Studies in the Basidiodendron caesiocinereum complex (Auriculariales, Basidiomycota)

Taxonomy of Basidiodendron caesiocinereum complex is revised based on morphological and molecular methods (with the use of nc LSU rDNA, ITS and TEF1 regions). The basidiospore ornamentation is justified as a key morphological character for the species recognition in the group. As redefined here, B. caesiocinereum is an angiosperm-dwelling species with smooth basidiospores. Bourdotia cinerella and B. cinerella var. trachyspora are proved to represent separate species with warted basidiospores; they are reintroduced as Basidiodendron cinerellum and B. trachysporum. Additionally, eight new species related to B. caesiocinereum are described based on material from Eurasia, North America and Africa, and identity of B. spinosum from Oceania is discussed.


Introduction
For almost two centuries, the basidiospore features, e.g. their shape, size, colour and ornamentation, have remained among the main features for morphological recognition of the basidiomycetous taxa. Almost all members of the Auriculariales (Basidiomycota) have small or medium-sized, colourless (hyaline), smooth basidiospores and three species with ornamented (warted or spiny) basidiospores have been so far detected in the genus Basidiodendron Rick (Wojewoda 1981). Two of them, Basidiodendron asperum (L.S. Olive) Wojewoda and B. spinosum (L.S. Olive) Wojewoda, are known from their type localities in Tahiti, while the third one, B. caesiocinereum (Höhn. & Litsch.) Luck-Allen, was reported from different geographic areas of the world (Wells and Raitviir 1975).

Morphological study
Type specimens and collections from herbaria H, O, GENT, LE, FH, PC, NY, TAAM, TU, CWU were studied. Herbarium acronyms are given according to Thiers (2020). Microscopic routine and terminology follow Spirin et al. (2020). All measurements were made from microscopic slides mounted in Cotton Blue, using phase contrast and oil immersion lens (Leitz Diaplan microscope, ×1250 magnification). At least 20 basidia, 10-20 gloeocystidia and 20-30 basidiospores were measured for each specimen studied. The following abbreviations are used in the taxonomic section: L, mean basidiospore length; W, mean basidiospore width; Q', L/W ratio; Q, mean L/W ratio; and n, number of measurements per specimens measured. Advances of phase contrast illumination versus bright-field microscopy are explained in Stein (1969). For microscopic study, we subjectively determined an accuracy of measurements as 0.1 µm. These measurements (including those for the basidiospore ornamentation elements, i.e. warts or spines) were subsequently calibrated with the scanning electronic microscope (SEM). For preparing SEM photos, basidiocarps were coated with a 25-mm layer of gold-palladium using an Eiko IB-3 sputter coater. Micrographs were taken using JSM-6380LA microscope at the Moscow State University (Russia) and JEOL JSM-7100FLV field emission microscope at Botanical Garden Meise (Belgium).

