Identity of the subalpine–subarctic corticioid fungus Megalocystidium leucoxanthum (Russulales, Basidiomycota) and six related species

Background and aims – To date, Megalocystidium leucoxanthum, a corticioid fungus originally described from the Italian Alps, was considered as a widely distributed species inhabiting numerous angiosperm hosts in the northern hemisphere. Its specimens collected in different geographic areas and from various host species revealed a high morphological variability and thus obfuscated differences from the closely related M. luridum. The objective of this study was to re-establish M. leucoxanthum based on newly collected and sequenced specimens and clarify the identity of morphologically deviating collections previously ascribed to this species. Material and methods – In total, 87 specimens of Megalocystidium spp. (including two historical types) were studied by morphological methods. Their phylogenetic relations were investigated based on DNA sequences (nrITS, nrLSU, and tef1) of 29 specimens. Key results – Based on morphological, ecological and DNA data, we showed M. leucoxanthum sensu typi is a rare species restricted to Alnus alnobetula in subalpine and subarctic zones. Consequently, records from other hosts (mostly representatives of Salicaceae) belong to three other species, M. olens, M. perticatum, and M. salicis, described as new to science. The fourth newly introduced species, M. pellitum, occurs on the same host tree as M. leucoxanthum but it can be separated from the latter due to distinctive morphological traits and DNA sequences. Additionally, Aleurodiscus diffissus is combined in Megalocystidium and the identity of M. luridum is clarified.


INTRODUCTION
Megalocystidium Jülich is a genus of corticioid fungi typified with Corticium leucoxanthum Bres. Initially introduced for three species, Megalocystidium currently embraces ten species (Jülich 1978;Ginns & Freeman 1994). As redefined in phylogenetic studies, it belongs to the family Stereaceae of the Russulales and encompasses crust-like fungi with clamped hyphae, long and deeply rooted gloeocystidia, as well as narrowly ellipsoid or cylindrical, smooth, strongly amyloid basidiospores (Larsson et al. 2004;Larsson 2007). The type species was described from the Italian Alps as growing on twigs of Alnus alnobetula subsp. alnobetula (= Alnus viridis) (Bresadola 1898).
Two European representatives of Megalocystidium, M. leucoxanthum (Bres.) Jülich, and M. luridum (Bres.) Jülich have been described in the literature as morphologically differentiated mainly due to the basidiospore length (above 10 μm and under 10 μm, respectively) and host preferences (Alnus or Salix for M. leucoxanthum versus mainly Fagus for M. luridum) (Bourdot & Galzin 1927;Eriksson & Ryvarden 1975;Bernicchia & Gorjón 2010). However, identity of North European specimens collected mostly from wood of Salicaceae and having basidiospores of an intermediate size has been interpreted in two different ways. Litschauer (in herb.) was apt to associate Corticium leucoxanthum (as Gloeocystidium leucoxanthum (Bres.) Höhn. & Litsch.) with specimens found on A. alnobetula in the subalpine zone of Europe. In turn, he labelled the problematic North European material as Gloeocystidium luridum (Bres.) Höhn. & Litsch. (see Eriksson & Ryvarden 1975). Litschauer's viewpoint was accepted by Eriksson (1958). On the other hand, Parmasto (1968) and Eriksson & Ryvarden (1975) assigned the North European collections to Gloeocystidiellum leucoxanthum (Bres.) Donk (= M. leucoxanthum) although they stressed morphological variability of the latter species. No attempts to verify these hypotheses with the use of phylogenetic methods have so far been performed.
In the present paper, we revise the taxonomy of the M. leucoxanthum-M. luridum complex in temperate-boreal Eurasia based on morphological, ecological/geographic, and DNA data. Additionally, we provide new evidence on the taxonomic position of Aleurodiscus diffissus (Sacc.) Burt.

Morphological study
Type material and specimens from herbaria H, S, O, GB, LE, PRM, TAAM, and OULU were studied. Herbarium acronyms are given according to Thiers (continuously updated). All measurements were made from microscopic slides mounted in Cotton Blue and Melzer's reagent (IKI), using phase contrast and oil immersion lens (Leitz Diaplan microscope, 1250× amplification). The following abbreviations are used in morphological descriptions: L -mean basidiospore length in the specimen measured, W -mean basidiospore width in the specimen measured, Q -mean length/width ratio in the specimen measured, n -number of measurements per specimens measured.

