Bacidiaalbogranulosa (Ramalinaceae, lichenized Ascomycota), a new sorediate lichen from European old-growth forests

Abstract A sterile sorediate member of the genus Bacidia s.str., B.albogranulosa, is described here as a new species. It is characterised by its very thin, pale grey thallus, white, farinose to granular soredia, the production of atranorin and the absence of ascomata and pycnidia. It grows on slightly acidic to subneutral bark of broad-leaved trees in old-growth forests in the Czech Republic, Poland, Ukraine and Russia (European part of the Caucasus). The new species is well characterised by its morphology, secondary chemistry and molecular (nrITS, mtSSU) traits. It is closely related to other atranorin-containing species in the genus, Bacidiadiffracta, B.polychroa and B.suffusa.


Introduction
Bacidia De Not. (Ramalinaceae, lichenised Ascomycota) is a genus of lichenised fungi with crustose thalli, a chlorococcoid photobiont, lecideine or biatorine apothecia and multiseptate oblong to acicular ascospores (Ekman 1996). Many of the species do not produce any lichen substances detectable by TLC, but one or more pigments in the apothecial tissues are known (Ekman 1996, Coppins and Aptroot 2009, Wirth et al. 2013. Names of five acetone-insoluble pigments are derived from Bacidia s.str., i.e. Arceutinayellow, Laurocerasi-brown, Polychroa-brown, Rubella-orange and Schweinitzii-red (Ekman 1996, Meyer andPrintzen 2000). The genus Bacidia includes approximately 230 species wordwide (Lücking et al. 2016). However, many species named 'Bacidia' belong to other genera or even other families, so the diversity of Bacidia, in its strict sense, is estimated to be 60-90 species (Ekman 1996, 2001, Coppins and Aptroot 2009. During field research in old-growth forests in Europe, we repeatedly collected a sterile sorediate crust, preliminarly assigned to the genus Lecanora Ach. due to the production of atranorin. Surprisingly, molecular data placed the species into Bacidia s.str. The currently known members of Bacidia s.str., except for B. sorediata Lendemer & R. C. Harris (Lendemer et al. 2016), do not produce isidia or soredia, but the thallus of some species consists of granules that very likely have a function as vegetative propagules (Ekman 1996). The new species is related to B. diffracta S. Ekman, B. polychroa (Th. Fr.) Körb., B. rubella (Hoffm.) A. Massal. and B. suffusa (Fr.) A. Schneid., which also produce atranorin as the main secondary metabolite (Culberson andCulberson 1969, Ekman 1996). Based on morphological, chemical and molecular characters, we describe this very distinct taxon as new to science.

Sampling, morphology and chemistry
Collected specimens are deposited in KTC, PRA, UGDA and the personal herbarium of J. Malíček. Microscopic descriptions are based on hand-cut sections mounted in water. Lichen secondary metabolites were identified using thin layer chromatography (TLC) in A, B' and C solvents (Orange et al. 2010). Figures were acquired by the stereomicroscope Olympus SZX 12 with the cooled colour digital camera Olympus DP 70 (resolution 12.5 Mpx) in the software QuickPHOTO MICRO 3.0 (Promicra), using an extended depth of field module Deep Focus.

DNA extraction, PCR amplification and sequencing
The Invisorb Spin Plant Mini Kit (Invitek) and CTAB protocol (Cubero et al. 1999) were used for DNA extractions. The fungal ITS rDNA (henceforth ITS) and mitochondrial SSU (mtSSU) were amplified with the following primers: ITS1F (Gardes Table 1. GenBank accession numbers and voucher information of specimens used in this study. New sequences are indicated in bold. and Bruns 1993) and ITS4 (White et al. 1990), mrSSU1, mr SSU2R and mrSSU3R (Zoller et al. 1999). PCR reactions of nrITS and mtSSU were prepared for a 20 µl final volume containing 14 µl double-distilled water, 4 µl MyTaq polymerase reaction buffer, 0.2 µl MyTaq DNA polymerase, 0.4 µl of each of the 25 mM primers and 1 µl of the sample. Amplifications of both loci consisted of an initial 1 min denaturation at 95 °C, followed by 35 cycles of 1 min at 95 °C, 1 min at 56 °C, 1 min at 72 °C and a final extension of 7 min at 72 °C. The PCR products were visualised on a 0.8% agarose gel and cleaned with GenElute PCR Clean-Up Kit (Sigma), according to the manufacturer's protocols. In total, 5 new ITS and 8 mtSSU sequences were generated (Table 1). Two short mtSSU sequences, containing ca. 400 positions, were exluded from the final analysis.