DNA study
In total, 80 specimens were selected for molecular sampling (Table 1). We performed PCR directly from small fragments of dried basidiocarps (without prior DNA extraction) using Phire Plant Direct PCR Kit (Thermo Scientific) according to the manufacturer's instructions. DNA extraction of Belgian and Dutch collections was done using the DNeasy Plant Mini kit (Qiagen).
The following primers were used for both amplification and sequencing: the primers ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) for the nrITS1-5.8S-ITS2 region, primers EF1-983F and EF1-1567R (Rehner and Buckley 2005) for a part of the TEF1 region and primers JS1 (Landvik 1996) and LR5 (Vilgalys and Hester 1990) for D1-D3 domains of nc LSU rDNA region. PCR products were purified applying the GeneJET Gel Extraction and DNA Cleanup Micro Kit (Thermo Scientific). Sequencing was performed with an ABI model 3500 Genetic Analyzer (Applied Biosystems). Raw data were edited and assembled in MEGA 7 (Kumar et al. 2018). Molecular studies were mostly carried out at the centre for collective use of scientific equipment "Cellular and molecular technology of studying plants and fungi" (Komarov Botanical Institute, Russian Academy of Sciences, St. Petersburg, Russia) and the centre for molecular phylogeny and evolution (CeMoFe) (Ghent University, Belgium).
Three datasets were prepared for the present study: (1) ITS + nc LSU rDNA dataset (1647 characters including gaps) used to reconstruct a general topology of the Auriculariales, with special focus on Basidiodendron spp.; (2) ITS + nc LSU rDNA + TEF1 (1884 characters including gaps); and (3) ITS only (628 characters including gaps) datasets for the B. caesiocinereum complex.
Phylogenetic reconstructions were performed with maximum likelihood (ML) and Bayesian (BI) analyses. Before the analyses, the best-fit substitution model for the alignment was estimated based on the Akaike Information Criterion (AIC) using FindModel web server (http:// www. hiv. lanl. gov/ conte nt/ seque nce/ findm odel/ findm odel. html). "K80 plus Gamma" model was chosen for ITS dataset, and "GTR  (Ronquist et al. 2012), for two independent runs, each with 5 million generations (for ITS + nc LSU rDNA + TEF1 dataset) and 10 million generations (for ITS and ITS + nc LSU rDNA datasets), under described models and four chains with sampling every 100 generations.
To check for convergence of MCMC analyses and to get estimates of the posterior distribution of parameter values Tracer v1.6 was used (Rambaut et al. 2014). We accepted the result where the ESS (effective sample size) was above 200 and the PSRF (potential scale reduction factor) was close to 1.
Newly generated sequences have been deposited in Gen-Bank with corresponding accession numbers (Table 1). Alignments have been deposited in TreeBASE (S27231).

ITS + nc LSU rDNA dataset
The overall topologies of the ML and BI trees were nearly identical ( Fig. 1). They uncovered all B. caesiocinereum s. lato specimens involved in the analyses in one strongly supported clade (bs =100, pp =1) with B. luteogriseum Rick (the generic type of Basidiodendron) and members of the B. eyrei complex (as defined by Spirin et al. 2020). Therefore, we interpreted all these taxa as belonging to one genus, Basidiodendron. In turn, the Basidiodendron clade was split into four strongly supported subclades. Two of them covered the Basidiodendron eyrei complex and contained fourteen species with smooth, ellipsoid or globose, usually small basidiospores. They all were dealt with in our previous publication (Spirin et al. 2020). Two remaining subclades encompassed the B. caesiocinereum complex. The larger subclade (designated in Fig. 1

ITS + nc LSU rDNA + TEF1 dataset
The final alignment contained 1884 characters (including gaps). The overall topologies of the ML and BI trees were nearly identical and in a good correspondence with the ITS + LSU phylogeny (Fig. 2). Both B. caesiocinereum s. str. and B. trachysporum lineages are strongly supported and therefore interpreted by us as representing single species each.

ITS dataset
Twenty additional environmental ITS sequences related to B. caesiocinereum complex were retrieved from GenBank and UNITE and used in the phylogenetic analyses (Supplement). Seven of them belonged to B. trachysporum and confirmed this species is widespread in temperate-boreal forests of Eurasia and North America. Basidiodendron caesiocinereum, B. cinerellum and B. walleynii were represented by three, two and one environmental sequences, respectively. Seven remaining sequences potentially represent four more species in the B. caesiocinereum complex: two from Canada (British Columbia) (KP889384, KP889562), one from temperate Europe (Austria) (JF519252, JF519305) and possibly one more represented by sequences from UK, Estonia and Alaska (AF504871, KF297103, UDB0141409). However, we could not connect these sequences with available herbarium material, and therefore their identity remains unresolved.

