Melanoleuca galbuserae, M. fontenlae and M. acystidiata—Three New Species in Subgenus Urticocystis (Pluteaceae, Basidiomycota) with Comments on M. castaneofusca and Related Species

Melanoleuca is one of the taxonomically most complicated genera of Agaricomycetes with several taxonomically lineages. The subgenus Urticocystis of the genus Melanoleuca contains species with either urticoid or absent cheilocystidia. In this paper, three new European species, Melanoleuca galbuserae, Melanoleuca fontenlae, and Melanoleuca acystidiata are described as new to science. Melanoleuca galbuserae, related to Melanoleuca stepposa and Melanoleuca tristis, was discovered in alpine grasslands in North Italy. The type specimens and recent collections of Melanoleuca angelesiana, Melanoleuca castaneofusca, Melanoleuca luteolosperma, Melanoleuca pseudopaedida, and Melanoleuca robertiana were sequenced and morphologically examined. Moreover, the related Melanoleuca microcephala and Melanoleuca paedida were included in morphological examination and DNA sequence analyses. All the species were delimited by macro- and micromorphological characters and the multigene phylogenetic analyses of a combined (ITS, rpb2, and tef1) dataset on the basis of the species tree estimation. In accordance with new molecular and morphological data, we suggest taxonomic reappraisal of M. pseudopaedida and M. robertiana, and M. fontenlae and M. acystidiata are proposed as new species. The differences between the type material of M. angelesiana from the USA and European M. angelesiana specimens are discussed.

This study represents a continuation of papers dealing with European taxa of Melanoleuca subgen. Urticocystis [2,3,5]. During mycological field excursions within the "43 • Comitato Scientifico Provinciale" held in Solda (Sulden), Bolzano-South Tyrol, Italy, in July 2018, and organized by the Bolzano group of the Associazione Micologica Bresadola, basidiomata of remarkable Melanoleuca were found in the alpine grassland with a dwarf willow (Salix herbacea). According to its macro-and micromorphological characters and the molecular data, it belongs to subgen. Urticocystis and represents a new species described here.
Melanoleuca castaneofusca Contu is a lesser-known species from Sardinia, Italy. The species has been known only from the type locality, but according to our records, it is distributed across several European countries. Melanoleuca castaneofusca is related to Melanoleuca luteolosperma (Britzelm.) Singer, Melanoleuca microcephala (P. Karst.) Singer, and Melanoleuca paedida (Fr.) Kühner & Maire. The subject of this paper is detailed taxonomical revision of these species including available type collections and recent specimens. The study is based on a macro-and micromorphological examination and a multigene phylogeny of both recent and type specimens. The information about ecology and distribution of all studied species are summarized here.

Morphological Dataset
Macroscopic descriptions, on the basis of fresh basidiomata, were made by the authors or collectors. Color abbreviations follow Kornerup and Wanscher [6], Munsell [7], and Küppers [8]; herbarium abbreviations follow Thiers [9]. Authors of fungal names are cited according to the Authors of Fungal Names page (http://www.indexfungorum.org/ AuthorsOfFungalNames.htm, accessed on 5 February 2021). The so-called finger test is used for the character of the pileus surface [10]. A positive test means that the finger leaves a clear imprint on the pileus surface. The caulohymenium, formed in particular on the stipe apex surface in some Melanoleuca species, is a layer composed of caulobasidioles, caulocystidia, and sporulating caulobasidia. It is comparable with the hymenium of the hymenophore in many respects [11]. Microscopic features were described from dried material mounted in KOH, Melzer's reagent, and Congo Red, using an Olympus BX-50 light microscope (Tokyo, Japan), Leica DM 1000 (Wetzlar, Germany), and Olympus BX41 with a magnification of 1000×. The SEM microphotograph of basidiospores was taken using a Tescan Mira 3 LMU electron microscope (Brno, Czech Republic). For basidiospores, the factors E (quotient of length and width in any one spore) and Q (mean of E-values) were used. Characters of cheilocystidia are defined according to Vizzini et al. [4] and Boekhout [12]. The notation [a/b/c] at the beginning of micromorphological data means (a) structures were measured from (b) basidiomata taken from (c) collections.

