we found several corticolous specimens of the genus Ramalina.Determination of taxonomic status of specimens revealed new species for Daghestan.In this paper, we provide detailed information on new records with a key for all Ramalina taxa occurring in Dagestan.

Materials and Methods
Light microscopy (LOMO MSP-2 microscope), chemical spot tests and UV light were used for specimen investigations.The secondary metabolites of species were identified using high performance thin layer chromatography (HPTLC) in solvent system A according to the methods summarized by Arup et al. (1993).Camag glass plates with F254 layer were used.
Genomic DNA was amplified directly from thalli pieces of dried Ramalina specimens with the Phire Plant Direct PCR Master Mix Kit (Thermo Fisher Scientific, Lithuania) without prior DNA purification according to the manufacturer's instructions.The internal transcribed spacer regions of the nuclear ribosomal DNA (ITS) were amplified with the primers pair ITS1F/ITS4 (White et al., 1990;Gardes, Bruns, 1993).Products of amplification were visualized by 1 % agarose gel electrophoresis, and then purified using the CleanMag DNA (Evrogen, Russia) purification kit.Sequencing was performed on an ABI model 3500 Genetic Analyzer (Applied Biosystems, Foster City, USA).Raw data were edited and assembled in MEGA X (Kumar et al., 2018).The newly obtained sequences were deposited in GenBank.
The sequences were aligned with ten additional sequences retrieved from GenBank database (Table ), using a MAFFT version 7 web tool (Katoh et al., 2019) with the E-INS-I option.The maximumlikelihood phylogenetic analysis using the IQ-TREE web server (Trifinopoulos et al., 2016) with 1000 ultra-fast bootstrap repeats was performed to identify the phylogenetic position of newly sequenced specimens.

Results and discussion
Based on morphological, anatomical and chemical identification of specimens supported by molecular analysis results, three Ramalina species new to Dagestan -R.europaea Gasparyan, Sipman et Lücking, R. lacera (With.)J. R. Laundon, and R. obtusata (Arnold) Bitter -have been revealed.For R. obtusata, we notice a minor concentration of obtusatic acid as a result of not favourable habitat conditions.
Molecular study on Ramalina species has been carried out using data on ITS nrDNA sequences.In this research, two complete ITS sequences for Ramalina lacera and R. obtusata were generated based on collections made in the Republic of Dagestan.Based on a megablast search of NCBIs GenBank nucleotide database, our R. lacera sequence showed 100 % identity (97 % query coverage) with all of 18 ITS sequences of R. lacera originated from Spain, and our R. obtusata sequence had 100 % identity (98 % query coverage) with OK333014 (voucher PRA-JV24089) from Czech Republic.
For phylogenetic analysis, a total of nine nrITS reference sequences of Ramalina species presented in recent molecular studies (Gasparyan et al., 2017;Kistenich et al., 2018;Marthinsen et al., 2019;Garrido-Benavent et al., 2022;Vondrák et al., 2022) and the sequence of Niebla robusta (R. Howe) Rundel et Bowler as an outgroup (GU827336) were chosen.The final dataset contained 12 nrITS sequences and consisted of 852 characters, including gaps.Tree topology obtained using Maximum Likelihood method for phylogenetic analysis is presented in Fig. 1.Our nrITS phylogeny supports separate clades corresponded to different Ramalina morphological species.Among them, there are two well-supported lineages Ramalina lacera and R. obtusata that include sequences originated from the Russian part of the Caucasus and generated for the first time from Ramalina specimens collected in riparian forests of the Republic of Dagestan on the territory of Samurskiy National Park.These molecular data are congruent to morphological and ecological features of species revealed.

