A new clitocyboid genus Spodocybe and a new subfamily Cuphophylloideae in the family Hygrophoraceae (Agaricales)

Abstract Phylogenetically, the genera Cuphophyllus, Ampulloclitocybe and Cantharocybe are treated as basal in the family Hygrophoraceae, despite weak support. However, the exact phylogenetic positions of the three genera have remained unresolved, and taxa related to these genera are poorly known. In this study, a new clitocyboid genus Spodocybe was proposed based on multigenic phylogenetic inference datasets and morphological evidence. The analyses of ITS as well as two combined datasets ITS-nrLSU-rpb2 and ITS-nrLSU-rpb1-rpb2-tef1-α-atp6 supported that (1) Spodocybe formed a well-supported monophyletic clade; and (2) sisters Spodocybe and Ampulloclitocybe, along with Cantharocybe and Cuphophyllus also formed a monophyletic lineage, as sister to the rest of the Hygrophoraceae. Meanwhile, two new species, namely S. rugosiceps and S. bispora, from southwestern China, were documented and illustrated. These results support the new proposed genus Spodocybe, and that Spodocybe, Ampulloclitocybe, Cantharocybe and Cuphophyllus should be retained in the Hygrophoraceae as a new subfamily Cuphophylloideae.


Introduction
The widespread genus Clitocybe (Fr.) Staude currently encompasses large numbers of species with clitocyboid habit, sharing the features of saprophytic nutrition, funnelshaped pileus, decurrent lamellae, a usually white, cream or pale colored spore-deposit and smooth and inamyloid spores (Singer 1986;Breitenbach and Kraenzlin 1991;Laessøe and Petersen 2019). As a consequence of the poor, broad and unrepresentative morphological characteristics, the genus appeared heterogeneous and was subsequently proven to be polyphyletic based on the phylogenetic analysis Harmaja 2003).
The molecular phylogenetic relationships among members of the Hygrophoraceae Lotsy were well studied by Lodge et al. (2014). In their work, the family was divided into subfamily Hygrophoroideae E. Larss., Lodge, Vizzini, Norvell & S.A. Redhead, Hygrocyboideae Padamsee & Lodge, Lichenomphalioideae Lücking & Redhead and Cuphophylloid grade. Meanwhile, the Cuphophylloid grade was retained in the Hygrophoraceae as the base comprising the genera Cuphophyllus (Donk) Bon, Ampulloclitocybe and Cantharocybe, despite weak phylogenetic support (Matheny et al. 2006;Binder et al. 2010;Lodge et al. 2014). Consequently, the taxonomic problem of the three genera on whether to be included or excluded in the Hygrophoraceae has remained unresolved.
Recently, some collections were shown to be closely related to Clitocybe trulliformis (Fr.) P. Karst. based on ITS-BLAST searches while at the same time they were surprisingly related to taxa of the genus Cuphophyllus based on nrLSU-BLAST searches. As far as we know, C. trulliformis and allied species were lacking taxonomic revision, especially regarding their molecular phylogenetic status. Furthermore, the phylogenetic delimitation of the Hygrophoraceae was ambiguous due to the uncertain positions of Cuphophyllus, Ampulloclitocybe and Cantharocybe. Hence, the aims of this study were (a) to propose and describe a new genus of the Hygrophoraceae for species related to C. trulliformis based on morphological and molecular analyses and (b) to reconstruct the phylogeny of the Hygrophoraceae for determining the exact phylogenetic placements of Cuphophyllus, Ampulloclitocybe and Cantharocybe with multi-gene data.

Specimens
Twenty-three specimens of species similar to C. trulliformis and related species were collected from southwestern and northeastern China and western Germany, during 2007 to 2020. The fresh fruitbodies were dried using heat or silica gel. Voucher specimens were deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS). Detail information of these specimens is given in Table 1.