Molecular Dataset
DNA from dried fungal material was isolated, and DNA of 3 genes (ITS region of ribosomal RNA gene = ITS; translation elongation factor 1-alpha = tef1; RNA polymerase II, the second largest subunit = rpb2) was amplified in accordance with the work of Antonín et al. [2,3]. In the case of older type specimens, the genus-specific primers for the Melanoleuca-targeting ITS2 region (MELITS2F/MELITS2R) developed by Antonín et al. [2] were applied for amplification. The 2 datasets were the subject of phylogenetic analyses. The ITS-only dataset contained of all specimens including the type specimens, whereas the combined multilocus ITS-tef1-rpb2 dataset contained selected representatives of each species. The sequences were aligned using MAFFT, version 7 online program, setting up the Q-INS-i option [13].

Phylogenetic Analysis
Phylogeny for both datasets, multilocus (tef1, rpb2, and ITS) and ITS only, was inferred by means of the maximum likelihood and Bayesian methods.
Within the maximum likelihood analysis, we estimated best-fitting partitioning schemes and evolutionary models for each subset with PartitionFinder 2 [19] according to the corrected Akaike Information Criterion (AICc). For both datasets, the analysis was carried out for both linked and unlinked branch lengths and the results were exactly the same in the case of both datasets. The program was set to test the largest set of models possible, including models with base frequencies estimated using maximum likelihood (84 models in total, the option models = allx), and all possible partitioning schemes (the option search = all).
Phylogenetic inference estimated on the basis of the maximum likelihood method was produced in the RAxML-NG software [20]. The sufficient number of bootstrap replicates was determined automatically using the MRE-based bootstopping test [21] with the cutoff value 0.01 (the option-bs-cutoff 0.01). The maximum number of replicates was set to 5000 (the option-autoMRE{5000}). When the convergence was not reached, the analysis was run again for another 5000 bootstraps, both log files were concatenated, and the convergence was tested post hoc using the command bsconverge. We repeatedly added 5000 replicates until the convergence was reached. For the multilocus dataset, the bootstopping test converged after 6200 replicates. For the ITS only dataset, the convergence was not reached even after 30,000 for the cutoff value 0.01, but converged after 1700 trees for the cut-off value 0.03.
We calculated 2 support metrics: (1) Felsenstein's bootstrap and (2) Transfer Bootstrap Expectation [22]. The latter better revealed support for some branches, and it is displayed in the final trees.
Bayesian inference was computed using the BEAST 2 software [23]. For the ITS only dataset, the analysis was carried out using the Standard template. For the multilocus dataset, the species tree inference was performed via the StarBEAST package [24]. Partitioning schemes selected by PartitionFinder2 (see above) were used as an input for both of these analyses. In both analyses, the site models were chosen automatically through the model averaging implemented in the bModelTest package [25]. The posterior estimates of the parameters were summarized with Tracer [26]. The quality of posterior estimates was evaluated on the basis of estimated sample size (ESS) value and visual analysis of the trace plots. In the case of the ITS only dataset, we used the Markov chain Monte Carlo (MCMC) chain length of 50,000,000, which led to all parameters showing evidence of thorough sampling (ESS >> 1000). For the StarBEAST analysis, the number of MCMC generations resulting in sufficient sampling was much higher-we gradually increased the chain length to up to 400,000,000 when the ESS for all parameters was high enough. All estimates had ESS > 200, and the majority of them >> 1000. Both analyses were set up to sample both the tree and trace every 5000th state. Operators were always adjusted according to the program's suggestions in the output of the previous run. As a consensus tree method, we opted for the maximum clade credibility tree and produced it using the TreeAnnotator program (a part of BEAST 2). Burn-in was set to 25% and the height of each node to the mean height across the entire sample of trees for that clade.

Molecular Dataset and Phylogeny
The phylograms resulted from both maximum likelihood and Bayesian methods had almost identical topologies. Within the Bayesian framework, convergence for both datasets was assessed on the basis of the visual assessment of the trace plot and the estimated sample size (ESS) of the posterior estimates of the parameters; in all cases, the value was higher than 200.
The specimens used in molecular analyses are summarized in Table 1. The phylogenetic analyses of both datasets (Figures 1 and 2) resulted in independent and well supported positions of several Melanoleuca spp. insufficiently included in previous phy-logenetic studies and one species new to science. We succeeded in ITS sequencing of type specimens of Melanoleuca angelesiana A.H. Sm., Melanoleuca castaneofusca, Melanoleuca luteolosperma, Melanoleuca pseudopaedida Bon, and Melanoleuca robertiana. The holotype of M. pseudopaedida is conspecific with M. luteolosperma, and therefore M. pseudopaedida in current concept [3] is described here as a new species, Melanoleuca fontenlae. Surprisingly, sequence of M. robertiana holotype fell among the subgenus Melanoleuca (species with macrocystidia), and therefore a taxonomic solution of this problem was proposed.   The species tree of ITS-tef1-rpb2 genes of Melanoleuca species conducted by multispecies coalescent analysis (for the legend to numbers, see Table 1). Numbers at branches indicate maximum likelihood bootstrap proportion and Bayesian posterior probability values. The asterisks (*) mark low support (<75 in maximum likelihood; <90 in Bayesian analysis). The bar indicates the number of expected substitutions per site.
