Introduction
The genus Biatora Fr. is a quite diverse group of predominantly epiphytic crustose lichens with biatorine apothecia and narrowly ellipsoid to bacilliform pale ascospores. In his revision, Printzen (Reference Printzen1995) used the ontogeny of apothecia as a useful criterion to delimit Biatora from similar looking taxa, such as Mycobilimbia Rehm. Almost two decades later, Printzen (Reference Printzen2014) performed a phylogenetic revision of the group (using three molecular markers: the internal transcribed spacer region of the nuclear rDNA (ITS), RNA polymerase II gene (RPB2) and the small subunit of the mitochondrial rDNA (mtSSU)) and the genus Biatora was emended by the inclusion of several species formerly classified in other genera. The lumping trend has continued by including Ivanpisutia oxneri S. Y. Kondr. et al. and the polysporic Myrionora albidula (Willey) R. C. Harris into Biatora (Kistenich et al. Reference Kistenich, Timdal, Bendiksby and Ekman2018).
In its current circumscription, the genus is almost impossible to define by morphological characters alone. Even some features formerly believed to be characteristic for Biatora, such as the Biatora-type of asci, were recently rejected as phylogenetically useful synapomorphic characters based on molecular evidence. For example, the species Biatora ligni-mollis T. Sprib. & Printzen has asci approaching the Micarea-type (Spribille et al. Reference Spribille, Björk, Ekman, Elix, Goward, Printzen, Tønsberg and Wheeler2009). One of the best features to characterize the genus is the heavily gelatinized chondroid exciple in which individual hyphae are hardly discernible, a character that may be connected with the specific mode of apothecial ontogeny (Printzen Reference Printzen1995).
Printzen (Reference Printzen2014) mentioned 42 species for the genus Biatora, among which several undescribed taxa were noted, and this number was followed in the last generic classification of lichens by Lücking et al. (Reference Lücking, Hodkinson and Leavitt2017). Further additions were recently published by Printzen et al. (Reference Printzen, Halda, McCarthy, Palice, Rodriguez-Flakus, Thor, Tønsberg and Vondrák2016), Kistenich et al. (Reference Kistenich, Timdal, Bendiksby and Ekman2018), Ekman & Tønsberg (Reference Ekman and Tønsberg2019) and Spribille et al. (Reference Spribille, Fryday, Pérez-Ortega, Svensson, Tønsberg, Ekman, Holien, Resl, Schneider and Stabentheiner2020). However, the exact number of species is hard to determine because some authors have recently started to simultaneously split off new genera from, and combine taxa from, outside Biatora into the genus based on largely unsupported phylogenies (Kondratyuk et al. Reference Kondratyuk, Lőkös, Farkas, Jang, Liu, Halda, Persson, Hansson, Kärnefelt and Thell2019). It is clear, however, that numerous taxa still await descriptions. One of these so far unrecognized species is described here. It has been known to the authors since 2015, and was listed as ‘Biatora amylacea ined.’ in two previous studies (Vondrák et al. Reference Vondrák, Malíček, Palice, Bouda, Berger, Sanderson, Acton, Pouska and Kish2018; Urbanavichus et al. Reference Urbanavichus, Vondrák, Urbanavichene, Palice and Malíček2020). In the former work, the most distinctive and diagnostic features of this taxon were summarized in the supplementary material of the paper. Mitochondrial SSU and nuclear ITS sequences were generated (in GenBank under Biatora sp.) in the latter publication for a Caucasian specimen of this species.
Material and Methods
Microscopic examinations were made on hand-cut sections and squash preparations mounted in water, Lugol's solution or lactophenol cotton blue (LCB) using a Zeiss Axioskop 2 equipped with a Zeiss Axiocam 305 for imaging, or using an Olympus CX41 and SZ12, the latter equipped with an Olympus DP70 camera for imaging. External features were studied through a Zeiss Stemi 11 and an Olympus BX60 dissecting microscopes. Habit images were made with a Zeiss Axiozoom V16 or using the Olympus DP70 camera's extended depth of field module, Deep Focus. Vouchers are deposited in BG, FR, PRA and the private herbarium of J. Malíček. Colour reactions of acetone-insoluble pigments in apothecia or prothalline hyphae were observed after application of a 10% aqueous solution of potassium hydroxide (K), a 50% aqueous solution of nitric acid (N) and a 10% aqueous solution of hydrochloric acid (HCl). Ascospore measurements are given as (smallest single measurement–) smallest mean-largest mean (–largest single measurement), (n = number of measurements). The values have been rounded to the nearest 0.5 μm. Thin-layer chromatography (TLC) was used for detection of secondary lichen metabolites, using solvents A, B′ and C (Orange et al. Reference Orange, James and White2010).
