Melanoleuca subgriseoflava and M. substridula—two new Melanoleuca species (Agaricales, Basidiomycota) described from China

Two new Melanoleuca species, Melanoleuca subgriseoflava and M. substridula, are originally reported and described in China based on both morphological and molecular methods. Melanoleuca subgriseoflava, collected in Liaoning province, is mainly characterized by its greyish-brown to yellowish-grey pileus, creamy to light orange lamellae, greyish-yellow context, round and warted basidiospores and fusiform hymenial cystidia. Melanoleuca substridula, discovered in Sichuan province, is mainly characterized by its light brown to dark brown pileus, whitish lamellae, light brown to greyish-brown stipe, round and warted basidiospores and lack of any forms of cystidia. The phylogenetic relationships as well as divergence-time estimation were analyzed using the combined data set (ITS-nrLSU-RPB2), and the results showed that the two Melanoleuca species formed two distinct lineages. Based on the combination of morphological and molecular data, M. subgriseoflava and M. substridula are confirmed as two new species to science. A theoretical basis is provided for the species diversity of Melanoleuca.

Despite considerable evidence that the Melanoleuca genus belongs to a monophyletic group, the infrageneric classification system of the genus has always been controversial. Singer (1986) divided the genus into four sections circumscribed only by pileus color and stipe ornamentations, i.e., sect. Alboflavidae Singer, sect. Humiles Singer, sect. Oreinae Singer and sect. Melanoleuca Pat. Bon (1978), the first to take micro-morphological characters into consideration, divided the genus into seven sections. As Boekhout (1988) emphasized the crucial role of cystidia, the genus was divided into three subgenera according to the absence/presence and shape of cystidia, i.e., subgen. Macrocystis Boekhout, subgen. Melanoleuca Pat. and subgen. Urticocystis Boekhout. However, these taxonomical units are not supported by molecular data. Vizzini et al. (2011), using a large number of ITS sequences to construct phylogenetic relationships of Melanoleuca, defined only two subgenera, i.e., subgen. Urticocystis Vizzini and subgen. Melanoleuca Vizzini. Then, follow-up studies on species of Melanoleuca support the classification opinion proposed by Vizzini et al. (2011) (Yu et al., 2014;Kalmer, Acar & Dizkirici, 2018;Xu et al., 2019).
Only 31 species of Melanoleuca have been reported in China (Bau & Li, 1999;Zhang, Li & Song, 2001;Chen, 2007;Mao, 2009;Sun et al., 2012;Wang, 2013;He et al., 2014;Yu et al., 2014;Zhao et al., 2014;Wei, Fan & Yan, 2015;Du et al., 2016;Tian et al., 2018;Xu et al., 2019;Pei et al., 2021). Although China has a complex climate and geographical conditions, species resources of Melanoleuca remain scarce. This study reports and describes two Melanoleuca species collected from Liaoning province and Sichuan province in China from 2019 to 2020. In order to confirm the two collections as new to science, both morphological and method analyses were conducted. The morphological similarities and differences between the two species and other related species are also discussed.

Specimens and morphological description
Fresh basidiomata were photographed in the field. Specimens were dried with an electric drier and deposited with silica gel. Dried specimens were preserved in the Fungal Herbarium of Shenyang Agricultural University (SYAU-FUNGI), Liaoning, China and Herbarium of Microbiology Institute of Guangdong (GDGM), Guangdong, China. Tissue blocks were removed from the inner part of the dried specimens for DNA analyses. Color abbreviations followed Kornerup & Wanscher (1963). Methods for morphological observation followed Pei et al. (2021). For observation of surface of the basidiospores, SEM microphotographs were performed using a scanning electron microscope (REGLUS 8100; Hitachi, Tokyo, Japan).

Nomenclature
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. In addition,  Matheny et al. (2007) b6F CCCATRGCYTGYTTMCCCATDGC Matheny et al. (2007) new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix ''http://www.mycobank.org/MycoTaxo.aspx?Link=T{&}Rec=''. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central SCIE, and CLOCKSS.''

Divergence time estimation within Melanoleuca
Divergence time was estimated using BEAST v2.6.3 (Bouckaert et al., 2014). BEAUTI v2.6.3 was used to construct an XML file. ModelFinder (Kalyaanamoorthy et al., 2017) was used to infer the best substitution model. The clock model and substitution model were chosen following Pei (2021) and Zhao et al. (2016). On the calibrated nodes, the offset ages of 98 and 110 Ma were set for the genus Melanoleuca and Pluteus, respectively (Pei, 2021). We

Phylogenetic analyses
The GenBank accession numbers of the sequences, determined in this study, are from ON262569 to ON262573 and ON220896 to ON220899 (Table 2). Maximum likelihood (ML) and Bayesian Inference (BI) showed almost identical topologies and the BI tree was selected for display ( Fig. 1). The phylogenetic result suggested that the Melanoleuca should belong to a monophyletic group (PP = 1, BS = 100), which is consistent with the previous studies (Yu et al., 2014;Vizzini et al., 2011). A total of five clades (Clade A to Clade E) can be recognized within Melanoleuca, which is in line with the result of Pei et al. (2021). MycoBank No. MB843803 (Fig. 3) Etymology The epithet ''subgriseoflava'' refers to the greyish-brown color of the pileus, which is similar to the species Melanoleuca griseoflava. Diagnosis: Pileus convex to applanate to depressed at center, greyish-brown to yellowishgrey pileus; lamellae adnate to sinuate, creamy to light orange; stipe yellowish-brown to Melanoleuca grammopodia JX429194 JX429179 -