Taxonomy
Distribution and ecology. Europe (Belgium, Estonia, Finland, Germany, Italy, Norway), Asia (China, Russia -Siberia); almost exclusively on rotten wood of deciduous trees, as a rule in excessively humid habitats.
Remarks. Höhnel and Litschauer (1908) described C. caesiocinereum based on a single collection from Germany. They overlooked inner septation of basidia as well as the presence of cystidia, and this was a reason for them to assign the new species to the genus Corticium s. lato. Bourdot and Galzin (1927) restudied the type material of C. caesiocinereum and found that it has gloeocystidia and fourcelled basidia. They concluded that C. caesiocinereum is an older name for Bourdotia cinerella Bourdot & Galzin. This viewpoint has persisted in the literature until the present day. However, we argue below that B. cinerella represents a warted-spored taxon, while the type specimen of C. caesiocinereum has smooth basidiospores. Therefore, the synonymy of C. caesiocinereum and B. cinerella should be abandoned.
As redefined here, B. caesiocinereum is most similar to B. glaucum. The two species are best separated by their ecological preferences. Basidiodendron glaucum is a northern species almost exclusively restricted to coniferous wood, while B. caesiocinereum is connected to angiosperm hosts, mainly in inundated habitats. However, B. glaucum may accidentally occur on deciduous trees, and one sequenced collection of B. caesiocinereum came from spruce. In these cases, B. caesiocinereum can be distinguished from B. glaucum due to longer basidia occasionally provided with a long stalk-like base. Basidiospores of B. caesiocinereum are on average larger than in B. glaucum, although their variation ranges Basidiocarps effused, smooth, first waxy, pruinosereticulate, greyish, then gelatinized, continuous, dirty-grey to pale ochraceous or brownish, occasionally tuberculate, often with irregularly scattered craters, 0.01-0.05 mm thick, covering a few cm, margin gradually thinning-out. Hyphal structure monomitic, hyphae clamped, glued together; subicular hyphae thin-walled, subparallel, 2.5-4 μm diam, subhymenial hyphae thin-walled, ascending or interwoven, occasionally twisted, 1.5-3 μm diam, basidia-bearing hyphae distinct in older parts of hymenium, slightly thickwalled, up to 12 × 2-3.5 μm. Gloeocystidia abundant, more or less clearly tapering, slightly projecting, hyaline or yellowish to brownish, (13.5) 14-34 (35)   Remarks. Bourdot and Maire (1920) introduced B. cinerella from a large set of specimens collected in the southern part of France. They described it as having smooth spherical basidiospores and later considered it a synonym of C. caesiocinereum. However, B. cinerella has remained untypified, and therefore its actual relation to C. caesiocinereum was obscure. We studied all (in total 29) specimens stored in Bourdot's herbarium (PC), which were labelled by him as 'Sebacina (Bourdotia) cinerella'. Of them, two collections (Bourdot 40882 and 9047) belong to B. cinereum (Bres.) Luck-Allen s. lato, a species with cylindrical-ellipsoid spores; they do not fit to the protologue and cannot be used for typification. The rest of the specimens have globose spores, in good accordance with the original description, but they are warted, not smooth. Among them, two specimens represent Bourdotia cinerella var. trachyspora described seven years later (Bourdot and Galzin 1927) and considered here as a separate species. They certainly were not the main source for the B. cinerella description. Twelve remaining specimens were collected from coniferous (Pinus) and thirteen were from angiosperm hosts. However, both deciduous trees and conifers were mentioned as substrates in the protologue of B. cinerella, and therefore the host indication alone is insufficient for understanding the original idea of the species. Fortunately, Bourdot and Galzin provided a good description of macroscopic traits of B. cinerella: basidiocarps were described as 'whitish, whitish-grey, often glancing, pale ochraceous and crustaceous when old' ('blanchâtre, blanc-gris souvent brillant, subocracé et crustacé sur le vieux' - Bourdot and Galzin 1920: 71). These indications fit the pine-dwelling specimens and preclude collections from angiosperm hosts with arid, opaque basidiocarps. We assign the latter ones to a new species B. walleynii and select the best-developed specimen from Pinus (Galzin 14526, herb. Bourdot 12419) as a lectotype of B. cinerella.
Basidiodendron cinerellum is one of three species with warted basidiospores distributed in Europe. The most striking microscopic feature, differentiating B. cinerellum from two other species (i.e., B. trachysporum and B. walleynii), is the presence of a cyanophilous gelatinous matter covering basidial cells. In juvenile specimens, this matter is visible at least in some basidia as an essential (1-2 μm) thickening of the basidial wall. In mature, and especially in senescent basidiocarps, the gelatinous matrix covers basidia up to the very top and glues them together in large, easily detectable groups. Moreover, basidiocarps of B. cinerellum often have dirty-greyish or ochraceous-brownish tints and, at least in some parts, they are more or less clearly gelatinized. In contrast, basidiocarps of B. walleynii remain arid and normally pale, while in B. trachysporum they are usually very thin, constantly whitish-greyish and occasionally gelatinized only when old. No signs of hymenial gelatinous matter so characteristic for B. cinerellum were detected in B. trachysporum or B. walleynii.
Basidiodendron cinerellum and B. trachysporum inhabit mostly coniferous hosts and sometimes occur in the same habitats. Our data suggest, however, that they may have quite different ecological specialization. Basidiodendron cinerellum seemingly prefers tough, often still corticated wood-mainly thick, still hanging or just fallen branches or small-to medium-sized logs. In turn, Basidiodendron trachysporum mostly occurs on well-decomposed wood, often on rotten logs lying on the ground or inside old stumps,    (Bourdot 19447 (O F-248006, isotype -H).
Distribution and ecology. Europe (France, Norway, North-West Russia, Sweden), Asia (Russian Far East); almost exclusively on rotten wood of conifers.
Remarks. Basidiodendron groningae is the only species with spiny basidiospores so far detected in Europe. It was collected three times around Groningen in the Netherlands and once in Belgium, but its actual distribution is unknown. Four other species with spiny spores dealt with in this paper were found in North America (B. mexicanum, B. spiculosum), Africa (B. widdringtoniae) and Oceania (B. spinosum). In the type specimen of B. groningae, basidia occasionally bear oblique or even transversal septa, and then only one or two apical cells produce sterigmata. This feature illustrates morphological flexibility of basidial cells most likely caused by environmental conditions and seems to have no taxonomic importance.
Distribution and ecology. So far known from the type locality; strongly decayed wood of conifers (Thuja plicata).