Morphological observation
Macroscopic characters of species were described based on the raw field record data and photographs. Colors used in description referred to Kornerup and Wanscher (1978). For the microscopic structure observation, tissue sections of dried specimens were mounted in 5% KOH solution or distilled water and structures of lamellar trama, pileipellis and stipitipellis, basidia and basidiospores were observed with a light microscopy. For the description of lamellar trama structure, seven types, including regular, subregular, divergent, pachypodial, bidirectional, tri-directional and interwoven, were used following Lodge et al. (2014). Besides, Melzer's reagent was applied to test the amyloidity of the basidiospores. In the description of basidiospores, the abbreviation [n/m/p] represent that the measurements were made on n basidiospores from m basidiomes of p collections. The range notation (a)b-c(d) stands for the dimensions of basidiospores in which b-c contains a minimum of 90% of the measured values while a and d in the brackets stand for the extreme values. In addition, a Q value show the length/width ratio of basidiospores and a Qm value for average Q ± standard deviation. All microstructures were illustrated by hand drawing.
The combined dataset ITS-nrLSU-rpb2 comprised 221 sequences from 88 samples with a total of 3135 positions. In the three-gene tree (Fig. 1), 11 specimens from four novel Spodocybe species collected in this study, C. cf. trulliformis and C. herbarum formed a strongly supported monophyletic clade (BP = 100%, PP = 1.0), as sister to Ampulloclitocybe (BP = 63%, PP = 0.98). The phylogenetic analysis showed that the new proposed genus Spodocybe should be placed within the Hygrophoraceae, although intergeneric branched orders among Spodocybe, Ampulloclitocybe, Cantharocybe and Cuphophyllus were unstable with low support values.
In order to accurately determine the position of Spodocybe in the family Hygrophoraceae and better clarify the phylogenetic relationships of Spodocybe, Ampulloclitocybe, Cantharocybe and Cuphophyllus, a further six-gene matrix ITS-nrLSU-rpb1-rpb2-tef1-α-atp6 composed of 179 sequences from 54 samples with 5405 positions was used to rebuild the Hygrophoraceae tree. As revealed by the six-gene phylogenetic analysis (Fig. 2), the branch support level of the six-gene tree was obviously improved, compared with that of the previous three-gene tree. The monophyly of Spodocybe clade was strongly supported (BP = 100%, PP = 1.00), including Spodocybe rugosiceps (BP = 100%, PP = 1.00), S. bispora (BP = 100%, PP = 1.00) and two unnamed Spodocybe species. Spodocybe and Ampulloclitocybe were sister clades (BP = 78%, PP = 0.99), then further clustered with Cantharocybe (BP = 59%, PP = 0.97) and finally together with Cuphophyllus formed an independ-  Lamellar trama type B for bidirectional, D for divergent, I for interwoven, P for pachypodial, R for regular, S for subregular, T for tri-directional. Lamellar trama types of specimens collected in this study were identified by ourselves and others referred to Lodge et al. (2014) and Hosen et al. (2016). In addition, an ITS dataset (23 sequences, 1053 positions) was applied to phylogenetic analysis for displaying the relationships among Spodocybe species from this study and species of Clitocybe treated from GenBank. In the ITS tree (Fig. 3), Spodocybe species formed a highly supported monophyletic clade with C. trulliformis and related species (BP = 100%, PP = 1.00), which was also a sister clade to Ampulloclitocybe with strong support (BP = 91%, PP = 0.99).  decurrent. Basidiospores ellipsoid, oblong or subglobose, thin-walled and inamyloid. Pileipellis usually a cutis, sometimes ixocutis or trichoderm. Lamellar trama regular, subregular, interwoven or bidirectional. Clamp connections present.

Taxonomy
Habitat, ecology and distribution. Usually gregarious or caespitose on ground, rarely on wood; widespread in temperate and tropical regions.
The genera Ampulloclitocybe, Cantharocybe, Cuphophyllus and Spodocybe are included in the subfamily Cuphophylloideae, which is in correspondence with Cuphophylloid grade of Lodge et al. (2014)  Diagnosis. Differs from Ampulloclitocybe by its small basidiomes and subregular lamellar trama rather than medium-sized basidiomes and bidirectional lamellar trama. Differs from Cuphophyllus in the ratio of basidia to basidiospore length less than 5, and lamellar trama subregular rather than interwoven. Differs from Cantharocybe in its absence of cheilo-and caulocystidia, having small basidiomes rather than large ones and having subregular lamellar trama rather than regular one.
Etymology. Spodo-refers to grey; -cybe refers to head; that is a Clitocybe-like genus with gray pileus.
Habitat, ecology and distribution. Saprophytic, usually gregarious or caespitose on the ground of coniferous or coniferous and broad-leaved mixed forest; distributed in the temperate and subtropical zones from June to November. Diagnosis. Differs from S. bispora in having a rugose pileus, smaller basidiospores and 4-spored rather than 2-spored basidia. Differs from C. trulliformis in having smaller basidiospores and a rugose rather than felty-squamulose pileus.
Habitat, ecology and distribution. Saprophytic, usually gregarious on the ground of coniferous or coniferous and broad-leaved mixed forest, known from Yunnan, China; July to September.
Additional specimens examined.

