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A preliminary overview of the corticioid Atractiellomycetes (Pucciniomycotina, Basidiomycetes)

The taxonomy of the corticioid fungi from the class Atractiellomycetes (Pucciniomycotina, Basidiomycetes) currently addressed to the genus Helicogloea, is revised based on morphological and nuclear ribosomal DNA (ITS and LSU) data. The genus is restricted to 25 species with semitranslucent, gelatinous basidiocarps lacking differentiated cystidia and clamps on hyphae, of which 11 are described as new to science. The asexual genus Leucogloea is placed as a synonym of Helicogloea s. str. Since the type species of Saccoblastia, S. ovispora, is combined to Helicogloea, a new genus, Saccosoma, is introduced to encompass Saccoblastia farinacea and six related species, one of which is described as new. In contrast to Helicogloea in the strict sense, the basidiocarps of Saccosoma are arid, not gelatinized, and hyphae are clamped. The third lineage of the corticioid Atractiellomycetes is represented by the Bourdotigloea vestita complex. Species of Bourdotigloea are devoid of clamps but often possess well-differentiated cystidia, as well as long, cylindrical-fusiform basidiospores. Bourdotigloea encompasses nine species, of which six are described here as new.


INTRODUCTION
The Atractiellomycetes is a class among the rust fungi (Pucciniomycotina), with about 50 species (Aime et al. 2014, Toome-Heller 2016. The presence of saccate probasidia and unique ultrastructural elements (atractosomes), as well as absence of a yeast stage are the main diagnostic features of this group. The Atractiellomycetes encompasses representatives with pycnidioid (Basidiopycnis, Basidiopycnides), gasteroid (Atractiella, Phleogena) and corticioid (Helicogloea, Saccoblastia) fructifications, as well as a number of asexual taxa (Hobsonia, Infundibura, Leucogloea, Proceropycnis) (Bauer et al. 2006, Oberwinkler et al. 2006, Aime et al. 2014, 2018. For now, most of them are considered saprotrophic, inhabiting plant remnants or beetle galleries in decaying wood (Bauer et al. 2006, Oberwinkler et al. 2006, with a few taxa detected in orchid mycorrhiza (Kottke et al. 2010) or associated with tree roots (Bonito et al. 2017). However, the genus-and species-level taxonomy of the class remains extremely poorly resolved, and DNA data available in public repositories are fragmentary. In this paper, we revise the taxonomy of the Atractiellomycetes species producing effused, crust-like (corticioid) basidiocarps currently addressed to the genus Helicogloea s. lat., using morphological and DNA methods.
The genus Helicogloea was described by Patouillard (in Patouillard & Lagerheim 1892) with a single species, H. lagerheimii, based on Lagerheim's collection from Ecuador. In the protologue, Patouillard compared the new genus with Platygloea and Helicobasidium; however, he overlooked saccate probasidia characteristic for this species and the genus as a whole. This seems to have been a reason to abandon Helicogloea in favour of Saccoblastia, another genus described three years later from Brazil (Möller 1895). Möller introduced his genus for two new species, S. ovispora and S. sphaerospora, with excellent descriptions and illustrations of basidial morphology. He emphasized the importance of saccate probasidia as the main generic feature. Four new species were added to Saccoblastia by Bourdot & Galzin (1909), Coker (1928) and Linder (1929). Bourdot & Galzin (1928) assumed that Möller's generic description refers to species with floccose basidiocarps, and therefore separated Saccoblastia sebacea, having gelatinous fructifications, to a newly introduced section Saccogloea. Baker's study (1936) was a crucial moment in the taxonomic history of the genus. She conducted cytological investigations of all species then addressed to Saccoblastia and showed that they all are microscopically similar. Baker studied the original specimen of H. lagerheimii, the type species of Helicogloea, and proved that it possesses saccate probasidia. Therefore, the genus name Saccoblastia was replaced by the older epithet Helicogloea. Donk (1966) raised objections to such a wide genus concept and proposed to limit Helicogloea to gelatinous species only (i.e. section Saccogloea) while species with floccose, nonhygroscopic basidiocarps were retained in Saccoblastia. Donk's arguments were accepted by Jülich (1976), Wojewoda (1982) and Kisimova-Horovitz et al. (2000). Phylogenetic studies by Aime et al. (2006) and Bauer et al. (2006)