To check whether the new species belonged to Biatora and find its closest relatives within the genus, a phylogenetic analysis was carried out using five samples, the data set used by Printzen (Reference Printzen2014) with the exception of Cliostomum and three species (Biatora chrysantha (Zahlbr.) Printzen, B. ementiens (Nyl.) Printzen and B. printzenii Tønsberg) responsible for conflicts among data sets, and a small number of additional sequences of species that were formerly not available for analysis (Table 1). DNA was extracted using either the Invisorb Spin Plant Mini Kit (Invitek) or a Chelex protocol (Ferencová et al. Reference Ferencová, Rico and Hawksworth2017). The following primers were used for amplification of the internal transcribed spacer region of the nuclear ribosomal DNA and the small subunit of the mitochondrial ribosomal DNA: ITS1F (Gardes & Bruns Reference Gardes and Bruns1993) and ITS4 (White et al. Reference White, Bruns, Lee, Taylor, Innis, Gelfand, Sninsky and White1990), mrSSU1, mrSSU2R, mrSSU3R (Zoller et al. Reference Zoller, Scheidegger and Sperisen1999) and MSU7 (Zhou & Stanosz Reference Zhou and Stanosz2001). PCR conditions followed protocols described in Printzen (Reference Printzen2014) and Malíček et al. (Reference Malíček, Berger, Palice and Vondrák2017). Newly generated DNA sequences were submitted to GenBank (Table 1).
Voucher information and GenBank Accession numbers for collections used for phylogenetic analyses. Accession numbers in bold indicate newly generated sequences.
Alignments of the single-locus data sets were created using an online application of MAFFT v. 7 (Katoh et al. Reference Katoh, Rozewicki and Yamada2019; https://mafft.cbrc.jp/alignment/server/) with default settings (‘Auto’ strategy, aligning gappy regions and the default guide tree). Based on these alignments, regions of uncertain alignment were identified using the GUIDANCE2 server (http://guidance.tau.ac.il; Landan & Graur Reference Landan and Graur2008; Penn et al. Reference Penn, Privman, Ashkenazy, Landan, Graur and Pupko2010; Sela et al. Reference Sela, Ashkenazy, Katoh and Pupko2015). Regions with GUIDANCE scores < 0.93 were removed from the analyses. Single-locus maximum likelihood (ML) trees were reconstructed using the IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/; Trifinopoulos et al. Reference Trifinopoulos, Nguyen, von Haeseler and Minh2016) with default search parameters (perturbation strength 0.5, stopping the analysis if no better tree was found after 100 random perturbations). The optimal substitution models and partitioning schemes for these data sets were inferred by IQ-TREE based on the Bayesian information criterion. For ITS, we suggested separate character sets for ITS1, 5.8S rRNA and ITS2. Branch support values were calculated using the ultrafast bootstrap algorithm with 1000 replications. Single-locus trees were checked for well-supported topological incongruencies. Since none were found, data sets were concatenated and an ML bootstrap tree reconstructed, this time suggesting four separate character sets (the above mentioned plus mtSSU; see Table 2). Branch support values for the concatenated data set were calculated using the standard bootstrap algorithm with 100 replications, the maximum possible on the web server.
Partitioning scheme and substitution models used in the maximum likelihood (ML) and Markov chain Monte Carlo (MCMC) analyses.
A Bayesian phylogeny was reconstructed using MrBayes v. 3.2.7 (Ronquist et al. Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012). We used the partitioning scheme inferred by IQ-TREE and, because the optimal models were not implemented in MrBayes, simplified substitution models as outlined in Table 2. Model parameters were unlinked between partitions. The mean of the branch length prior was inferred as outlined in Printzen (Reference Printzen2014), based on branch lengths in the standard bootstrap ML tree. MrBayes was set to sample every 200th tree out of 40 M generations using two independent runs, each with four chains that were incrementally heated by a factor of 0.15. To infer convergence of the Markov chains, the average standard deviation of bipartition frequencies among runs was calculated every 100 000th generation, discarding the first 50% of the sampled trees as burn-in and including only those bipartitions with a frequency of at least 10%. The analysis was stopped after 3.5 M generations when the standard deviation had dropped below 0.01.