DNA extraction, amplification, and sequencing
DNA extractions were performed from small fragments of herbarium specimens using the NucleoSpin Plant II Kit ( Macherey-Nagel GmbH and Co. KG, Düren, Germany) according to the manufacturer's protocols. The following primers were used for both amplification and sequencing: the primers ITS1F and ITS4B (Gardes & Bruns 1993) for the ITS1-5.8S-ITS2 region, primers EF1-983F andEF1-1567R (Rehner &Buckley 2005) for a part of the tef1 region, and primers LROR-LR5 (White et al. 1990;Vilgalys & Hester 1990) for D1-D3 domains of the nrLSU region. Purification of the PCR products was done with the GeneJET PCR Purification Kit (Thermo Fisher Scientific, Lithuania). Sequencing was performed with an ABI model 3130 Genetic Analyzer (Applied Biosystems, CA, USA). The raw data were edited and assembled in MEGA v.7 (Kumar et al. 2016).
Phylogenetic reconstructions were performed with Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. Before the analyses, the best-fit substitution model for each alignment was estimated based on the Akaike Information Criterion (AIC) using the FindModel web server (http://www.hiv.lanl.gov/content/sequence/findmodel/ findmodel.html). The GTR model was chosen for all datasets. Maximum likelihood analysis was run on RAxML servers, v.0.9.0 (Kozlov et al. 2019) with 1000 rapid bootstrap replicates. Bayesian analyses was performed with MrBayes v.3.2.5 software (Ronquist et al. 2012), for two independent runs, each with 5 million generations, under described model, and four chains with sampling every 100 generations.
To check for convergence of the MCMC analyses and to get estimates of the posterior distribution of parameter values, Tracer v.1.6 was used (Rambaut et al. 2014). We accepted the result where the ESS (Effective Sample Size) was 9782 for nrITS dataset, 11435 for nrITS + nrLSU dataset, 11303 for nrITS + tef1 dataset, and 14736 for tef1 dataset, and the PSRF (Potential Scale Reduction Factor) was close to 1.
Newly generated sequences have been deposited in GenBank with corresponding accession numbers (table  1). Sequenced specimens are marked in the Taxonomic Treatment by an asterisk.
(1) nrITS + nrLSU dataset for ten genera accepted in Stereaceae (as outlined by Larsson et al. 2004 andLarsson 2007). The final alignment contained 1696 characters (including gaps). The overall topologies of the ML and BI trees were identical and recovered Aleurodiscus diffissus (Sacc.) Burt as a member of a strongly supported Megalocystidium clade (BS = 99, PP = 1) ( fig. 1). The latter species was first introduced as Peniophora diffissa (Saccardo 1889) and then moved to Aleurodiscus due to the presence of acanthophyses, sterile hymenial cells with characteristic thorn-like outgrowths. However, this feature alone is not enough for justifying generic limits in Stereaceae because acanthophyses have been detected in almost all other genera of this family. Otherwise, A. diffissus is microscopically highly similar to M. leucoxanthum and M. luridum, and therefore we combine it in Megalocystidium.

Species
Specimen (     (2) ITS only dataset for Megalocystidium spp. The final alignment contained 776 characters (including gaps). The overall topologies of the ML and BI trees clearly divided all included specimens in two strongly supported groups, i.e. Megalocystidium diffissum clade and M. leucoxanthumluridum clade. Within the latter one, two strongly supported lineages corresponding to M. leucoxanthum s.s. (i.e. containing Alnus-dwelling specimens from subalpine or subarctic areas) and M. luridum were detected ( fig. 2). However, the rest of ITS sequences derived mainly from specimens collected from Salix spp. do not group into wellsupported clades. Morphological and genetic variability of this lineage prompted us to use one more marker (tef1).
(3) tef1 only dataset for Megalocystidium spp. The final alignment contained 595 characters (including gaps). The overall topologies of the ML and BI trees were identical. They split the aforementioned M. leucoxanthumluridum clade into six strongly supported lineages that we taxonomically interpret as separate species ( fig. 3). This interpretation is supported by morphological (both macroscopic and anatomical), ecological (host specificity) and, to some degree, geographic data as discussed below. Four of these species (M. olens, M. pellitum, M. perticatum, and M. salicis) are described as new to science.
The final alignment contained 1371 characters (including gaps). The overall topologies of the ML and BI trees were identical and they support our conclusions based on tef1 only dataset ( fig. 4).