Diagnosis. Melanoleuca stepposa differs by a dark brown or gray-brown context in the stipe base; a well-developed caulohymenium; and different sequences of ITS, tef1, and rpb2 genes.
Ecology. On soil in alpine grasslands with Salix herbacea. Distribution. Thus far, M. galbuserae has been found only in three localities in North Italy, but its occurrence in neighboring alpine countries (Austria and Switzerland) is expected.
Melanoleuca fontenlae is identical with M. pseudopaedida sensu Vizzini et al. [3]. However, the type specimen of M. pseudopaedida, phylogenetically tallies to M. luteolosperma. Therefore, we consider M. pseudopaedida in the original sense as a synonymum of M. luteolosperma.
Melanoleuca Remarks. Bon [28] described Melanoleuca robertiana as an acystidiate taxon with basidiospores of (6.5)7-8.5(9) × (5)5.5-6(6.5) µm. The holotype was preserved in the Herbarium M. Bon, deposited in LIP (LIP 72092034). The majority of holotype material was not found in LIP, but a pocket with a small piece of holotype was glued to the original author's sheet with a description of the holotype, consisting of roughly one-quarter of one small basidioma. Bon mentioned the absence of any cystidia and basidiospores of 6-7.5(8) × 5-6 µm on this description accompanying the holotype material. Macro-and microscopic characters of the Melanoleuca robertiana published in the literature [28,29] indicates the possibility this species belonging to the M. castaneofusca group. However, our holotype revision revealed the presence of 60-69 × 14-18 µm large cheilocystidia and basidiospores of 8.0-9.5(10) × 4.5-5.3 µm in size. The ITS sequencing of the holotype was successful, and ITS sequence agreed with that of macrocystidioid M. pallidicutis Bresinsky holotype (TAAM 178616, MT270846) [30] belonging to the subgenus Melanoleuca. The obvious disagreements between these features of the studied piece of the type material and the characters described in the original protologue of M. robertiana indicate that the original description and holotype specimen refer to two different taxa. It is almost certain that a mycologist as experienced as M. Bon described in the protologue a different collection than that which represents the type material of M. robertiana. In the literature, M. robertiana has always been considered an acystidiate species [4,10,31,32] (the last as synonym of M. melaleuca). We are convinced that the material was mistakenly confused and a wrong basidioma was deposited in the herbarium envelope. Melanoleuca pallidicutis is a taxon based on an unambiguous well-defined description and whose morphological characters in protologue match with that of the holotype and the DNA sequence supports its expected taxonomic position (Antonín et al. in prep.). On the basis of these facts, we considered the name Melanoleuca robertiana Bon a nomen confusum because there is a substantial conflict in crucial characters (the presence/absence and character of cystidia and spores size) between descriptions published in the protologue, other literature [10,28,29], and the type specimen.
Vizzini et al. [4] included, under the name M. robertiana, the sequence of a fungus collected in Italy and with macro-and microscopic agreement with the original description by Bon [28]. It is different from any other species of the Urticocystis Boekhout [2][3][4][5]. We decided to describe it as a new species here.
Distribution. Melanoleuca acystidiata has been found only in three montane localities in the north of Italy and Switzerland.
Additional specimens examined. Italy: Piemonte, Cuneo Distr., Bosco delle Navette di Ormea, alt. Remarks. Melanoleuca acystidata is characterized by a more or less dark brown pileus; whitish lamellae; a stipe shorter or longer than the pileus width that is whitish when young, then light brown; a white context; subglobose; broadly ellipsoid or ovoid basidiospores, 7.2-9 × 6-7.2 µm in size; the absence of any hymenial cystidia; a pileipellis in the form of an (ixo)cutis with adpressed to erect terminal cells; and one type of clavate or cylindrical caulocystidia.
Ecology. On soil under Ulmus (BRNM 761900), in a greenhouse on a bare soil (BRNM 761901), on sandy soil under Picea (LIP RC86021), in a flowerpot with Mentha sp. in an urban apartment (SLO 1639), on composted soil in a cemetery [35], and on soil and shredded bark mulch in a botanic garden (K(M) 92562); holotype on basic sandy soil near Cactaceae in the botanic garden (CAG 921103-01). The species may be associated with commercial soil and compost substrates.