Results
Phylogeny
The concatenated alignment comprised 1277 bp (ITS: 433, mtSSU: 844). Overall support for the phylogenetic tree is low, probably due to the low number of gene loci. The genus Biatora is well supported as a monophyletic group (Fig. 1). Within Biatora, of the groups previously defined by Printzen (Reference Printzen2014), the beckhausii-, meiocarpa-, rufidula- and hertelii-groups (including B. mendax Anzi) are recovered, some of them appearing unsupported (Fig. 1). The vernalis-group is distributed over two clades, with the meiocarpa-group in between. However, these relationships were not statistically supported. Close relationships were supported between B. flavopunctata (Tønsberg) Hinter. & Printzen and B. vacciniicola (Tønsberg) Printzen (BP = 100, PP = 1.0), B. hemipolia (Nyl.) S. Ekman & Printzen and B. globulosa (Flörke) Fr. (BP = 85, PP = 1.0), B. hypophaea Printzen & Tønsberg, B. ocelliformis (Nyl.) Arnold and B. oxneri (S. Y. Kondr. et al.) Printzen & Kistenich (BP = 98, PP = 1.0), five taxa of the meiocarpa-group (BP = 91, PP = 1.0), six species from the vernalis-group 1 (BP = 89, PP = 1.0), as well as B. radicicola Printzen et al. and five specimens of the hitherto undescribed Biatora (BP = 97, PP = 1.0). These latter form a strongly supported monophyletic clade (BP = 100, PP = 1.0) which supports their status as a separate, new species.
Maximum likelihood (ML) tree based on the concatenated data set of ITS and mtSSU sequences of Biatora species and related taxa. Standard non-parametric bootstrap support values (BP) from the ML analysis and posterior probabilities (PP) are given below or above branches. Branches with BP > 70 and PP > 0.95 are in bold. Infrageneric groups previously identified by Printzen (Reference Printzen2014) are highlighted. The close relationship between Biatora amylacea (in bold) and B. radicicola is well supported in both analyses.
Taxonomy
Biatora amylacea Palice, Malíček, Vondrák & Printzen sp. nov.
MycoBank No.: MB 849563
Recognizable within the genus Biatora by the combination of an immersed blue-green tinted or episubstratal creamy white to pale grey or pale ochre thallus, possessing minute dark green to blue-grey pigmented soralia, sparsely present dark grey-bluish, pale-rimmed apothecia and an absence of secondary lichen metabolites. The characteristic feature is a distinct dark violaceous reaction of the apothecial exciple and thalline hyphae with Lugol's solution.
Type: Norway, Sogn og Fjordane, Gloppen, Våtedalen valley, forest with Betula, Alnus and Sorbus on W-facing slope just above the road E39, 61°40ʹ42.6ʺN, 6°31ʹ16.8ʺE, alt. 140 m, on bark of Sorbus aucuparia, 8 September 2015, Z. Palice 19999 & T. Tønsberg (BG—holotype!). GenBank Accession no.: OQ682881 (as Biatora sp.).
Habit (A & B) and microscopical characters (C–G) of generative structures of Biatora amylacea. A, flat apothecia with pale margin (Palice 19363). B, irregularly deformed, convex apothecia without margin (Palice 19170). C, apothecial section in water (Palice 19363). D, same section stained with Lugol's solution; the hymenium stains dark blue, the exciple, hypothecium and parts of the interalgal hyphae dark violaceous. E, irregularly branching excipular hyphae and paraphyses in lactophenol cotton blue (Palice 19999 & Tønsberg). F & G, Biatora-type ascus and ascospores in Lugol's solution (Palice 19999 & Tønsberg). Scales: A & B = 0.5 mm; C & D = 50 μm; E & F = 10 μm; G = 5 μm. In colour online.
