Two new agaricoid species of the family Clavariaceae (Agaricales, Basidiomycota) from China, representing two newly recorded genera to the country

Abstract Two new lamellar species, Camarophyllopsis olivaceogrisea and Hodophilus glaberripes, of the family Clavariaceae were discovered in the subtropical zone of China. Camarophyllopsis olivaceogrisea is morphologically characterized by its hygrophanous basidiomata, greenish gray to dull green pileus, shortly decurrent lamellae, broadly elliptic basidiospores 4–5.5 × 3.5–4.5 μm in size, and cutis-like pileipellis composed of cylindrical cells. Hodophilus glaberripes is mainly characterized by its white to brownish pileus, glabrous stipe, slight yam bean smell, broadly elliptic basidiospores 5–6.5 × 4–5 μm in size, and epithelium-like pileipellis composed of inflated cells. Phylogenetic placement of the two species was determined by the combined analyses of a DNA data matrix containing ITS and LSU, and showed that collections of the two species formed two independent lineages in the Camarophyllopsis and Hodophilus clades respectively. The delimitation of C. olivaceogrisea and H. glaberripes were evaluated using molecular, morphological, and ecological methods. This is the first report of the genera Camarophyllopsis and Hodophilus in China.

Camarophyllopsis can be easily distinguished from other genera in the family by its small agaricoid basidiomata, hygrophanous pileus, subglobose to broadly ellipsoidal basidiospores, and epithelium pileipellis composed of chains of erect, ascending or repent, subcylindrical to ellipsoid terminal cells (Arnolds 1986;Young 2005). Hodophilus differs from Camarophyllopsis in the hymeniderm pileipellis composed of typically perpendicular, broadly inflated, globose, and obpyriform to sphaero-pendunculate terminal elements . Lamelloclavaria can be distinguished from the above genera by its rimulose non-hygrophanous pileus and cutis pileipellis .
In this study, some agaricoid collections were identified in China. Morphological observation and phylogenetic analyses confirmed that they are two novel taxa in the genera Camarophyllopsis and Hodophilus. This is the first report of these two genera in China.

Morphological studies
Photographs of basidiomata were taken in type localities when collected. Macro-morphological characteristics were recorded for fresh specimen. Specimens were dried and then deposited in the Fungal Herbarium of Guangdong Institute of Microbiology (GDGM). Methods used for morphological descriptions were followed Zhang et al. (2015). Colors were recorded and described in general terms according to the method of Kornerup and Wanscher (1978). Microstructures were observed from rehydrated materials, and the notation "basidiospores (n/m/p)" indicates that the measurements were conducted for n basidiospores from m basidiomata of p collections. Line drawings were prepared by free hand.

DNA extraction, PCR amplification, and sequencing
Total genomic DNA of each voucher specimen was extracted from silica-gel-dried materials using the Sangon Fungus Genomic DNA Extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. Primer pairs ITS1/ ITS4 (White et al. 1990) and LR0R/LR5 (Vilgalys and Hester 1990) were used to amplify the internal transcribed spacer (ITS) region and the large subunit nuclear ribosomal RNA (nrLSU) region, respectively. PCR protocol and sequencing were conducted following the method of Zhang et al. (2015).

Phylogenetic analyses
Newly generated sequences, related sequences used in previous studies (Adamčík et al. 2018) and a few sequences retrieved from GenBank by a Blast search were used to reconstruct phylogenetic trees. Detailed information on the newly sequenced samples, including the taxon names, voucher numbers, localities and GenBank accession numbers, is shown in Table 1. ITS and LSU sequences were respectively aligned using Clustal X v1.81 (Thompson et al. 1997) and manually modified where necessary in Bioedit v7.0.9 (Hall 1999), and a combined matrix of ITS and LSU sequences was obtained. The combined dataset was then analyzed using RAxML v7.2.6 (Stamatakis 2006) and MrBayes v3.1.2 software (Ronquist and Huelsenbeck 2003) for maximum likelihood (ML) and Bayesian inference (BI) analyses, respectively. For both BI and ML analyses, the substitution model for the two gene partitions was individually determined using the Akaike Information Criterion (AIC) complemented in MrModeltest v2.3 (Nylander 2004). For ML analysis, all parameters were kept at default values, except for choosing the GTRGAMMAI model, and statistical support was obtained using rapid nonparametric bootstrapping with 1000 replicates. BI analysis using selected models and 4 chains was conducted by setting the number of generations to 3 million and stoprul com- mand with the stopval value set to 0.01. Trees were sampled every 100 generations. The first 25% of generations were discarded as burn-ins and posterior probabilities (PP) were calculated from the posterior distribution of the retained Bayesian trees.