Morphology, ecology, and geography
In the M. leucoxanthum (1889) described this species as Peniophora diffissa based on a single specimen collected by Nikolai Martiyanov in Siberia, seemingly in the present-day Krasnoyarsk Region. The collection was evidently sterile but its identity is doubtless due to the peculiar basidiocarp shape, presence of acanthophyses, and the specific host (Rhododendron dauricum). We could not trace any authentic material of P. diffissa in public herbaria, and therefore we select Saccardo's illustration as a lectotype of this species. The holotype of Aleurodiscus sajanensis (Pilát 1931) was collected in the same geographic area as P. diffissa and agrees perfectly with the protologue of the latter species and our newly collected specimens. Megalocystidium diffissum is one of the most common species inhabiting dead but still attached branches of Rhododendron dauricum in the mountain regions of East Asia. Its basidiocarps die soon after branches are detached. Description -Basidiocarps annual or persistent, resupinate, first orbicular or frustulate, then fusing and producing crustaceous fructifications, 0.5-10 cm in widest dimension, 0.3-1 mm thick, leathery. Margin abruptly delimited from the substrate, adnate, 0.5-1 mm wide, first white, in older basidiocarps concolorous with the hymenial surface. Subiculum white, leathery, 0.1-0.2 mm thick. Hymenial surface cream-coloured to beige or ochraceous-brownish, often distinctly tuberculate, irregularly cracking with age. Smell weak, anise-like, or absent. Hyphal structure monomitic; hyphae clamped, 4-5(-5.5) μm in diam., thinto moderately thick-walled in subhymenium, thick-walled (wall up to 2 μm thick) in subiculum. Gloeocystidia usually moniliform, rarely clavate, thin-to clearly thick-walled, 60-120 × 7-8.5 μm. Hyphidia simple or bi-or trifurcate, rare, embedded in or slightly projecting above the hymenial layer, 4-5.5 μm in diam. Basidia clavate, 45-68 × 8-11 μm. Basidiospores hyaline, thin-walled, cylindrical to narrowly ellipsoid, (12.8-)13. 1-18.1 (-18.2) × (4.7-)4.9-7.2 (-8.0) μm (n = 120/4), L = 14. 54-16.81 Notes -According to our results, M. leucoxanthum is a species inhabiting dead, often still attached branches of A. alnobetula in subalpine and subarctic zones. It was found once on A. hirsuta but this record comes from an area where A. alnobetula is present, too. We interpret this finding as an accidental host change. Macroscopically, M. leucoxanthum is indistinguishable from M. pellitum but the latter species can be easily recognized due to clearly larger basidia and wider basidiospores. Microscopically, M. leucoxanthum is almost identical to M. perticatum. However, M. perticatum produces thinner basidiocarps with an indistinct subicular layer and it occurs on Salix spp. Eriksson (in Eriksson & Ryvarden 1975) studied and depicted original material from the Bresadola collection at S but did not give any details on the specimen. From a fragment preserved in GB we know that the specimen Eriksson studied and called type specimen is the one we here select as lectotype. Ginns & Freeman (1994) referred to another specimen from herb S as the type but a specimen with the label information they reported ("Italy: Alpes, viii.1894, G. Bresadola s.n.") does not exist in S, neither in FH where an isotype should be stored according to them. It is not possible to decide what material Ginns & Freeman studied and we therefore regard their selected type as lost.

CONCLUSION
In the present paper, we re-described M. leucoxanthum as a species restricted to a particular host tree, A. alnobetula, and introduced six other taxa, four of them as new to science. These results were based on newly collected material from Eurasia studied by morphological and molecular methods. Using tef1 region was crucial for the species delimitation in this group, and ecological and geographic data provided additional arguments for our species-level taxonomic conclusions. At the generic level, Megalocystidium was emendated to encompass one species with acanthophyses, M. diffissum.
However, the species diversity in M. leucoxanthum complex would be higher if the North American specimens are taken into account. We tentatively named a few Canadian collections available to us as belonging either to M. leucoxanthum s.s. or to M. pellitum. These specimens show no essential morphological differences from the Eurasian material so labelled but unfortunately our attempts to sequence them failed. Ginns & Lefebvre (1993) and Ginns & Freeman (1994) reported M. leucoxanthum from many geographic regions of North America. In these publications, the host list included not only various deciduous trees but also conifers (Picea engelmannii). This is a clear indication that the M. leucoxanthum complex in North America requires a closer look.

SUPPLEMENTARY FILE
Supplementary file 1 -Geographic distribution of Megalocystidium spp. https://doi.org/10.5091/plecevo.2021.1857.2457 sequencing several specimens. The research of the author SV was supported by the Grant of the President of the Russian Federation (grant no. МK-3216.2019.11) and the institutional research project of Komarov Botanical Institute RAS (project АААА- А19-119020890079-6).