Biatoraalnetorum (Ramalinaceae, Lecanorales), a new lichen species from western North America

Abstract Biatoraalnetorum S. Ekman & Tønsberg, a lichenised ascomycete in the family Ramalinaceae (Lecanorales, Lecanoromycetes), is described as new to science. It is distinct from other species of Biatora in the combination of mainly three-septate ascospores, a crustose thallus forming distinctly delimited soralia that develop by disintegration of convex pustules and the production of atranorin in the thallus and apothecia. The species is known from the Pacific Northwest of North America, where it inhabits the smooth bark of Alnusalnobetulasubsp.sinuata and A.rubra. Biatoraalnetorum is also a new host for the lichenicolous ascomycete Sclerococcumtoensbergii Diederich.


Introduction
During field work in the Pacific Northwest of the United States and Canada in 1995-2018, the second author came across a distinct crustose and sorediate lichen on the smooth bark of alders. Ascospores produced in the scattered pale-coloured apothecia turned out to be mostly three-septate, which prompted a search amongst the many names once described in or combined into the genus Bacidia De Not. As we were unable to find any previous description of a species fitting this morphology, it is described here as new to science. Morphological characteristics, primarily the combination of the structure of the proper exciple, sorediate thallus and ascospore shape, led us to suspect the new species to be a member of the genus Biatora Fr. (Printzen 1995(Printzen , 2014.

Materials and methods
Microscopic quantitative characters were investigated either in a 10% aqueous solution of potassium hydroxide (KOH) (ascospores, paraphyses) or in pure water (all other microscopic characters). Pigments were investigated and characterised using a 10% aqueous solution of KOH and a 50% v/v aqueous solution of commercial-grade nitric acid (70% HNO 3 ). Measurements of quantitative characters are given either as 'minimum valuemaximum value' or 'minimum value -arithmetic mean value -maximum value (s = sample standard deviation, n = sample size)'. Lichen substances were screened with Thin Layer Chromatography (TLC) in solvent systems A, B' and C following Culberson and Kristinsson (1970), Culberson (1972) and Menlove (1974). Aluminium plates were used in systems A and B' and glass plates in system C, the latter to allow the detection of fatty acids.
In order to obtain some indication of relationships from other than morphological data, we obtained a complete sequence from the internal transcribed spacer (ITS) region of the ribosomal DNA using the laboratory approach described by Ekman and Blaalid (2011). Subsequently, we downloaded the data (S15023) of Printzen (2014) from TreeBase (https://treebase.org) and excised the ITS region. For reasons of computational tractability, we removed Cliostomum griffithii (Sm.) Coppins (shown by Kistenich et al. 2018 to be more closely related to Ramalina Ach.), Mycobilimbia pilularis (Hepp ex Körb.) Hafellner & Türk (the genus already being well represented by two other species), sequences not definitively referred to any taxon (marked "cf.") and all but one sequence from taxa represented by multiple accessions. Question-mark symbols were either removed (when they were terminal) or replaced by "N" (when they were internal). Finally, all gaps were stripped. To this data, we added our own ITS sequence of Biatora alnetorum (MH818375), generated from Tønsberg 27500 (BG), resulting in a dataset with 45 sequences. We carried out a joint estimation of alignment and phylogeny using BAli-Phy version 3.1.4 (Suchard and Redelings 2006). We set the substitution model to a single GTR+I+Γ (the gamma distribution divided into four categories) and the gap model to a single RS07 model (Redelings and Suchard 2007) without partitioning the data. Priors were kept at their default values. The analysis consisted of 10 parallel runs and included a pre-burn-in of 10 iterations followed by 75000 cycles of Markov chain Monte Carlo (MCMC), sampling states every 50 cycles. The first 25000 cycles of each run were removed as burn-in. A more precise estimate of the time to convergence was obtained with the statreport tool of BAli-Phy.

Results
All numerical parameters of the BAli-Phy analysis had converged after 16650 cycles, but we anyway excluded the first 25000 cycles (resulting in a posterior sample of size 10×(75000-25000)/50 = 10000). In the posterior sample, the average standard deviation of split frequencies at or above 0.1 was 0.015. A majority-rule consensus tree with all compatible groups provided 0.97 posterior probability for the genus Biatora, including B. alnetorum (Fig. 1)  Diagnosis. Similar to Biatora pallens (Kullh.) Printzen in having 3-septate ascospores and crystals in the exciple, but differs from that species primarily by the sorediate thallus, the production of atranorin in the thallus and proper exciple and in sometimes producing up to 7-septate ascospores. Etymology. The epithet, alnetorum, means 'of the alder stands' and is a reference to the fact that Biatora alnetorum prefers thickets dominated by Alnus alnobetula subsp. sinuata.
Apothecia absent or sparse, sometimes abundant, biatorine, 0.3-0.5-0.9 mm diam. (s = 0.1, n = 50), at first flat, later moderately convex, epruinose or thinly pruinose on edge. Disc pale pink (or pale yellowish with age in the herbarium). Margin pale pink to almost white, concolorous with disc or slightly paler, thick, distinct, raised above disc in young apothecia, soon level with the disc, persistent.
Pigments: No pigments or small amounts of Rubella-orange (Meyer and Printzen 2000) in proper exciple, hypothecium and/or hymenium.
Distribution and ecology. Biatora alnetorum is known from the Pacific Northwest of North America in Washington and Alaska (U.S.A.) and British Columbia (Canada). Its vertical distribution ranges from 620 to 1450 m a.s.l. It occurs in openings in humid old-growth coniferous forest and Alnus woodlands and in the alpine scrub zone. B. alnetorum inhabits smooth bark of trunks or, occasionally, branches. The phorophyte is almost exclusively Alnus alnobetula subsp. sinuata (also known as Alnus viridis subsp.

