Introduction

According to recent phylogenetic studies, Megasporaceae Lumbsch is monophyletic (Nordin & al. 2010). They are mostly saxicolous crustose lichens (Valadbeigi & al. 2011). In Armenia, they are among the more common lichen families, in species diversity but especially in abundance, covering large parts of most siliceous rock faces and also present on limestone. The genus Megaspora (Clauzade & Cl. Roux) Hafellner & V. Wirth is closely related to the genus Circinaria Link (Nordin & al. 2010). It is an exception within the family, in that it is predominantly corticolous, with two species on trees, one of which is also occasionally terricolous. Both currently accepted species, M. rimisorediata Valadbeigi & A. Nordin and M. verrucosa (Ach.) Hafellner & V. Wirth (Valadbeigi & al. 2011), occur in Armenia (Gasparyan & Sipman 2013; Harutyunyan & al. 2011).

During a lichenological excursion to Armenia, organized by the second author, we collected a sorediate crustose lichen at the bases of trees of Juniperus polycarpos K. Koch in the Khosrov Forest State Reserve. The territory of the Reserve was already considered as a protected area in the fourth century C.E. by the Armenian king Khosrov Kotak (Khanjyan 2004). In 1958, the Khosrov Forest was officially declared as a state reserve (Anonymous 2008). The natural landscapes of phryganoid vegetation, open arid forests and montane steppes have high biological diversity and are recognized as a priority area for conservation. So far, 1849 species of vascular plants (including 24 endemic species) and 176 lichenized and lichenicolous fungi have been registered in the reserve (Anonymous 2008; Gasparyan & al. 2015).

While in the field it was not possible to recognize the collected specimens as representatives of Megasporaceae; rather they gave the impression of a species of the Caloplaca albolutescens (Nyl.) H. Olivier / C. teicholyta (Ach.) J. Steiner group or, less likely, a species of Lepraria Ach., but subsequent examination of the material revealed a few black apothecia immersed in the thallus, with large, thin-walled ascospores and a greenish epihymenium, suggesting Megasporaceae.

In the framework of a phylogenetic study of Asian Megasporaceae, the first author sequenced the material and found that it clusters inside Megaspora as a sister species to M. rimisorediata. Therefore, we describe it as a new species in this genus.

Megaspora rimisorediata has a restricted distribution. It was described from Iran (Valadbeigi & al. 2011) and later found also in S Armenia (Gasparyan & Sipman 2013; Gasparyan & al. 2015). Megaspora verrucosa has been reported from Europe, Africa, Asia, North and South America, New Zealand and Antarctica (Smith & al. 2009).

Currently, Armenia is the centre of diversity of the genus, with all three currently known species present. The new species has been reported from two localities. Further comprehensive studies are required to explore distributional and ecological patterns of the new species.

Material and methods

Identification and descriptive work was carried out in Soest and BGBM using an Olympus SZX7 stereomicroscope and an Olympus BX50 compound microscope with interference contrast, connected to a Nikon Coolpix digital camera. Sections were mounted in tap water, in which also all measurements were taken. The specimens from this study are preserved in ABL and B (herbarium codes after Thiers 2016+). The chemistry of the type specimen was investigated by thin-layer chromatography (TLC) using solvent A (Orange & al. 2001).

DNA extraction — We used nuclear ITS 1-5.8S-ITS2rDNA sequences of specimens in the molecular study because it has been shown that among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for a range of fungi (Schoch & al. 2012; Divakar & al. 2015). Total DNA was extracted from freshly collected material according to Park & al. (2014). We followed the instructions given in that paper except for the following steps: we used a 1 × 1 mm² piece of medulla and mixed it with beadbeader without liquid nitrogen; instead of chloroform we used Roti®-C/I (chloroform/isoamy 1 alcohol at a ratio of 24:1); and at the end we used only 30 µL TE buffer instead of 100 µL because of the low quantity of DNA.

