Three new species of EntolomasubgenusPouzarella from China based on morphological and molecular data.

In the present paper, three additional species of EntolomasubgenusPouzarella viz. E.erectoides, E.griseocarpum and E.rubropilosum are described from China. E.rubropilosum is a typical species in section Pouzarella; E.griseocarpum and E.erectoides are members of sect. Dysthales. The taxa are further confirmed by ITS, RPB2, LSU and mtSSU analyses and phylogenetic relationships with other Entolomasubgen.Pouzarella species are also discussed. ITS sequence analysis showed that the sizes of the entire ITS region and ITS1 are remarkably divergent, while the ITS2 is conserved in length within Entolomasubgen.Pouzarella. Molecular analyses, based on the combined dataset, demonstrated that species diversity of subgen.Pouzarella in China is much higher than previously thought, in the present study twenty phylogenetic species from China are taken into consideration. On the other hand, morphological and molecular analyses suggested that classification of Entolomasubgen.Pouzarella probably has to be fundamentally re-adjusted based on additional data.


Introduction
Pouzarella Mazzer is a distinctive group of entolomatoid species that was accepted as a genus by some researchers (Horak 2008;Largent 1994;Mazzer 1976). Others consider it as a subgenus of Entoloma P. Kumm. (Noordeloos 1979(Noordeloos , 1992(Noordeloos , 2004Noordeloos and Gates 2012). Recent molecular phylogenetic studies (Co-David et al. 2009;Baroni and Matheny 2011;He et al. 2013;Largent and Bergemann 2015) based on multiloci showed that Pouzarella actually represent a distinct monophyletic group separated from the other entolomatoid groups. In addition, taxa of Pouzarella are easily recognised both by macro-and micromorphological characters. However, here we still treat it as a subgneus of Entoloma s.l. because accepting Pouzarella as a separate genus would make Entoloma s.l. paraphyletic. Taxonomical revision of other well supported, distinct clades of Entoloma s.l. is therefore needed before a formal decision on the generic status of Pouzarella can be made. When such a revision is achieved, we predict, that Pouzarella will be a well-defined genus based on morphological and molecular evidence. In the field, species of Entoloma subgen. Pouzarella are often overlooked due to their small and dull-coloured basidiomes. However, the inconspicuous species are widespread and have been reported from tropical to temperate regions. So far, more than seventy species have been described worldwide (Mazzer 1976;Horak 1980Horak , 1983Horak , 2008Desjardin and Baroni 1991;Noordeloos 1992Noordeloos , 2004Noordeloos et al. 1992;Manimohan et al. 1995;Baroni and Ortiz 2002;Manimohan et al. 2006;Karstedt et al. 2007; Baroni et al. 2008;Largent et al. 2011;Baroni et al. 2012;Noordeloos and Gates 2012;He et al. 2013;Largent and Bergemann 2015;Raj and Manimohan 2017).
Basidiomes of members in subgen. Pouzarella are easy to recognise. However, many species have in common small basidiome size and greyish colours and, therefore, it is difficult to distinguish them to species by morphological characters only. Accordingly, both morphological and molecular data are needed to refine the species concept and understand the diversity of these small agarics in Entoloma subgen. Pouzarella.
In previous studies, seven species of Entoloma subgen. Pouzarella were reported to occur in China (Ying 1995;He et al. 2013). However, we believe that subgen. Pouzarella remains poorly understood in this region, rich in many diverse ecological habitats. In continuation of previous surveys, further field work was carried out in southwest and northeast China. More than 50 samples matching the concept of Entoloma subgen. Pouzarella were collected and many turned out to be different from the locally already known species. As a first step, three distinctive new taxa are described in the present study whereas the other specimens were shelved for the moment because of scarcity of material. To further confirm the three new taxa and infer the affinities amongst representative species of Entoloma subgen. Pouzarella, phylogenetic analysis was carried out based on the combination of ITS, RPB2, mtSSU and nLSU sequences.