Discussion
Morphologically, the new species stands out on account of its combination of pale pinkish apothecia with a proper exciple inspersed with small crystals, mostly 3-septate ascospores and green soralia formed from distinct pustules on the thallus and containing soredia that are remarkably even in size and shape. It agrees with other species of Biatora with regard to the radiating and (in lateral view) almost parallel excipular hyphae, the generally strongly gelatinised apothecial tissues, as well as the Biatora-type ascus (Hafellner 1984, Printzen 2014. Recent molecular phylogenies (e.g. Printzen 2014, Kistenich et al. 2018 have resulted in an increasingly inclusive delimitation of the genus compared to the first modern circumscription proposed by Printzen (1995). Concomitantly, the morphological amplitude accepted in the genus (tissue structures, chemistry, pigmentation etc.) has expanded. We adhere here to the most recent and quite broad circumscription of Biatora advocated by Printzen (2014) and confirmed by Kistenich et al. (2018) as monophyletic, the latter assuming that the genus Myrionora R. C. Harris is also included.
Our phylogeny (Fig. 1), which is based only on the internal transcribed spacer region, confirms that B. alnetorum is a member of the genus Biatora but does not allow any definitive conclusions beyond that. In the consensus tree, the new species appears as sister species to B. sphaeroidiza in a poorly supported group also including B. chrysanthoides, B. pallens and an undescribed species. Taking into account differences in taxon sampling, there are, however, different levels of support but no supported contradictions between our ITS phylogeny and the phylogeny of Printzen (2014), the latter based on more extensive DNA sequence data.
Amongst the previously known species of the genus, Biatora alnetorum (Figs 2, 3) is morphologically most likely to be confused with the esorediate B. pallens and the sorediate B. flavopunctata. B. pallens and B. alnetorum share the 3-septate ascospores and the presence of crystals in the exciple. B. alnetorum is, however, different from that species in forming large, sorediate thalli, the occurrence of atranorin in the thallus and apothecia, larger apothecia that do not become as markedly convex and longer ascospores, with sometimes up to five or seven septa. In B. pallens, thalli are esorediate and often small, apothecia become convex with an excluded margin early during development and ascospores are consistently three-septate and short-bacilliform. Small amounts of usnic acid and zeorin occur in the thallus, whereas large amounts of usnic acid form crystals in the proper exciple and upper part of the hymenium (Ekman 1997). In the field, however, Biatora alnetorum is most likely to be confused with B. flavopunctata (Fig. 3B), which is widespread on alpine shrubs in the Pacific Northwest. Both species are sorediate and often co-occur, forming mosaics on Alnus branches. In such situations, B. alnetorum is set apart by the presence of discrete and conspicuously yellowish grass-green soralia formed from convex pustules, whereas B. flavopunctata possesses pale (yellowish) green soralia not formed from convex pustules. Under the microscope, the soredia of B. flavopunctata are mostly globose and fragile, easily disintegrating in squash preparations. Ascospores are non-septate in B. flavopunctata (Tønsberg 1992;Printzen 1995) and chemical constituents include usnic, isousnic and often also stictic and cryptostictic acids in addition to atranorin (Tønsberg 1992, Printzen 1995. The anatomical and chemical differences between B. flavopunctata and B. alnetorum reflect the fact that the two species are unlikely to be closely related within the genus (Fig. 1). In addition, B. alnetorum shares the presence of punctiform soralia and more or less long-bacilliform, mainly three-septate ascospores with B. bacidioides Printzen & Tønsberg (Printzen and Tønsberg 2003). The latter, however, lacks crystals in the exciple, has near-black apothecia and produces argopsin, norargopsin and gyrophoric acid in the soralia.
The distribution of Biatora alnetorum (Fig. 4) coincides with the Vancouverian Subprovince of the Cordilleran-Arctic Province of McLaughlin (2007). To our knowledge, it does not occur in the coastal or inland rain forest zones (Schoonmaker et al. 1997, Goward andSpribille 2005) but seems to prefer somewhat inland conditions. Having said that, B. alnetorum is, although unlikely to be common, probably overlooked and its distribution underestimated.