Remarks:
The main features of M. subgriseoflava are its greyish-brown to yellowish-grey pileus, creamy to light orange lamellae, greyish-yellow context, basidiospores with scattered warts and fusiform hymenial cystidia. On account of the pileus color, M. subgriseoflava is closely related to M. griseoflava originally described in northeastern China by Pei et al. (2021). Nevertheless, M. griseoflava, differs from M. subgriseoflava by its adnate to adnexed and white lamellae. Besides, M. griseoflava differs by the presence of whitish tomentum at the stipe base . Micromorphologically, M. griseoflava is also distinct from M. subgriseoflava by its almost reticulate surface ornamentations of basidiospores and the presence of caulocystidia .  Diagnosis: Basidiomata slightly small; pileus umbonate, brown to dark brown; lamellae sinuate to adnate, white; stipe light brown in upper part and grey-brown in lower part; basidiospores with round and scattered warts and lack of any forms of cystidia. Holotype: CHINA. Sichuan Province: Jiuzhaigou valley reserve, on the soil in meadows, 19 Sep 2020, Ming Zhang (GDGM 84648). Description: Basidiomata slightly small-sized. Pileus 21-38 mm diam.; umbonate at first; margin first slightly inflexed, soon becoming straight, depressed when mature and dry; surface glabrous, light brown at first (5D5 to 5D7), becoming brown to dark brown (7E8 to 7F8) when mature, often darker at margin, with a conspicuous dark brown (6F8 to 7F8) umbo at centre. Lamellae crowded, sinuate to adnate, 3.0-4.0 mm broad, white, edge entire and concolorous, with lamellulae of two or four lengths. Stipe cylindrical and somewhat broadened downwards, 26-38 mm long × 4.0-−5.0 mm diam., central, solid, light brown (6D4 to 6D8) in upper part, becoming grey-brown (5E3 to 6E3) towards base, with whitish flocculose apex, longitudinally striate, with whitish basal tomentum. Context up to 30-50 mm thick near stipe attachment, thinner at margin, white. Odor faint. Spore deposit whitish.

Remarks:
The most distinctive characteristics of Melanoleuca substridula are its slightly small-sized basidiocarp, light brown to dark brown pileus with a prominent umbo, sinuate to adnate lamellae, light brown to greyish-brown stipe and lack of cystidia. On account of the pileus color, M. substridula is closely related to M. stridula originally described by Singer (1943). However, M. stridula is featured by a slightly larger pileus (15-60 mm in diameter) (Singer, 1943). Additionally, M. stridula is often characterized by a pileus with a center depression, which differs by the umbonate pileus of M. substridula. Microscopically, M. stridula can be distinguishable from M. substridula by the presence of subcylindrical cystidia-like cells at the apex of the stipe (Metrod, 1949).

DISCUSSION
Two new species, Melanoleuca subgriseoflava and Melanoleuca substridula, discovered and collected in Liaoning province and Sichuan province respectively, were originally reported and described in this study. Morphologically, the most distinctive features of M. subgriseoflava are a grey-brown to yellowish-grey pileus, creamy lamellae, fusiform hymenial cystidia and warted basidiospores. According to the classification system of Boekhout (1988), M. subgriseoflava should belong to the section Strictipedes in the subgenus Macrocystidia because of the presence of grey-brown pileus and fusiform hymenial cystidia (Boekhout, 1988 (Boekhout, 1988).
Melanoleuca substridula is easily recognized by its light brown to dark brown pileus, prominent umbo, adnate to sinuate lamellae, light brown to grey-brown stipe and acystidiate micromorphology. For lack of any forms of cystidia, M. substridula belongs to the subgenus Melanoleuca (Boekhout, 1988 (Boekhout, 1988). The latter three species also differ by their larger basidiomata, with a pileus diameter of 35-65 mm. Moreover, M. striimarginat a differs by a striate margin of the pileus (Metrod, 1942).
Both the phylogenetic relationships and the divergence-time estimation, based on three regions (ITS-nrLSU-RPB2), showed that there are five clades in the genus Melanoleuca ( Figs. 1 and 2), which was corroborated by Pei et al. (2021). According to the phylogram, M. subgriseoflava is closely related to the other three species with high support in clade E, i.e., M. arcuata, M. heterocystidiosa and M. griseoflava. Melanoleuca arcuata differs by its brick-red pileus and decurrent lamellae (Fries, 1821). Melanoleuca heterocystidiosa can be easily separated from M. subgriseoflava based on its smaller basidiomata, with a pileus diameter of 15 mm (Singer, 1939;Bon, 1984). Melanoleuca griseoflava differs from M. subgriseoflava as elaborated above. In clade A, with the exception of M. microcephala, M. substridula is far away from the other species of Melanoleuca. However, M. microcephala can easily distinguish from M. substridula by its longer stipe with a length of 105 mm. Besides, caulocystidia in groups can be observed in M. microcephala, but not any forms of cystidia in M. substridula (Antonín et al., 2021).
In the present study, five clades can be recognized in both the BI tree (Fig. 1) and the MCC tree (Fig. 2). However, using phylogenetic analyses, species of Melanoleuca were divided into two clades in former research (Vizzini et al., 2011;Yu et al., 2014;Nawaz, Jabeen & Khalid, 2017;Xu et al., 2019). The species of Melanoleuca within each clade have lacked uniform characteristics to work in identification. In order to clarify the infrageneric classification, taxonomic treatments should be performed based on additional materials and complete morphological descriptions in later studies. Two new Melanoleuca species have been confirmed and a key for further studies on the Melanoleuca genus has been provided in this study.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This study was supported by the National Natural Science Foundation of China (No. 31770014) and the Science and Technology Plan Project of Liaoning Province (2020-MZLH-33). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.