DNA extraction, amplification (PCR) and phylogeny
Extraction of genomic DNA was carried out using the NucleoSpin Plant II Kit (Macherey-Nagel GmbH & Co. KG), according to the manufacturer's instructions. ITS regions were amplified and sequencing with primers ITS1F-ITS4 (Gardes & Bruns 1993, White et al. 1990) and the nrLSU with primers JS1and LR5 (Landvik 1996; http://www.biology.duke.edu/fungi/mycolab/ primers.htm). PCR products were purified applying the GeneJET Gel Extraction Kit (Thermo Scientific, Thermo Fisher Scientific Inc., MA, USA). Sequencing was performed with an ABI model 3130 Genetic Analyzer (Applied Biosystems, CA, USA). Raw data were edited and assembled in MEGA v. 6 (Tamura et al. 2013). All steps of molecular studies were carried out at the Center for collective use of scientific equipment "Cellular and molecular technology of studying plants and fungi" (Komarov Botanical Institute, Russian Academy of Sciences, St. Petersburg). In case of specimens processed at the molecular lab of the mycology chair in the University of Tartu, the protocols described in Pärtel et al. (2016), were followed.

Phylogenetic analyses:
For this study, 51 nrITS and 34 nrLSU sequences were generated ( Table 1). The new sequences were aligned with additional related sequences downloaded from GenBank (Table 1) using MAFFT v. 7 web tool (http://mafft.cbrc.jp/alignment/server/) under the Q-INS-i option for both markers; the alignments were manually adjusted in MEGA v. 6.
Phylogenetic reconstructions were performed with Maximum likelihood (ML) and Bayesian (BA) analyses for two datasets (nrITS+nrLSU and nrITS). Before the analyses, the bestfit substitution model for the alignment was estimated based on the Akaike Information Criterion (AIC) using FindModel web server (http://www.hiv.lanl.gov/content/sequence/findmodel/ findmodel.html). GTR model was chosen for all datasets.
Maximum likelihood analysis (ML) was run on PhyML server (http://www.atgc-montpellier.fr/phyml/), under one hundred rapid bootstrap replicates. Bayesian analysis (BA) was performed with MrBayes v. 3.1 software (Ronquist & Huelsenbeck 2003), for two independent runs, each with 3 million generations with sampling every 100 generations, under described model and four chains. To quickly diagnose convergence of MCMC analyses and to get estimates of the posterior distribution of parameter values Tracer v. 1.6 was used (Rambaut et al. 2014).
Newly generated sequences have been deposited in GenBank with accession numbers listed in Table 1. The alignments have been deposited in TreeBASE (S22969 -nrITS + nrLSU and S22760 -nrITS dataset dataset).