3
Distribution and ecology. So far known from the type locality; decorticated coniferous wood in a highland forest.
Remarks. Basidiodendron mexicanum is one of the species with spiny basidiospores introduced here. It differs from B. spiculosum, also found in Mexico, in having thinner basidiocarps, shorter gloeocystidia and smaller basidiospores, as well as by the presence of hyphidia. Phylogenetically, B. mexicanum is closely related to B. groningae so far detected only in Europe (Figs. 1, 2). The latter species possesses basidiospores with more pronounced spines than in B. mexicanum, and it lacks hyphidia. ITS sequences of these species show 2.7-3.9% distance (the infraspecific differences within B. groningae are under 1.6%). Morphologically, B. mexicanum is most similar to B. widdringtoniae (see remarks under the latter species). Basidiodendron widdringtoniae is so far known from Malawi only and phylogenetically closer to B. walleynii than to B. mexicanum or B. spiculosum.  (H).
Distribution and ecology. North America (Canada -Ontario, USA -New York, Tennessee); rotten wood of deciduous trees.
Remarks. Basidiodendron robenae is a smooth-spored species distributed in the north-eastern part of North America Etymology: Spiculosus (Lat., adj.) -spiculate, in reference to the basidiospore ornamentation.
Distribution and ecology. So far known from the type locality; dry stem of a tree fern.
Distribution and ecology. So far known from the type locality; rotten wood of an unidentified tree.
Remarks. This species was initially introduced as a warted-spored variety of B. cinerella (Bourdot and Galzin 1927), while the latter was erroneously described as a smooth-spored species (see discussion under B. cinerellum). The single authentic specimen labelled by Bourdot as 'Sebacina (Bourdotia) cinerella f. trachyspora' is an extensive collection in good condition, and it is designated here as a lectotype. Morphological differences of B. trachysporum from B. cinerellum are listed under the latter species. Another similarly looking European species, B. walleynii, occurs on deciduous hosts and has thicker basidiocarps and differently shaped, wider and clearly projecting cystidia. Basidiodendron trachysporum is certainly the most common species of the genus in the middle-and north-boreal forests of North Europe.