Distribution. This species is now confirmed from the Czech Republic, France, Great Britain (England), Italy, and Slovakia until now. However, we suppose it is more broadly distributed in similar habitats. Remarks. Melanoleuca castaneofusca, described from Sardinia [36], is characterized by rather robust basidiomata with a dark-colored pileus; cream lamellae; an apically distinctly floccose to floccose-tomentose, brownish to gray-brown stipe; whitish and brownish yellow tinged context in the stipe base; rather small basidiospores; urticoid cheilocystidia of the brevipes-and exscissa-type; a pileipellis in the form of an ixocutis; and well-developed caulohymenium. This species produces its basidiomata in late autumn in habitats strongly influenced by humans or at completely artificial places (e.g., greenhouse, cemetery, botanic garden). In recent literature, only one collection (Great Britain, Surrey, Morden Cemetery, October 2012 leg. T. Brown) was published [35]; it was identified on the basis of our sequences.
Melanoleuca malenconii Bon is macroscopically somewhat similar but differs by pale to grayish yellow lamellae when mature; a slightly pruinose-pubescent to distinctly floccose, sometimes especially in lower part floccose-hairy stipe that is rarely subglabrous at apex; the presence of pleurocystidia; longer basidiospores (up to 10 µm); and sometimes also by relatively smaller, paler basidiomata [2,28]. Melanoleuca humilis (Pers.) Pat. is morphologically similar species different in its grayish or beige lamellae in mature basidiomata, a (dark) brown context in the stipe base, longer basidiospores (up to 10 µm), and often also a smaller pileus [2]. Lectotype. Britzelmayr, Bericht der Naturhistorischen Vereins in Augsburg 31: Table  647, 1894 [37].
Ecology. On soil, under Picea abies and Fagus sylvatica in a montane forest and on the riverbank under Populus, Sambucus, and Alnus (Slovakia), on the riverbank among grass, Dryas, and Picea (Slovenia, along the path in decaying remnants of grasses; under Juglans regia, Rosa, and Clematis (Italy); sandy soil under Populus (Italy); under Cistus ent from M. pseudopaedida sensu Vizzini et al. [3], which is described as a new species M. fontenlae above.
Ecology. On soil in the montane and alpine belt, on a pasture with scattered Picea, in an alpine meadow, in mosses and vegetation along the montane stream, in grass in spruce forest, and along the forest path. It seems to be a montane calciphilous species.
Distribution. It is known from the montane and alpine belt on altitudes of ≈1500 m or more a.s.l. It is only confirmed from Finland (lectotype), Italy, and Slovakia until now.
Additional specimens examined. Italy: South Tyrol, Schlern-Rosengarten Naturpark Remarks. Melanoleuca microcephala is characterized by moderately large basidiomata with a (grayish) brown, beige-gray, grayish, or uniformly grayish brown pileus; whitish to pale cream lamellae; a gray-brown to dark gray-brown stipe; a dark (reddish)brown to black-brown context in the stipe base and the absence of cheilo-and pleurocystidia and the absence of caulohymenium. Two cystidioid elements, 35-36 × 7.0-8.0 µm, on the lamellar edge were found only in the Italian collection (Italy, Tiers, BRNM 817786). However, they were not typically urticoid cells, but structures such as a transient form between cystidia and marginal cells. An atypical pale orangish yellow coloring of the stipe after touching appeared in the Slovak collection (Ružomberok, BRNM 817788). Caroti et al. [41] published a collection of M. microcephala with a small, 15-20 mm broad, dark brown pileus with a white outermost margin and scattered urticoid cheilo-and pleurocystidia. However, this collection was not confirmed by phylogenetic studies and its identity is unclear.
In comparison with phylogenetically similar species, Melanoleuca paedida differs by an ochraceous fawn to pale or dark gray-brown pileus, a stipe of the same length or shorter than the pileus diameter, a slightly darker context in the stipe base, and slightly smaller basidiospores; the collection with cystidia as well as the acystidiate collection are known in this species. Melanoleuca stridula has a uniformly rather dark, yellow-brown, (gray-)brown, or dark brown pileus and smaller basidiospores, 6.5-8(9) × 5-6 µm, average 7.5 × 5.5 µm (our unpublished studies, [31,33,34]). Melanoleuca acystidiata has a dark brown pileus; a whitish, then light brown stipe; and a dark brown context in the stipe base. Among other species without cystidia, M. brachyspora Harmaja differs by a differently colored, brownish gray or brown to dark brown pileus; a white context in the stipe base; and slightly smaller basidiospores, (6.5)7.0-9.0 × 4.5-6.5 µm, average = 7.5 × 5.5 µm. The European collections of M. angelesiana differs by a whitish context in the stipe base and large basidia and basidioles (30-55 × 10-12 µm) (our unpublished studies, see also above).