Habit (A) and microscopical characters (B) of vegetative structures of Biatora amylacea. A, detail of several excavate soralia with blue-green pigmented external soredia (Vondrák 22719). B, squash preparations of corticate soredia with uneven surface in water; note the distinct Cinereorufa-green pigment in part of their cortex (Malíček 13765). Scales: A = 0.5 mm; B = 20 μm. In colour online.
Thallus immersed (endosubstratal) to distinctly superficial, continuous to ±rimose-areolate, sometimes cracked into irregularly delimited, strongly convex, minute areoles, surface creamy white to pale grey or pale ochre, matt, becoming scurfy with age; pigments in immersed parts of thalli staining the substratum blue-green. Soredia 12–35(–40) μm, corticate, outside with a dark greenish to bluish grey pigment (in more exposed parts) or pale, whitish-greenish or dull yellowish, covered by a continuous and uneven one-layered cortex composed of intricately interwoven hyphae, 2–3 μm thick. Soralia delimited, small, usually up to 0.2 mm, rarely exceeding 0.3 mm diam., rounded or irregular, occasionally prolonged, excavate or tuberculate, rarely confluenting, usually containing soredia in the order of tens, sometimes as tiny aggregations of several soredia. Hypothallus not clearly developed, usually seen as a prothallus composed of blue-green patches of a loose fine net of pigmented hyphae, 2–3 μm thick. Cortex absent or indistinct, up to c. 10 μm high, usually not distinguishable from a variably thick epinecral, largely amorphous layer, which may exceed 60 μm in height in extreme cases (aged thalli). Algal layer 60–90 μm high (when well developed), often discontinuous, in smaller patches or colonies, disrupted by the host tissue and/or bundles of medullary hyphae, medullary/interalgal hyphae 2–3 μm thick, largely or patchily ILugol+ dark violaceous (Fig. 2D). Photobiont chlorococcoid, cells broadly ellipsoid to globose, 5–14 μm diam. Medulla lacking or 35–40 μm high.
Apothecia (Fig. 2A & B) few, single or in groups of up to three, 0.15–0.60 (mean 0.3–0.4) mm diam., rounded or deformed, sessile with a slightly constricted base. Disc flat to moderately convex, surface sometimes irregular, different shades of bluish grey (Fig. 2A), sometimes partly beige, epruinose, matt. Margin level with disc when young, rarely becoming slightly prominent or excluded (Fig. 2B), white to pale grey, matt or slightly glossy. Proper exciple strongly gelatinized, laterally 30–40 μm, basally 30–50 μm wide, mostly colourless, but sometimes pale turquoise or purplish near hymenium and subhymenium, ILugol+ dark violaceous (Fig. 2D), composed of radiating, apically branched hyphae (Fig. 2E), lumina 1–2 μm (apically up to 3 μm) wide. Hypothecium 25–70 μm high, colourless or pale yellowish. Subhymenium 10–30(–50) μm high, pale greenish grey to brown with a faint pink to violaceous hue. Hymenium 30–40 μm high, colourless or pale grey to brown with a faint violaceous hue, ILugol+ medium to dark blue. Epihymenium c. 5 μm high, colourless or greyish/greenish black (Fig. 2C) with a pinkish hue, K+ violaceous, KC+ green, in parts also dark green or purplish and K+ intensifying, pigment amorphous, rarely as patchy granules around the ends of paraphyses or slightly spreading as vertical streaks. Paraphyses mostly simple, but apically branched, lumina 1.0–1.5 μm (apically 1.0–2.0 μm) wide, mostly colourless, but some with pigmented apical cells. Asci of Biatora-type (Fig. 2F), 8-spored. Ascospores (Fig. 2G) colourless, usually simple or rarely 1–3-septate, narrowly ellipsoid, straight, (9–)12.0–16.0(–20) × (2.5–)3.5–5.0 (–6.0) μm (n = 50).
Pycnidia not seen.
Chemistry
Thalli K−, C−, KC−, Pd−, UV−. No lichen metabolites detected by TLC. Apothecial pigments refer to Laurocerasi-brown and Cinereorufa-green as described by Meyer & Printzen (Reference Meyer and Printzen2000). In addition, Cinereorufa-green is present in protothalline and cortical hyphae of external soredia.
Etymology
The specific epithet refers to the typical violaceous colour reaction of the exciple and thalline hyphae with Lugol's iodine, resembling that of starch.