Sequence alignment and phylogenetic analysis
The newly produced sequences were edited in BioEdit 7.2.5 (Hall 1999). The final analyses included the newly generated sequences, the most similar Bacidia sequences (identity > 90%) according to a BLASTN search (Altschul et al. 1990) in the GenBank database and sequences of chemically and morphologically similar species (B. schweinitzii (Fr. ex Tuck.) A. Schneid., B. sorediata) to demonstrate their distant position in the tree. Bacidina arnoldiana s.lat. and Toninia sedifolia (Scop.) Timdal were selected as an outgroup. The ITS and mtSSU regions were aligned separately using MAFFT 7 (Katoh and Standley 2013) with L-INS-i method (Katoh et al. 2005). Ambiguous positions were excluded from the analysis using Gblocks 0.91b (Castresana 2000), with a less stringent selection, on the Phylogeny.fr server (Dereeper et al. 2008). The final ITS alignment contained 443 positions and 29 sequences; the mtSSU alignment had 730 positions and 28 sequences. Gaps were coded in SeqState by simple coding (Simmons and Ochoterena 2000). We concatenated the alignments and inferred a phylogeny using MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001;Ronquist et al. 2012). Results of MrModeltest 2.0 (Nylander 2004) suggested the general time reversible model, including gamma-distributed rates across sites modelled with four discrete categories and a proportion of invariant sites (GTR+G+I), as the best substitution model for both regions. Each analysis was performed with two runs, each with four MCMC chains (temperature 0.05). Trees were sampled every 500 th generation. Analyses were stopped when the average standard deviation of the split frequencies between the simultaneous runs was below 0.01. To eliminate trees sampled before reaching apparent stationarity, the first 25% of entries were discarded as burn-in and the rest were used to compute a majority-rule consensus tree with Bayesian posterior probabilities for the branches.
A maximum likelihood analysis was performed using RAxML-HPC v. 8.2.10 (Stamatakis 2014) with the GTR+G+I model on the CIPRES Science Gateway (Miller et al. 2010). Non-parametric bootstrap analysis was performed with 1000 bootstrap replicates. The maximum likelihood consensus tree is not shown, but bootstrap values are indicated at branches in the Bayesian tree (Fig. 2).  Diagnosis. The species is characterised by a grey-white hypothallus or very thin thallus covered by groups of white, farinose to granular soredia or by being completely sorediate. Ascomata and pycnidia are unknown. Atranorin is the only secondary metabolite. The species occurs in old-growth forests on bark of broad-leaved trees with high bark pH (> 5).