Molecular phylogenetic results
For phylogenetic analyses, 25 sequences (13 ITS and 12 LSU) were newly generated from 13 collections, 219 related sequences (123 ITS and 96 LSU) were retrieved from GenBank, and Ramariopsis corniculata (Schaeff.) R.H. Petersen selected as an outgroup based on previous studies Adamčík et al. 2016Adamčík et al. , 2018. The combined matrix of 137 samples with 1614 nucleotide sites was constructed for phylogenetic analyses and the final alignment was submitted to TreeBASE (Submission ID 24440). SYM+I and SYM+G were chosen as the best substitution models for ITS and LSU, respectively. The ML and BI analyses generated nearly identical tree topologies with minimal variation in statistical support values; thus, a ML tree was selected for display ( Figure 1). The tree topologies generated in this study are similar to those obtained by Adamčík et al. (2018) and are therefore not described in detail here, except for the results relevant to the two new species. The two monophyletic genera Camarophyllopsis and Hodophilus were highly supported ( Figure 1). Two collections (GDGM44497 and GDGM44519) formed an independent lineage with strong statistical support (BS = 100, PP = 1), located within the Camarophyllopsis clade, and presented as a sister group to a collection numbered as GDGM44501 from China (with low statistical support). Ten collections (GDGM45940, GDGM52374, GDGM52530, GDGM52545, GDGM52583, GDGM55689, GDGM70329, GDGM70331, GDGM72434 and GDGM72518) were grouped together with strong statistical supports (BS = 100, PP = 1) and formed an independent lineage in the Hodophilus clade, and were revealed as a sister group to H. indicus K.N.A. Raj, K.P.D. Latha & Manim. with significant statistical support (BS = 100, PP = 1). Etymology. The epithet "olivaceogrisea" refers to the olive-gray pileus color.
Diagnosis. This species is easily distinguished from other species in the genus Hodophilus by its larger basidiomata, white, brownish orange to brown pileus, glabrous stipe, slightly yam bean smell and broadly elliptic basidiospores.
Description. Basidiomata small to medium-sized. Pileus 15-50 mm broad, hemispherical, convex to plano-convex at first, then becoming broadly convex or plano-convex but hardly fully expanded to plane, often depressed at disc when old; white to yellowish white at first, then gradually becoming orange white, pale yellow, pale orange, brownish orange, light brown, brown to reddish brown (5A2, 3A3-5A3, 5C4-7C4, 5D5-9D5) when mature and dry, hygrophanous; margin first slightly inflexed, soon straight, slightly crenate when mature, non-striate or indistinctly translucently striate up to one third when wet; surface matt, velvety and later with fine and darker granules or pruina, at first even, but becoming rugose or rough towards the center when mature, often concentrically cracked in dry conditions. Flesh 0.5-2 mm thick in half radius of the pileus, white, pinkish white to pale beige; Lamellae 3-5 mm deep, distant to subdistant, L = 21-32, l = 1-3, short decurrent, notched, orange white to pinkish white (5A2-10A2) when young, brownish orange, light brown, reddish brown to brownish red (5C4-7C4, 5D6-10D6) when mature, unchanging when bruised; edge entire, concolorous or slightly paler than lamella sides. Stipe (50) 80-100 × 3-5 mm, cental, usually flexuous, cylindrical and slightly narrower downwards; glabrous smooth and shiny, hygrophanous, white to yellowish white at first, becoming pale yellow to pale orange when mature and in dry condition. Odor none or slight yam bean smell, taste mild.

Key to the species of Camarophyllopsis and Hodophilus
According to the phylogram (Figure 1), C. olivaceogrisea nested well into the Camarophyllopsis clade and formed a sister group of an unidentified Chinese collection (GDGM44501) with low statistical support. Because of the low number of specimens, GDGM44501 was not described here, but it can be easily separated from C. olivaceogrisea by branch distance. The other three species in the phylogenetic tree, C. atrovelutina, C. deceptive and C. schulzeri, also can be easily separated from C. olivaceogrisea. The closest relatives of the new species remain unresolved in this study because of the lack of significant statistical supports and the few available sequences of Camarophyllopsis used in phylogenetic analysis.
Ecologically, very little is known about the ecology of Camarophyllopsis species, as for most species only a few verified collections are known and little molecular data is available. Camarophyllopsis species are widely distributed in the southern and northern hemispheres, from tropical zones to cool temperate zones, and in monsoon forest, bushy forest, and grassland habitats, and some species have been shown to be saprotrophic (Young 1999(Young , 2005Boertmann 2002;Kovalenko et al. 2012). Camarophyllopsis olivaceogrisea was collected from subtropical regions in southwest China at altitudes of over 1000 m, and is typically found in wet areas with moss under mixed forest, which is mainly dominated by broadleaf plants (Castanopsis spp., Fagus spp. and Schima spp.) with few conifer (Pinus massoniana Lamb. and Pinus spp.). This study expands the geographic distribution of the genus Camarophyllopsis to China.
Hodophilus taxa were mainly reported in temperate to boreal zones of the northern hemisphere and can be found in forest, bushy forest margin, grassland and bare soil habitats (Adamčík et al. , 2017a(Adamčík et al. , 2017b(Adamčík et al. , 2018, and a recent study reported a new tropical distribution in India (Crous et al. 2017). In this study, collections of H. glaberripes were distributed from 23°N to 26°N in the subtropical zone of southern China and at altitudes of 200-800 m, mostly occurred in the margin of broadleaf forest (mainly dominated by Fagaceae, Hamamelidaceae and Theaceae plants) and mixed forest (dominated by broadleaf tree mixed with few conifer as Pinus massoniana Lamb., Pinus spp. and Cunninghamia spp.), commonly along the sides of cement road in the forest and preferentially in heavy clay soil to humus. This study revealed an expanded geographic distribution of Hodophilus species to subtropical regions.