Ecology and distribution
Biatora amylacea is widely distributed and so far known from montane temperate and boreal Europe and the Caucasus. It occurs in preserved woodlands and old-growth forest habitats at elevations between 140 m a. s. l. (type locality in Norway) and 1870 m (Caucasus). It is an inhabitant of smooth to slightly roughened, mildly acidic bark of deciduous trees. In a Caucasian specimen (Vondrák 22719), the thallus was also found to spread to neighbouring epiphytic liverworts of the genus Frullania. The species has been recorded from Carpinus betulus, Fagus orientalis, F. sylvatica and Sorbus aucuparia. Virtually all specimens were found in well-preserved unmanaged forests in meso-/microclimatically stable and humid areas.
Additional specimens examined (paratypes)
Ukraine: Zakarpatska Oblast Region: Eastern Carpathians, Khust, Velyka Uhol'ka, E–ESE descending limestone ridge, mixed deciduous forest on steep SSW–S-facing slope 0.9 km WNW of the rock Molochnyi kamen, 48°15ʹ22ʺN, 23°39ʹ40ʺE, alt. 820 m, on bark of Fagus sylvatica and Carpinus betulus, 2015, Z. Palice 19170 (PRA), 19363 (FR).—Russia: Republic of Adygea: Caucasus Mts, Caucasian Biosphere Reserve, Mezmay, KPP Lagonaki, mixed primeval forest (Abies, Acer, Sorbus, Ulmus) on limestone bedrock, 44°04ʹ40ʺN, 40°00ʹ50ʺE, alt. 1830 m, on bark of Sorbus aucuparia, 2016, J. Malíček 11048, Z. Palice, J. Vondrák & G. Urbanavichus (hb. Malíček). Republic of Kabardino-Balkaria: Caucasus, Baksan, Elbrus, mixed forest on left slope above River Adyl-Su, 43.23549, 42.64539, alt. 1870 m, on mossy bark of Sorbus aucuparia, 2018, J. Vondrák 22719 (PRA, sterile). Krasnodar Territory: Caucasus, Sochi, Krasnaya Polyana, Estosadok, 4 km SSW of Mt Pik Geomorfologov [2665], well-lit hornbeam/beech/oak forest on W-descending crest, WNW of the point 1106,6, between the streams of Achipse and Assara, 43°43ʹ19ʺN, 40°15ʹ40ʺE, alt. 820–850 m, on bark of young Fagus orientalis, 2019, Z. Palice 35475, S. Svoboda, G. Urbanavichus, I. Urbanavichene & J. Vondrák (PRA, sterile).—Czech Republic: Southern Bohemia: Novohradské hory Mts, Horní Stropnice, Hojná Voda National Nature Monument, fragment of primeval forest predominated by beech above road, 48°42ʹ27ʺN, 14°45ʹ05ʺE, alt. 830–880 m, on bark of Fagus sylvatica, 2020, J. Malíček 13765 (hb. Malíček); ibid., below road, 48°42ʹ21ʺN, 14°45ʹ09.9ʺE, alt. 857 m, Z. Palice 28991 (PRA, sterile). Western Bohemia: Šumava Mts, Prášily: Mt Ždanidla, SW–SSW-facing slope, remnant of montane mixed forest, 49°06ʹ02.5ʺN, 13°20ʹ41.4ʺE, alt. 1198 m, on bark of old hollow Fagus sylvatica, 2021, Z. Palice 32822 (PRA, FR, sterile).
Specimens of other species examined
Bacidia caesiovirens. Norway: Nordland: Sømna, Kvaløya Island, forest on steep NE–NNE-facing slope, along a small stream, 0.4–0.5km SW–WSW from Vennesund, 65°12ʹ50.1ʺN, 12°02ʹ00.5ʺE, alt. 90 m, on bark of Sorbus aucuparia, 2016, Z. Palice 31265 (PRA).
Caloplaca ahtii. Russia: Orenburg Region: Saraktash, village of Andreevka, protected area ‘Andreevskie Shishki hills’ at village, 51°56ʹ49ʺN, 56°39ʹ12ʺE, alt. c. 250–350 m, on bark of Ulmus laevis, 2011, J. Vondrák 13014 (PRA).