PCR amplifications and sequencing — The primer pair ITS1F (Gardes & Bruns 1993) and ITS4 (White & al. 1990) was used for the PCR amplifications. PCR amplifications were performed in a 12.5 µL volume containing 2 µL undiluted DNA, 0.5 µL of each primer (10 mM), 6.4 µL of sterile water, 1 µL dNTP (2 mM), 1 µL s-buffer, 1 µL MgC12, 0.1 µL Taq-polymerase. Thermal cycling parameters were initial denaturation for 5 min at 95 °C, followed by 30 cycles of 30 sec at 95 °C, 30 sec at 54 °C, and 1 min at 72 °C; following the last cycle a final extensions for 3 min at 72 °C was included. Amplification product was viewed by electrophoresis on 1% agarose gels and stained with ethidiumboromide and was purified by adding 2 µL ExoSAP-IT™ (Exonuclease 1-shrimp alkaline phosphatase) to 5 µL of the PCR products, followed by a heat treatment of 15 min at 37 °C and 15 min at 80 °C. The PCR product was sequenced in both directions by Bik-F Laboratory in Frankfurt am Main. For the reconstruction of a phylogenetic tree, all ITS sequences of Megasporaceae from Valadbeigi & al. (2011) were used as well as seven accessible sequences of Megaspora from NCBI GenBank ( http://www.ncbi.nlm.nih.gov/genbank/). Two sequences were obtained from the new species and submitted to the NCBI GenBank (Table 1). The sequences were aligned through the Muscle V4 program web server (Edgar 2004) with the default settings. The aligned sequences were adjusted manually in PhyDE software (Müller & al. 2010). Gblocks 0.91b ( http://molevol.cmima.csic.es/castresana/Gblocks_server.html) was used to eliminate ambiguously aligned positions, applying settings allowing for smaller final blocks, gap position within the final blocks and less strict flanking position (Castresana 2000).

Phylogenetic analyses — MrModeltest (Nylander 2004) was used to determine the most appropriate model using AIC, with GTR + I + G found to be the best-fitting model of nucleotide evolution. Bayesian inference of phylogeny with Markov chain Monte Carlo sampling was performed on the Bayesian inference of phylogeny with Markov chain Monte Carlo sampling was performed on the 477 unambiguously aligned nucleotide positions. Bayesian analyses were conducted with Mr-Bayes v. 3.2.2 (Ronquist & Huelsenbeck 2003) using the GTR model of nucleotide substitution including a proportion of invariable sites and a discrete gamma distribution with six rate categories. Two independent runs, each with four Metropolis-Coupled Markov Chain Monte Carlo chains and a temperature of 0.2 were initiated and run for 1 000 000 generations, with tree and parameter sampling every 100 generations. Burn-in was set to discard 25 % of samples. Maximum parsimonious trees (MPs) were reconstructed in PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2003) using the heuristic search option with 100 random sequence additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm. Alignment gaps were treated as missing and all characters were unordered and of equal weight. The robustness of the trees obtained was evaluated by 1000 bootstrap replications with ten random sequence additions. Molecular Evolutionary Genetics Analysis software (MEGA version 7.0) was used to reconstruct the Maximum Likelihood phylogenetic tree based on the GTR + I + G model (Nei & Kumar 2000; Kumar & al. 2016). Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A GTR model of nucleotide substitution including a proportion of invariable sites and a discrete gamma distribution with five rate categories (GTR + I + G) were used in Maximum Likelihood approach.

Table 1.

Voucher specimens and NCBI GenBank accession numbers of the ITS sequences used in the phylogenetic analyses.

Table 2.

The main distinguishing characteristics of Megaspora cretacea, M. rimisorediata and M. verrucosa.

Results

Phylogeny

The maximum parsimony analysis resulted 12 most parsimonious trees with 513 steps, consistency index (CI) = 0.591, retention index (RI) = 0.690, rescaled consistency index (RC) = 0.407 and homoplasy index (HI) = 0.409. The Maximum Likelihood analysis resulted a tree with the highest log likelihood (-2014.3274). Majority rule consensus tree for maximum parsimony analysis was congruent with the tree obtained by Bayesian and maximum likelihood phylogenetic inference. The majority rule consensus tree of Bayesian analysis is shown here (Fig. 1 ) with posterior probabilities of Bayesian analysis and bootstrap numbers of Maximum Parsimony and Maximum Likelihood analysis.

Fig. 1.

Phylogenetic relationships of family Megasporaceae showing the consensus tree of the Bayesian analysis of the ITS dataset. Bayesian posterior probabilities are shown above the branches and MP/ML bootstrap values ≥ 50 are shown below the lines adjacent to the branches. Distance of outgroup and ingroup root is shortened three times.

