Additions to tribe Chromosereae (Basidiomycota, Hygrophoraceae) from China, including Sinohygrocybe gen. nov. and a first report of Gloioxanthomycesnitidus

Abstract Sinohygrocybegen. nov., typified by S.tomentosipessp. nov., is described upon morphological and molecular evidence. The new genus is characterised by its sinuate to subdecurrent or short deccurent, usually furcate and interveined and relatively distant lamellae, dry and whitish tomentose stipe, thin-walled ellipsoid to oviod, non-constricted basidiospores and particularly elongated basidia and a ratio of basidiospore to basidium length of >5 to 8; it is close to genera Chromosera and Gloioxanthomyces of the tribe Chromosereae, but morphologically differs from Chromosera in less umbilicate basidiomata, tomentose stipe and usually longer basidia and differs from Gloioxanthomyces in more robust basidioma and less glutinous pileus and/or stipe surface. Phylogenetic analyses, with ITS-LSU-RPB2 data, also indicate that Sinohygrocybe forms a very distinct and independent clade at the generic level. In addition, a Chinese new record G.nitidus is described here.


Introduction
Hygrophoraceae Lotsy (Hymenomycetes, Basidiomycota) is a large family in Agaricales, including 26 genera and over 600 species (Lodge et al. 2014). In a six-gene phylogenetic tree of Agaricales, Hygrophoraceae, Pterulaceae Corner, Typhulaceae Jülich and some small groups formed a Hygrophoroid clade, which is one of the six largest clades in Agaricales (Matheny et al. 2006); and in a genome based mushroom tree of life, Hygrophoraceae and Clavariaceae Chevall. are representative families of the suborder Hygrophorineae Aime, Dentinger & Gaya, which is one of the seven suborders of the Agaricales (Dentinger et al. 2016). Traditionally, the family Hygrophoraceae referred to a group of agaricoid, waxy-gilled and white-spored mushrooms; and a majority of the members are classified in the type genus Hygrophorus Fr. and genus Hygrocybe (Fr.) P. Kumm. Morphological characters of the Hygrophoraceae taxa are relatively simple (usually without annulus or volva and a cystidiate) amongst the agaric fungi and their basidioma colours are often very susceptible to the environmental conditions and developmental stages, making their classification and identification difficult, so it is often challenging to make correct identification and taxonomy of them just according to morphological recognition (Young 2005). Modern molecular techniques have been revolutionising the taxonomy and phylogeny of Hygrophoraceae. Lodge et al. (2014) had conducted the most comprehensive molecular phylogenetic study on the family until now, therefore their systematic viewpoint on Hygrophoraceae is adopted in this paper. According to their study, the family could be divided into four groups at subfamily level, i.e. subfamily Hygrophoroideae E. Larss., Lodge, Vizzini, Norvell & S.A. Redhead, Hygrocyboideae Padamsee & Lodge, Lichenomphalioideae Lücking & Redhead and Cuphophylloid grade. The subfamily Hygrocyboideae could be divided into three tribes, i.e. tribe Chromosereae, Humidicuteae and Hygrocybeae; and the tribe Chromosereae included two sister genera, Chromosera Redhead, Ammirati & Norvell and Gloioxanthomyces Lodge, Vizzini, Ercole & Boertm. Chromosera, the type genus of the tribe Chromosereae, was erected to accommodate Omphalina cyanophylla (Fr.) Quél. which was originally described from Sweden and combined as C. cyanophylla (Fr.) Redhead, Ammirati & Norvell (Redhead et al. 1995, 2012 Lodge et al. 2014). Before the recognition of Gloioxanthomyces, those two species were usually placed in the genus Hygrocybe as H. vitellina (Fr.) P. Karst and H. nitida (Berk. & M.A. Curtis) Murrill, respectively. Morphologi-cally, the main differences between the two species were in their basidiospore sizes: G. nitidus had ellipsoid to oblong basidiospores, measuring 7-10 × 5-6 μm with Q = 1.3-1.8; while G. vitellinus had subglobose basidiospores, measuring 6.5-8.5 × 5-7 μm with Q=1.1-1.6 (Boertmann 1990). Since their differences were limited, the two taxa seemed to be conspecific (Boertmann 2011). However, according to the phylogenetic analyses with ITS data by Boertmann (2012), the European collections clearly clustered together as the G. vitellinus species clade, while the North American materials independently formed another group as the G. nitidus species clade, thus they could actually be sharply defined as two separated sister species.
During the studies on the Chinese Hygrophoraceae in recent years, some collections morphologically corresponding to tribe Chromosereae were collected. Comprehensive observation and analyses revealed some interesting findings, which can contribute to the taxonomic knowledge of the tribe. In this paper, we aim to: 1) formally describe a new genus of tribe Chromosereae from East Asia based upon morphological and molecular analyses and present a Chinese new record of Gloioxanthomyces nitidus; 2) reconstruct the phylogeny of the family Hygrophoraceae using 3 gene regions, i.e. the internal transcribed spacer region (ITS), the large subunit nuclear ribosomal RNA region (nrLSU) and the nuclear RPB2 6F to 7.1R region (RPB2). Detailed studies were therefore conducted and the results are presented as follows.