Caloplaca turkuensis. Austria: Salzburg: Hohe Tauern, Bucheben, Hüttwinkltal valley, a small alder wood among pastures on W-facing slope, 47°07ʹ29.3ʺN, 12°59ʹ11.2ʺE, alt. 1210 m, on bark of Alnus incana, 2016, F. Bouda, Z. Palice 18571 & O. Peksa (PRA).
Parvoplaca nigroblastidiata. Russia: Krasnodar Territory: Caucasus, Sochi, Krasnaya Polyana, Estosadok, 4 km SSW of Mt Pik Geomorfologov [2665], well-lit hornbeam/beech/oak forest on W-descending crest, WNW of the point 1106,6, between the streams of Achipse and Assara, 43°43ʹ19ʺN, 40°15ʹ40ʺE, alt. 820–850 m, on bark of Fagus orientalis, 2019, Z. Palice 27012, S. Svoboda, G. Urbanavichus, I. Urbanavichene & J. Vondrák (PRA).
Discussion
The description of this so far unrecognized, morphologically distinctive and identifiable species (without the help of molecular data) highlights the fact that even lichenologically relatively well-studied areas of temperate and boreal Europe are still under-explored. The new species is quite well recognizable within the genus Biatora, when richly developed and fertile. It is therefore unlikely to be a frequent species but is rather a rare forest lichen and niche specialist with high bioindication potential for well-preserved old-growth forests. The first three authors have undertaken numerous, very detailed lichen surveys in a number of valuable old forest reserves, mainly across Central Europe, and Biatora amylacea was detected only rarely. It was never prominent in epiphytic crustose lichen communities, and usually only scanty specimens were found.
Phylogenetically, the closest relative of B. amylacea is B. radicicola, another rare specialist species preferentially growing in places subjected to water (on roots of trees at river banks) or to snow (bases of trees in montane forests with a long snow cover; Printzen et al. Reference Printzen, Halda, McCarthy, Palice, Rodriguez-Flakus, Thor, Tønsberg and Vondrák2016). The latter species has recently also been found saxicolous on humid rocks in Sweden (Ekman et al. Reference Ekman, Svensson, Westberg and Zamora2019).
Other Biatora species with bluish grey apothecia and lacking secondary lichen metabolites, such as B. beckhausii, B. globulosa, B. hemipolia and B. radicicola, have a non-amyloid exciple and cannot be confused with B. amylacea. The problem is rather that B. amylacea is apparently only rarely fully developed and richly fertile, and therefore easily missed by lichenologists. The characteristic amyloid exciple, visible as a dark violaceous reaction after adding iodine solution, is well known from Biatora aegrefaciens Printzen and B. rufidula (Graewe) S. Ekman & Printzen (Printzen Reference Printzen1995, Reference Printzen2014; Printzen & Tønsberg Reference Printzen and Tønsberg2000). These taxa are distinguishable macroscopically by the orange to reddish brown colour of their apothecia, and microscopically by the broader, usually 3-septate ascospores. The amyloid exciple is also typical for the poorly known and phylogenetically unrelated taxon Lecidea betulicola f. endamylea (Hedl.) Hinter., which also has dark grey apothecia but produces more than 8 spores in the asci and has a more pronounced exciple, formed by radiating and anastomosing hyphae with thin lumina (see Printzen & Tønsberg Reference Printzen and Tønsberg2000).