The molecular phylogenetic results confirmed affiliation of the new species to the genus Megaspora. It is clusters in a phylogenetic tree in Megaspora, as sister to M. rimisorediata (PP = 1; MP/ML BS= 100/100). The phylogenetic trees resulting from the three different analyses also confirmed Megaspora clade as a monophyletic group even after adding the new species samples (M. cretacea) with a high posterior probability and bootstrapping values (PP = 1; MP/ML BS = 99/99). Monophyly of species M. verrucosa and M. rimisorediata were confirmed with a high supporting values (PP = 1; MP/ML BS = 100/100 for M. verrucosa and PP = 0.94; MP/ML BS = 99/98 for M. rimisorediata).

Taxonomy

Megaspora cretacea Gasparayan, Zakeri & Aptroot, sp. nov. — MycoBank #817072 — Fig. 2A–C.

Holotype: Armenia, Ararat, Vedi, Urtsadzor, Khosrov Forest State Reserve, 40°00′42″N, 44°54′04″E, 1600 m, on Juniperus polycarpos bark, 17 Jun 2015, A. Aptroot 73835 (B 600200932; isotypes: ABL, GLM).

Diagnosis — Megaspora with thallus whitish grey, cretaceous, fully sorediate with soredia c. 0.1 mm in diam.; apothecia sparse, immersed; ascospores 4 per ascus, broadly ellipsoid, 27–31 × 18–21 µm, hyaline, thin-walled.

Description — Thallus whitish grey, crustose, ecorticate, to 0.2 mm thick, irregularly delimited to almost lobate, occupying areas up to 5 cm in diam. Medulla white, cretaceous. Soralia covering most of thallus surface, pale bluish grey; soredia c. 100 µm in diam. Photobiont chlorococcoid. Apothecia sparse, dispersed, immersed in thallus, round, 0.3–0.5 mm in diam.; disc black, concave; margin black, raised above disc, incurved, c. 0.1 mm wide, with some crenations. Hymenium IKI+ blue, c. 150 µm high, not inspersed with oil droplets. Subhymenium hyaline. Epihymenium greenish, colour unchanged in KOH. Hypothecium hyaline. Paraphyses 2–2.5 µm thick, not branched. Asci clavate, 125–140 × 25–31 µm. Ascospores 4 per ascus, broadly ellipsoid, 27–31 × 18–21 µm, hyaline, thin-walled (less than 1 µm). Pycnidia not observed. Conidia not observed.

Fig. 2.

A–C: Megaspora cretacea, holotype; A: thallus with soredia and apothecia; B: ascospore; C: hymenium (with excipulum at left). — D: M. verrucosa thallus with apothecia. — E: M. rimisorediata thallus with net of cracks and soredia. — Scale bars: A, D, E = 2 mm; B, C = 20 µm.

Chemistry — Thallus KOH-, C-, Pd-, UV-. TLC: No lichen substances detected.

Distribution and ecology — The species is known from two separate localities within the Khosrov Forest State Reserve, Armenia. It occurs on bases of trees of Juniperus polycarpos K. Koch in arid, open, montane forests. The forest ecosystems in the Khosrov Forest State Reserve, at 1400–2300 m, are generally dominated by oak trees (Quercus macranthera Fisch. & C. A. Mey. ex Hohen.) and sparse juniper (J. polycarpos) formations, accompanied by Fraxinus excelsior L., Sorbus aucuparia L., and species of Acer L. and Pyrus L. (Khanjyan 2004).

Etymology — The epithet is derived from word cretaceus (resembling chalk) in reference to the colour and texture of the thallus.

Additional specimen examined—Armenia: Ararat, Vedi, Urtsadzor, Khosrov Forest State Reserve, 39°59′07″N, 44°53′51″E, 1390 m, on Juniperus polycarpos bark, 17 Jun 2015, A. Gasparyan (B 600199170).

Discussion

Megaspora cretacea is a morphologically distinctive species, from which the two other species of the genus, M. verrucosa (Fig. 2D) and M. rimisorediata (Fig. 2E), can be separated as follows (Table 2): M. verrucosa has no soredia, whereas M. cretacea and M. rimisorediata are both sorediate; the closely related M. rimisorediata differs from M. cretacea by the presence of a dense net of elongate cracks over the thallus, dark bluish green soredia, branched paraphyses and larger ascospores.