The new genus Spodocybe
In our current study, the new clitocyboid species were clustered into a monophyletic lineage (BP = 100%, PP = 1.00) in the Hygrophoraceae according to the multi-gene phylogenetic analysis (Figs 1, 2). As a result, the new generic name Spodocybe is proposed here to accommodate the new lineage, which is irrelevant to Clitocybeae of the Tricholomatoid clade (Matheny et al. 2006;Alvarado et al. 2015). The three-gene tree of the Hygrophoraceae (Fig. 1) in this study presented basically consistent topological structure with Lodge et al. (2014), and showed that Spodocybe was a sister to Ampulloclitocybe located within the family Hygrophoraceae and further confirmed by a six-gene tree (Fig. 2).
Besides the molecular analyses, morphological data also support its separation from the relative genera. Spodocybe shares clitocyboid basidiomes, decurrent lamellae, inamyloid basidiospores and the presence of clamps with the other genera Ampulloclitocybe, Cuphophyllus and Cantharocybe. However, the genus Ampulloclitocybe, typified by A. clavipes, differs from Spodocybe in having medium-sized basidiomes and bidirectional lamellar trama (Harmaja 2002;Lodge et al. 2014). Afterwards, Cuphophyllus differs from Spodocybe in having long basidia, typically 7−8 (rarely 5−6) times the length of the basidiospores, highly interwoven lamellar trama, rarely subregular (Voitk et al. 2020). Finally, Cantharocybe differs from Spodocybe in having large basidiomes, broad lamellae, cheilo-and caulocystidia, clamps but not on all hyphal septa or at the base of every basidium and more regular lamellar trama (Ovrebo 2011;Hosen et al. 2016). In view of the four genera above with different structures in lamellar trama (Fig. 2), the type of lamellar trama can become a good distinguishing microscopic character for them.
For a long time, C. trulliformis has been placed in the genus Clitocybe based on the clitocyboid feature and habit since 1879 (Karsten 1879). However, C. trulliformis shares many morphological characteristics with Spodocybe, such as the small basidioma with applanate to infundibuliform pileus, grey-brown pileus and stipe, decurrent and whitish lamellae, and smooth and inamyloid basidiospores (Bas et al. 1995). Besides, the ITS phylogenetic analysis in our study (Fig. 3) showed that C. trulliformis and related Clitocybe species were involved in the Spodocybe clade as well, indicating that C. trulliformis and related species should be placed with Spodocybe. In consequence, it is foreseeable that C. trulliformis and other related clitocyboid species will eventually be moved to Spodocybe. Accordingly, more taxonomic work is needed in future.

The placements of Spodocybe, Cuphophyllus, Ampulloclitocybe and Cantharocybe
In previous studies, Cuphophyllus, Ampulloclitocybe and Cantharocybe were treated as basal in Hygrophoraceae (Lodge et al. 2014), but their phylogenetic placements were not resolved. In a six-gene phylogenetic analysis by Binder et al. (2010) and a threegene analysis by Wang et al. (2018), Ampulloclitocybe and Cantharocybe were located between Cuphophyllus and the rest of the Hygrophoraceae, but without support. While two four-gene analyses by Lodge et al. (2014) showed that Ampulloclitocybe and Cantharocybe were sister clades as basal to Cuphophyllus along with the rest of the Hygrophoraceae with weak support. However, in our six-gene analysis (Fig. 2), the new proposed genus Spodocybe and Ampulloclitocybe were sisters (BP = 78%, PP = 0.99) and they clustered with Cantharocybe followed by Cuphophyllus, forming a supported monophyletic sister clade to the rest of the Hygrophoraceae (BP = 83%, PP = 1.00). Hence, Spodocybe, Ampulloclitocybe, Cantharocybe and Cuphophyllus should be retained in Hygrophoraceae, and a new subfamily, Cuphophylloideae, is proposed to accommodate the lineage.