Ramalina europaea
Description: thallus corticolous, fruticose, yellowish-green to green (greyish green in herbarium), erect to subupendulous, up to 3 cm long and 4 cm wide.Lobes flattened, up to 3 mm wide and 1-2 cm long, solid, developing numerous tiny, irregular, sometimes spine-like lateral lobules, on top of which developing soralia as punctiform, granular structures.Soralia lateral and terminal, never  Distribution: species with Eurasian distribution.Widespread in Europe.Easternmost locality is known from the south of the Ural Mountains (Gasparyan et al., 2017).Also common in broadleaved forests of the Caucasus.
Notes: From close Ramalina pollinaria it differs by the punctiform soralia borne terminally on tiny  spine-like, lateral branches, and by the granular soredia.Our specimens are close in morphology to other Caucasian and Russian specimens summarized in study describing this species (Gasparyan et al., 2017).
Distribution: mainly Mediterranean-Atlantic species with broad but somewhat spotted distribution.Known from Europe, west of Asia, Africa, southwest of North America, South America (Gumboski et al., 2014).For Russia and the Caucasus, R. lacera is known from several references.Within Russia previously reported only from Crimea (Krasnaya kniga, 2015).Doubtful record of this species from Western Caucasus (Krivorotov, 1997), because the report was not confirmed by the specimen.For the East Caucasus R. lacera is known only by our record.
Notes: R. lacera is polymorphic species with a wide ecological amplitude.It is growing on trees and rocks in areas with a humid-warm climate, in rather dry, well-lit sites of manmade habitats, with strong coastal affinities (Gumboski et al., 2014;Nimis, 2016).Habitus variates from deep lacerate to more or less foliose-fruticose forms.Our specimens with a deep lacerate thallus grew as epiphyte in habitats with sparse vegetation along the forest roads and in manmade pine plantings on the Caspian Sea coast.
Description: Thallus fruticose, greenish grey, shrubby, 1 cm high and 2 cm wide, growing from a narrow holdfast.Branches several arising from the base.Laciniae fistulose, inflated, ridged, foveolate, with some side branches.Medulla hollow.Soralia common, labriform to helmet-shaped, developed within terminal or subterminal vesicles.Soredia farinose.Apothecia not observed in Dagestan material.Photobiont chlorococcoid.Spot tests: all negative.Distribution: a cool-temperate to southern boreal species widespread in Europe and North America (Walker, Pystina, 2005) and quite common in Russia (Urbanavichus, 2010).Location in Dagestan is the southernmost in the European part of Russia.
Notes: Ramalina obtusata mainly grows in wellpreserved humid boreal forests, more rarely in coldmoist, open montane forests.Probably, our record from dry, lowland conditions is extreme habitat for the species.This is also indicated by the small size of the thallus of our specimen and minor concentration of obtusatic acid.R. obtusata is morphologically and chemically similar to close R. baltica, but the latter one doesn't occur within the Caucasus.
Including new records presented in this paper, the genus Ramalina in Dagestan comprises 13 taxa.The key for all taxa known in Dagestan is given below.+ Thallus 2-7 cm long, erect to pendent.Lobes often strongly or slightly branching, flat, rather thin, with outgrowths along the edges, lower surface with protruding ribs and slit-like white pseudocyphellae between them.Apothecia terminal or subterminal.Spores broad ellipsoid 12-14 × 4-6 μm ... R. sinensis

An identification key to
Ismailov A. B., Volobuev S. V., Kataeva O. A. New records of the genus Ramalina (Lecanorales, Ascomycota) in Dagestan with a key to species becoming large and labriform.Soredia granular.Medulla white, compact.Apothecia not observed in Dagestan material.Photobiont chlorococcoid.Spot tests: all negative.Substrate and ecology: on trunk of Fagus orientalis Lipsky in broad-leaved beech-hornbeam forest in the gorge of the Akhtichay River.Secondary metabolites: usnic and evernic acid.Specimen examined: "Russia, Republic of Dagestan, Kazbekovskiy district, vicinity of Almak village, beech forest in gorge of the Akhtichay River, on Fagus orientalis, 950 m above the sea level.42°57ʹ51ʺN, 46°34ʹ53ʺE.08 V 2018. A. B. Ismailov" (DAG 1435).

Fig. 1 .
Fig. 1.Phylogenetic position of ITS of nrDNA Ramalina sequences newly generated in this study (in bold face) based on Maximum Likelihood method.Bootstrap support values are shown above branches (BS ≥ 75 %).The scale bar represents the expected number of nucleotide changes per site.

Table
Voucher information and GenBank accession numbers for ITS sequences used in this study