Pileus 30-60 mm broad, convex, soon flattened, then with depressed center, with absent or very reduced umbo and entire margin, glabrous (silky under lens, positive finger test), dry, opaque, ochraceous fawn to pale or dark gray-brown, with darker center. Lamellae moderately close to close, with numerous lamellulae of various lengths, emarginate and attached with tooth, high, straight, whitish or brownish gray, with concolorous edge.
Ecology Remarks. Melanoleuca paedida is characterized by an ochraceous fawn to pale or dark gray-brown pileus with darker center; whitish or brownish gray lamellae; a stipe shorter or of the same length as the pileus diameter; brownish, ochraceous orange, or concolorous with the pileus; a slightly darker context in the stipe base, moderately large, ellipsoid, or subglobose basidiospores; and lacking caulocystidia. Cheilo-and pleurocystidia are either present (ANC M0189) or absent (010907-02). The variability of the latter character requires further study. Therefore, the epitype is not proposed here. The identity of this species is based on the studies by Vizzini et al. [4].

Discussion
Melanoleuca is an example of an agaricomycete genus where molecular revision of type specimens is crucial for species concept and species recognition [42]. The several European Melanoleuca species proposed by Bon (e.g., [10,28]) were later synonymized [2][3][4] due to results of DNA sequence analyses. Although taxonomy of Melanoleuca in Europe has a long tradition, a new can be discovered here, e.g. Melanoleuca juliannae Rimóczi et al., was described recently [5]. Both recently described species M. galbuserae and M. juliannae share a preference for the specific grassland habitats (alpine grasslands or Pannonian sand grasslands), although their habitat preferences may be broader. Ecological differences can be found also among other species. Melanoleuca fontenlae prefers sandy soil and dunes, M. acystidiata and M. microcephala seem to be restricted to montane/alpine habitats, M. paedida prefers soils with coniferous litter, and M. luteolosperma occurs in a wide range of habitats. M. castaneofusca has been found in many habitats affected by human activity and may be dispersed by commercial substrates.
Melanoleuca is an extremely difficult genus for species identification due to overlapping morphological characters; the most useful distinguishing characters are noted here. The context color at the stipe base is the most important macroscopic characters in Melanoleucain the M. castaneofusca group, M. acystidiata and M. paedida have a white context, while M. fontenlae, M. microcephala, and M. luteolosperma differ by a darker context; M. castaneofusca has a whitish context with a brownish yellow or pale orange tinge. In the M. exscissa group, Melanoleuca galbuserae has a white to only slightly grayish context in the stipe base, which is similar to Melanoleuca exscissa and Melanoleuca rasilis; other European species (Melanoleuca diverticulata, Melanoleuca stepposa Vacek, and Melanoleuca tristis) have a dark-colored stipe base context [3]. The white stipe base context is also known in M. porphyropoda described from China, but has also been reported in the UK on the basis of their morphological features [16]. Melanoleuca galbuserae and M. porphyropoda seem to be related according to ITS data (Figure 1). Moreover, caulohymenium [11] is a useful distinguishing character in Melanoleuca-M. stepposa and M. tristis differ from M. galbuserae and M. griseobrunnea by the well-developed caulohymenium [3]. Such caulohymenium can be found also in M. castaneofusca, M. fontenlae, and M. luteolosperma.
Melanoleuca castaneofusca can be well distinguished also by a distinctly floccose to floccose-tomentose stipe apex and a pale-colored stipe; other related species (M. luteolosperma, M. paedida, M. fontenlae) have a darker-colored stipe with only a finely pruinose to pruinose-flocculose apex.
The basidiospore dimensions are crucial for identification in many agaricomycetes, but in Melanoleuca, this character may be confusing, because both, four-and two-spored basidia can occur in one hymenium and respective spores differ in their dimensions [43]. Melanoleuca paedida has both two-and four-spored basidia in the hymenium; only fourspored or only rare two-spored basidia were found in other species. Two species, M. microcephala and M. acystidiata, consistently lack cheilocystidia, but both cystidiate and acystidiate collections are known in M. paedida.