Morphological studies
Specimens were photographed and annotated in the field and then dried in an electric drier. Macroscopic descriptions were gained from the original field notes and photographs. Colour descriptions followed Kornerup and Wanscher (1978). Tissue sections were immersed in 5% potassium hydroxide (KOH) and/or 1% Congo Red solution for microscopical examinations, but in distilled water for colour descriptions of basidia, pileipellis and stipitipellis. From a mature specimen, over 40 basidiospores and 20 basidia were randomly selected and measured under a light microscope in KOH. The notation (a)b-c(d) was used to describe dimensions where the range b-c representing 90% or more of the measured values and a, d were the extreme values. The length/width ratio of spores was presented as Q and the mean ratio was presented as Q m . The studied specimens were deposited in the Fungal Herbarium of Guangdong Institute of Microbiology (GDGM), Guangzhou, China.

Molecular studies
Genomic DNA was extracted from the herbarium specimens using the Sangon Fungus Genomic DNA Extraction kit (Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The ITS, LSU and RPB2 gene regions were ampli-fied by Polymerase Chain Reaction, using universal primers ITS1F/ITS5 and ITS4 (White et al. 1990;Gardes and Bruns 1993), LR0R and LR5 (http://biology.duke. edu/fungi/mycolab/primers.htm) and RPB2-6F and RPB2-7.1R (Matheny 2005), respectively. Amplified products were sequenced by Beijing Genomic Institute (BGI) using the same primers. The abi format sequences were assembled by SeqMan version 7.1.0 (DNAStar, Inc.) and then the assembled sequences were submitted to GenBank.
In this study, two datasets were constructed. The first one is an ITS-LSU-RPB2 matrix of the family Hygrophorceaeae for making a comprehensive phylogenetic tree and analysing the positions of the new taxa; most known species of Hygrophoraceae with available sequences from reliable sources were included in the dataset, each of them having at least an LSU sequence and Typhula phacorrhiza (Reichard) Fr. was selected as the outgroup referred from Yang et al. (2013) and Lodge et al. (2014). The second dataset is an ITS matrix of the tribe Chromosereae and Hygrocybe conica (Schaeff.) P. Kumm. and H. conica var. conicoides (P.D. Orton) Boertm. were chosen as outgroups. Each gene was independently aligned on the online MAFFT service (Katoh et al. 2017), then combined by the Geneious software (Biomatters Ltd.) for the first dataset. Maximum likelihood phylogenetic trees were generated by the RAxML software (Stamatakis 2014) on the CIPRES service (Miller et al. 2010) with 1000 bootstrap replications using the default options.

Molecular phylogenetic results
The combined 3-gene dataset composed of 120 samples (Table 1), including 5 newly sequenced samples and 115 published ones. In the final matrix, the ITS, LSU and RPB2 regions comprised positions 1 to 1751, 1752 to 2873, 2874 to 3759, respectively. In the 3-gene Maximum Likelihood tree ( Fig. 1), the four Chinese collections (GDGM43351 and GDGM43347 from Sichuan province, GDGM50075 and GDGM50149 from Hunan province) formed a strong monophyletic clade with 100% bootstrap support, which was near the Chromosera-Gloioxanthomyces clade composed of members of Chromosera and Gloioxanthomyces with 76% bootstrap support.
The ITS dataset included 30 samples of all known taxa of tribe Chromosereae and 2 Hygrocybe sequences chosen as the outgroups, the matrix length is 679 bp. In the ITS Maximum Likelihood tree (Fig. 2), collections of the species G. nitidus and G. vitellinus were clustered together with 93% and 100% support values, respectively and the North American and the East Asian G. nitidus were clustered as sister groups with 93% support value; all the members of Chromosera (except C. viola), Gloioxanthomyces and Sinohygrocybe were clustered together with 95%, 93% and 100% support values, respectively; and the Chromosera-Gloioxanthomyces clade was presented as the sister clade of the Sinohygrocybe clade with strong support value (100%). Description. Basidiomata medium-sized, subcaespiotose. Pileus convex to applanate, slightly depressed in the centre, yellow, orangish-yellow to orange, dry to subviscid, slightly when wet, never strongly gelatinised or glutinous. Lamellae adnate to decurrent, concolorous with pileus, with usually furcate and interveined lamellulae. Stipe yellow to whitish or almost concolorous with pileus, yellow or covered by white to yellowish-white tomentum. Basidiospores ellipsoid to oblong, ovoid, Qm = 1.6-1.7,  Diagnosis. Differs from the other members of the tribe Chromosereae by its larger and more robust basidiomata, concolorous yellow pileus, lamellae and the subsurface of stipe, usually furcate and interveined lamellae and lamellulae, white fibrillose stipe surface, long basidia (up to 80 μm), ratio of basidia to basidiospore length over 5 and even up to 8.  Description. Basidiomata small to medium-sized. Pileus 2.5-6 cm diam., convex to applanate, usually slightly depressed in the centre, smooth, dry but subviscid when wet, light yellow to vivid yellow (3A5-8) or to deep yellow (4A5-8), or light orange to dark orange (5A5-8), becoming paler when dry; margin even, straight or upturned and occasionally split when mature. Lamellae up to 7 mm wide, adnate to sinuate or decurrent, distant, 17-22 lamellae per pileus, with 1-3 lamellulae between two complete lamellae, usually furcate, often interveined or anastomosing at lamella base, thick, concolorous with the pileus; lamellar base and lamellulae irregular and occasionally the whole hymenophore irregular; lamellar edge even and concolorous. Context concolorous with lamellae and pileus, unchanged when cut. Stipe 4-6.5 × 0.6-1.2 cm, central or occasionally eccentric, subcylindrical, moderately to densely covered with white tiny adpressed fibres. Odour indistinct.
Habitat and known distribution. Gregarious, caespitose, or scattered in broadleaf forest in subtropical temperate transition zone, so far known only from Sichuan and Hunan Provinces in China.
Habitat and known distribution. Solitary or scattered, on moist ground in a mixed forest with mosses in North-eastern China, so far known in North America and East Asia.