Results and discussion
Etymology. The name refers to the white rough (granular) soredia that are often present.
Description. The thallus consists of a hypothallus (i.e. without photobiont cells) or, in some parts, a lichenised and thinly episubstratal thallus (up to 100 µm high), which is smooth or partially areolate, pustulate or granular, grey-white to grey, sorediate. A prothallus is absent or very thin and white. Soredia are not produced in clearly delimited soralia, but dispersed in groups or forming a more or less continuous layer, white or, when fresh, yellowish-white, farinose to granular, simple, (25-)35-65 µm in diam., or in consoredia up to 125 µm in diam. Soredia are enclosed by a colourless, more or less compact "wall" without projecting hyphae. The photobiont is trebouxioid, and 5-16 µm in diameter. Ascomata and pycnidia are unknown.
Distribution and ecology. The new species is reported from the Czech Republic, Poland, Russia (European part of the Caucasus) and Ukraine. It has already been published under a provisional name, Bacidia albogranulosa ined. from the Czech Republic (Vondrák et al. 2016) and the Ukrainian Carpathians .
Bacidia albogranulosa grows abundantly in old-growth floodplain and scree forests in the Czech Republic and old-growth ash or hornbeam dominated broad-leaved forests in Poland. It rarely occurs in old-growth beech (Ukraine) and mixed forests (Russia). It has usually been found on a dry and coarse bark of broad-leaved trees with a relatively high bark pH (approximately > 5). The most frequent phorophytes are Acer campestre (n=5), A. platanoides (11) and Fagus orientalis/sylvatica (4; overmature or dying trees due to a fungal infection). A few specimens were recorded also on Fraxinus angustifolia (2), F. excelsior (2), Carpinus orientalis (1), Euonymus europaeus (1) and Quercus sp.
(2). The species prefers rather shaded trunks and places not directly exposed to rain, similar to many Lepraria species (Saag et al. 2009).
Alyxoria varia (Pers.) Ertz & Tehler, Bacidia rubella and the non-lichenised fungus Dendrothele acerina (Pers.) P.A. Lemke (on Acer spp.) are the most commonly recorded, co-occurring species. In the Czech Republic, the new species was repeatedly found on weathered bark with the red-listed Gyalecta flotowii Körb. or G. ulmi (Sw.) Zahlbr. It co-occurred also with Acrocordia gemmata (Ach. Phylogeny. The new species is strongly supported as a distinct clade in the ITS and mtSSU phylogeny (Fig. 2) and belongs to Bacidia s.str. sensu Ekman (2001). According to the ITS data, it is closely related to Bacidia diffracta, B. suffusa and B. polychroa. These four species form a well supported group, characterised by the presence of the pigments Laurocerasi-brown and Polychroa-brown in the apothecia. Bacidia albogranulosa is also related to B. rubella, a species it frequently co-ocurrs with. The only sorediate member of Bacidia s.str., the North American B. sorediata, seems not to be closely related to the new species, based on the ITS and mtSSU sequence data (Fig. 2).
Notes. Although apothecia and pycnidia are unknown, B. albogranulosa can be recognised in the field by its white-grey hypothallus or very thin thallus covered by groups of white to yellowish-white soredia that often extend across the entire thallus. Ecologically, the species prefers trees with rough and slightly acidic or subneutral bark in old-growth forests.
Bacidia albogranulosa may macroscopically resemble some Lepraria species or poorly developed Phlyctis argena (Ach.) Flot., but it clearly differs by having a noncontinuous, locally developed thallus, composed of dispersed granular aggregates that disintegrate into soralia at an early stage and by the lack of a fibrous prothallus. Additionally, atranorin alone is not known from any described Lepraria species (Saag et al. 2009). Similarly, sorediate European Lecanora species contain other substances in addition to atranorin, such as aliphatic acids, depsides/depsidones or terpenoids and usually form thicker thalli or at least a distinct fibrous hypothallus . A slightly similar appearance is typical for a few other Lecanora species (e.g. L. compallens Herk & Aptroot, L. stanislai Guzow-Krzem., Łubek, Malíček & Kukwa), producing usnic acid and zeorin and forming a yellowish-greenish to greenish-grey sorediate thallus (Guzow-Krzemińska et al. 2017).
Initial stages of the new species may resemble sterile thalli of Caloplaca substerilis Vondrák, Palice & van den Boom. This taxon lacks atranorin and tends to form thin areolate-squamulose, almost evanescent thalli with occassional sulcate or marginal soralia (Vondrák et al. 2013). The closely related species B. diffracta produces a similar, finely granular grey thallus and contains atranorin in addition to traces of zeorin. Nevertheless, this species is richly fertile, has larger thalline granules (40-100 µm diam.) and is so far only known from eastern North America (Ekman 1996). The only presently known sorediate member of Bacidia s.str., B. sorediata, differs in having a better developed, grey-green to dark green thallus, diffuse, rarely confluent soralia and fine soredia. It occurs only in south-eastern North America (Lendemer et al. 2016) and it is not phylogenetically closely related to B. albogranulosa (Fig. 2).