Phylogeny
The phylogenetic analyses resulted in two trees for nrITS+nrLSU and nrITS datasets (Figs 1, 2). The phylogeny derived from the nrITS+nrLSU dataset (1 675 characters, including gaps) (Fig. 1) shows that members of Helicogloea s. lato (as advocated by Baker 1936) are spread among three lineages within the class. In general, these phylogenetic reconstructions follow the results of earlier studies (in particular, Aime et al. 2006, 2018, Bauer et al. 2006) demonstrating that Helicogloea is polyphyletic and Platygloea vestita (= Helicogloea vestita) belongs to the Atractiellomycetes. Most species of Helicogloea s. lat. included in the analyses form a strongly supported clade with a reference specimen of H. lagerheimii, the generic type ( Fig. 1, pp = 0.99, bs = 75 %), thus constituting Helicogloea s. str. The single representatives of Leucogloea (L. compressa) and Neogloea (N. variabilis) belong to Helicogloea, too, and therefore we consider these generic names taxonomic synonyms. The second clade is represented by Helicogloea farinacea and its closest relatives ( Fig. 1, pp = 1, bs = 100 %). Traditionally, this group was associated with the generic name Saccoblastia. However, we argue below that the latter generic epithet is not applicable to the H. farinacea complex, and a new genus Saccosoma is described to encompass it. Helicogloea vestita and its siblings form a third lineage in the phylogeny (Fig. 1, pp = 1, bs = 100 %) only distantly related to both Helicogloea s. str. and Saccosoma. As a consequence, they are separated into the recently described genus, Bourdotigloea. ITS dataset for the corticioid Atractiellomycetes (972 characters, including gaps) included sequences from 54 fungal samples (Fig. 2). The three major clades (Saccosoma, Bourdotigloea, Helicogloea) are highly supported and the  internal topology of these clades is well resolved and support the distinction of 26 accepted species.
DNA data obtained in the present study correspond well to the morphological evidence. All species of Helicogloea s. str. and all but one of Bourdotigloea spp. possess gelatinous, semitranslucent basidiocarps that turn to a vernicose crust after drying, while Saccosoma spp. have sturdier, arid fructifications, unchanged in dry condition (Fig. 3). In microscopic terms, presence of clamps and shape of basidia are of crucial value for recognizing crust-like genera of the Atractiellomycetes. Both Helicogloea and Bourdotigloea spp. are totally devoid of clamps; basidia are variably curved, with a terminal cell bearing   (Figs 4,5). However, almost all Bourdotigloea species treated below have differentiated cystidia and distinctly coloured (brownish), very thick-walled subicular hyphae, whereas Helicogloea lacks cystidia and hyphal colouration (subicular hyphae are in general less differentiated from subhymenial ones). In contrast, all hyphae in Saccosoma spp. are clamped, basidia are straight (not curved) and their terminal cell bears an apical sterigma (Fig. 6). Sporodochial asexual morphs (Leucogloea, Infundibura) have been so far detected in Helicogloea s. str. only (see Kirschner 2004 for a detailed discussion).
Even when Bourdotigloea and Saccosoma are separated from Helicogloea, the latter genus shows rather high morphological diversity and divergency of nrITS and LSU sequences. Therefore, we do not exclude that further splitting may become necessary. Fulfilling this task, however, implies much wider sampling of the Atractiellomycetes, especially of asexual taxa, and use of additional genetic markers. Basidiocarps resupinate, very thin, at first pruinose, then waxy to ceraceous. Hyphae clampless, in many cases rather shortcelled and variably inflated. Basal hyphae coloured (brownish), in a few species hyaline, slightly to very thick-walled, 7-19 μm  A B C diam, strongly cyanophilous. Subhymenial hyphae hyaline, thin-to slightly thick-walled, often easily collapsing, 3.5-8 μm diam, slightly to moderately cyanophilous. Basidia four-celled, tubular-clavate, straight or somewhat curved, with sharppointed, thick sterigmata; probasidia saccate. Cystidia as a rule present, tubular, more or less straight to somewhat sinuous or moniliform, decumbent but often with ascending apical part. Basidiospores cylindrical, fusiform or sigmoid, 12-36 × 4-10 μm, germinating by repetition.
On decayed wood, dead fructifications of other basidiomycetes and plant debris.

Type species: Bourdotigloea vestita (Bourdot & Galzin) Aime.
Notes: This genus is re-introduced here to embrace P. vestita and its closest relatives. The species of Bourdotigloea are morphologically most similar to Helicogloea spp. The latter ones differ in having narrower and not inflated, colourless hyphae, and their basidia bear more slender sterigmata. No differentiated cystidia are detected among Helicogloea species. Moreover, basidiospores of all known Helicogloea spp. (except H. angustispora) are ellipsoid/subglobose or broadly cylindrical, not fusiform.

Type species: Helicogloea lagerheimii Pat. (selected by Clements & Shear 1931).
Notes: As defined here, Helicogloea embraces 25 species with waxy or gelatinous, semitranslucent basidiocarps and clampless hyphae. Basal hyphae of almost all Helicogloea species treated here are wider than subhymenial ones, and they possess thicker walls. They are, however, colourless, and this feature, as well as absence of differentiated cystidia, differtiates Helicogloea from Bourdotigloea. Saccosoma species have floccose or dense, arid (non-gelatinized) basidiocarps and clamped hyphae, as well as straight (not curved) basidia.
Distribution and ecology: South America (Brazil -Santa Catarina); still corticated logs and branches.

Helicogloea pellucida
Notes: Höhnel (1907) described this species as Helicobasidium farinaceum based on single collection from Austria. It was almost forgotten until D.P. Rogers (in Martin 1944) studied the type and concluded it is the same species as Saccoblastia pinicola. Rogers moved H. farinaceum to Helicogloea, in accordance with the genus concept introduced by Baker (1936). It seems that Höhnel's original specimen does not exist anymore: it could not be located in FH and our request to W remained unanswered. Therefore, the only source for judging H. farinaceum sensu orig. is its protologue. Höhnel's description referred to a palecoloured, non-gelatinized, resupinate fungus of fine-granular, mealy consistence ("feinkörnig-mehlig") and possessing clamped hyphae. These statements preclude Helicogloea s.