Biatora amylacea regularly produces small, often blue-green-grey pigmented vegetative propagules containing one or more photobiont cells enveloped by a single-layered cortex with a bulging surface (Fig. 3B), frequently originating in small delimited crater-like areas developing from ruptures in the thin outermost layer of the thallus (Fig. 3A). Similar vegetative structures have been called soredia (arising in soralia) in descriptions of taxa such as Gyalideopsis helvetica van den Boom & Vězda (van den Boom & Vězda Reference van den Boom and Vězda2000) or Caloplaca ahtii Søchting (Søchting Reference Søchting1994). Both these microlichens may resemble our species in the sterile state. Interestingly, some subsequent authors referred to the same propagules as goniocysts (produced in goniocystangia) in the former species (e.g. Spribille & Björk Reference Spribille and Björk2008) or as blastidia (Arup et al. Reference Arup, Vondrák and Halıcı2015) in the latter species, apparently based on presumed differences in the ontogeny of these propagules. More recently, Ekman (Reference Ekman2023) used the more universal term ‘granule’ for the fine soredia-like propagules in some members of the genus Bacidina, interpreting the tiny particles as the result of gradual splitting of the thallus. Earlier, Printzen (Reference Printzen1995: 24) had explained the development of vegetative, soredia-like propagules in Biatora fallax Hepp in a similar way. In B. amylacea, establishment of propagules has not been studied in much detail because it is beyond the scope of this primarily taxonomic contribution. However, on the hand-cut sections of thalli and adjacent bark it was evident that the propagules were being formed before they were released. In one specimen (Malíček 13765), the soralia were observed in ±endophloedic parts of thalli where pigmented hyphae of the mycobiont predominated (prothallus). Propagule formation may therefore be likely to occur in early stages of development, after the first interactions of the fungal hyphae with the photobiont. This implies that the propagules in B. amylacea are not referrable to the granules formed secondarily by some representatives of the genus Bacidina (Ekman Reference Ekman2023) or to those of Biatora fallax (Printzen Reference Printzen1995). Hence, we prefer to call the propagules in the new species soredia in a broad sense (sensu Tønsberg Reference Tønsberg1992), although the pigmented soredia formed in tiny, often excavate soralia (Fig. 3A) of the new species differ somewhat from the soredia produced by most representatives of the genus Biatora.
When considering sterile specimens, species similar to Biatora amylacea (lacking lichen substances and possessing blue-green to blue-grey vegetative propagules in delimited soralia) include several taxa of Teloschistaceae, such as Caloplaca ahtii, C. turkuensis (Vain.) Zahlbr. or Parvoplaca nigroblastidiata Arup et al. (Søchting Reference Søchting1994; Šoun et al. Reference Šoun, Vondrák, Søchting, Hrouzek, Khodosovtsev and Arup2011; Arup et al. Reference Arup, Vondrák and Halıcı2015). All these species contain a blue-grey pigment which is, however, referable to Sedifolia-grey (K+ purple). A habitually similar lichen also exists among Ramalinaceae. The Cinereorufa-green pigment in the propagules and hypothallus is shared by the predominantly north-western European Bacidia caesiovirens S. Ekman & Holien. This taxon can be easily distinguished by the larger isidioid granules, usually exceeding 40 μm in diameter, occasionally showing projecting hyphae, and sometimes containing traces of atranorin (Ekman & Holien Reference Ekman and Holien1995).
The presence of the amyloid reaction in thalline hyphae of the newly described species is a good character for the identification of sterile specimens of Biatora amylacea in combination with the content of the Cinereorufa-green pigment, absence of secondary metabolites and the general character of the thallus. Among the epiphytic crustose lichens that occur in the boreal and temperate zones of Europe, we know of only one lecideoid species with an I+ dark violaceous medulla, Lecidea roseotincta Coppins & Tønsberg (Coppins & Tønsberg Reference Coppins and Tønsberg1988). An amyloid reaction of thalline hyphae was seen by us in all examined specimens of B. amylacea. This reaction was particularly conspicuous in the mycelium surrounding the algal layer or between photobiont colonies, and less obvious in pigmented hyphae of the prothallus and cortex of soredia, where the violaceous colour was visible in larger aggregations of soredia or hyphae. Conversely, the amyloid reaction was not observed in any of the examined samples of the potentially confusable lichens mentioned above (see ‘Specimens of other species examined’).
Acknowledgements
This paper is dedicated to Pier Luigi Nimis who has contributed so much to the various aspects of lichenology, in particular to the documentation of lichen diversity in Southern Europe. ZP is grateful to Tor Tønsberg for organizing a field trip to Norway and bringing him to the type locality of Lecanora flavoleprosa, where coincidentally also the holotype of Biatora amylacea was collected. ZP, JM and JV acknowledge the long-term research development project RVO 67985939. Jiří Machač helped photograph vegetative structures. Måns Svensson is thanked for his valuable comments on the manuscript.
Author ORCIDs
Zdeněk Palice, 0000-0003-4984-8654; Jiří Malíček, 0000-0002-3119-8967; Jan Vondrák, 0000-0001-7568-6711; Christian Printzen, 0000-0002-0871-0803.
Competing Interests
The authors declare none.