Discussion
Phylogenetically, the distinction of the three subfamilies (Lodge et al. 2014) within Hygrophoraceae has very convincing support in the multi-locus tree of this study ( Fig. 1). In addition, the establishment of the three well-defined monophyletic tribes in subfamily Hygrocyboideae is supported in this phylogenetic frame where the tribe Hygrocybeae with 73% support values and the tribe Humidicuteae with low support value are sister clades, while the tribe Chromosereae with 76% support values is located at their base. However, the cuphophylloid grade appears not to be monophyletic, thus more studies are still needed to understand the phylogenetic positions of Ampulloclitocybe, Cantharocybe H.E. Bigelow & A.H. Sm. and Cuphophyllus (Donk) Bon.
In the multi-gene analyses, Sinohygrocybe is placed together with two other genera in Chromoserae. Chromosera and Gloioxanthomyces are sister genera under the monophyletic tribe Chromosereae, while Sinohygrocybe is an independent generic lineage; and the distances between Sinohygrocybe and Chromosera or Gloioxanthomyces are further than the distance between Chromosera and Gloioxanthomyces. Such results are confirmed in the ITS phylogenetic tree (Fig. 2). According to the Blastn results, the ITS and LSU sequence identities of the new species to the known taxa are not more than 76% and 96%, respectively, with the Chromosera and Gloioxanthomyces sequences in GenBank. Thus, it is clear the new genus is independent of those two existed genera.
Beside the molecular analyses, morphological data also support its recognition within tribe Chromosereae. Sinohygrocybe shares a bright pileus colour and decurrent lamellae with the other genera Chromosera and Gloioxanthomyces (Table 2). However, the genus Chromosera, typified by C. cyanophylla (Fr.) Redhead, Ammirati & Norvell, differs from Sinohygrocybe in having omphaloid basidiomata, ephemeral dextrinoid reactions in the context, ratio of basidiospore to basidium length <5, ephemeral pigment bodies in the pileipellis and lilac pigments sometimes present (Redhead et al. 1995, Candusso 1997, Lodge et al. 2014; while Gloioxanthomyces differs from Sinohygrocybe by having weaker/ delicate basidiomata, viscid pileus and stipe surface, gelatinised lamellar edge and cheilocystidia, shorter basidia (Boertmann 1990(Boertmann , 2012 with a length ratio of basidium to basidiospore 4-5. Sinohygrocybe shares some macroscopic characters with Hygrocybe, typified by Xin Zhang for their help during the field trips in Gesala, Taoyuandong and Changbai Mountains, respectively; to Dr. Tolger Bau for trying to find some additional samples in HMJAU; to Dr. David Boertmann, Dr. Md. Iqbal Hosen and Dr. Wang-Qiu Deng for their improvements and constructive comments on an earlier version of this paper; to Dr. David Hibbett and the International Exchange Scholarship of the University of Chinese Academy of Sciences for providing an opportunity to the first author to learn molecular data analyses. This study was financed by the Ministry of Science and Technology of China (Nos. 2013FY111500, 2013FY111200), the Science and Technology Program of Guangzhou, China (No. 201607020017), the GDAS' Special Project of Science and Technology Development (2018GDASCX-0907) and the Science and Technology Project of Guangdong Province (2017A030303050).