Morphological descriptions
Fresh basidiomes were photographed in the field and described macroscopically. Colour notations follow Kornerup and Wanscher (1978). Microscopic examination was done using a Leica DM5000B microscope. Basidiospores, basidia and pileipellis were mounted and measured in 5% potassium hydroxide (KOH) and/or 1% Congo Red. Pigmentation of the micro-structures was observed in distilled water. Measurements of the basidiospores excluded hilar appendix (apiculus) and at least 30 basidiospores of each specimen were measured. Q represents the length to width ratio of a basidiospore in profile view; Q represents the average Q of all basidiospores and is given ± standard deviation; x represents the means of basidiospore length and width ± standard deviation. All cited collections, including the holotypes, are deposited at the Mycological Herbarium of Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences (SAAS), Chengdu, China and the Herbarium ZT of ETH Zurich, Switzerland.

Sequence alignment and phylogenetic analyses
Sequences used in phylogenetic analyses are listed in Table 1 and aligned in muscle 3.6 (Edgar 2004). The aligned sequences were manually modified where necessary in Mega 6.0 (Tamura et al. 2013). Phylogenetic analyses were based on the combined ITS, nLSU, RPB2 and mtSSU sequences. For the ITS region, only ITS1 and ITS2 were kept for further analyses. Conflicts between the ITS, nLSU, RPB2 and mtSSU datasets were evaluated by comparing the topologies resulting from the phylogenetic analysis of the single gene. As no conflict was detected amongst the well supported clades of the different trees, sequences of the four genes were combined for further analyses.
Maximum likelihood analysis -ML analysis was carried out by the web RAxML Version 8 (http://www.phylo.org/sub_sections/portal/) under the GTRGAMMAI model with 1000 bootstrap replicates (Stamatakis 2014). "Find best tree using maximum likelihood search" option was selected when analysis was undertaken.
Maximum parsimony analysis -MP analysis was performed using PAUP* version 4.0b10 (Swofford 2003). All characters were treated as unordered and of equal weight. Gaps were treated as missing data. Bootstrap values (BS) were obtained from 1000 replicates.
Bayesian analysis -Bayesian analysis was performed using MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003). The best substitution models for each marker were selected using the Akaike Information Criterion (AIC) in jModelTest 2.1.7 (Darriba Table 1. A list of taxa, specimens and GenBank accession numbers of sequences used in this study.  Sequences in bold and marked with "KU" and "MH" are newly generated in this study. Sequences marked with "GQ" were from Co-David et al. (2009). Sequences marked with "HQ" were from Largent et al. (2011). KC710056, KC710073 and KC710117 were from Morgado et al. (2013). KC678996, KC678997 and sequences marked with "JQ" and "JX" were from He et al. (2013). KY643747 and KY643724 were from Raj and Manimohan (2017). JF908004 was from Osmundson et al. (2013). KJ523135 was from Kim et al. (2015). KY744163 and MF797654 are unpublished. et al. 2012). GTR+I+G model was selected for nLSU, GTR+G for mtSSU and ITS and SYM+G for RPB2. Two runs of six Markov chains were run from random starting trees for 6 million generations and sampled every 100 generations. Every time the diagnostics were calculated, 25% of the samples from the beginning of the chain were discarded. Runs were stopped after the average standard deviation was below 0.01. Bayesian posterior probabilities (BPP) were determined after calculating a 75% majority rule consensus tree. Etymology. Erectoides, refers to the suberect to erect fibrils on the pileus. Description. Pileus 5-15 mm broad, bluntly conic, convex or campanulate, dry, slightly hygrophanous, greyish-brown to brown (5C2-5D2), densely covered with suberect fibrils or minutely fibrillose squamules; fibrils silvery greyish, striate from entire margin to near centre. Lamellae sinuate, ventricose, distant, up to 3.5 mm wide, moderately thick, with two tiers of lamellulae, dark grey to brownish-grey, with entire and concolorous edge. Stipe 40-60 × 1-2.5 mm, central, cylindrical, equal, dry, concolorous with pileus, densely covered with grey to greyish fibrils, hollow, surface dry, with a pale yellow brownish to pale brownish strigose base. Context thin, concolorous with pileus. Odour and taste not distinctive.
Habitat. Scattered on soil amongst decaying litter in broadleaf forest dominated by Quercus or in mixed forest with Quercus, Betula, Rhododendron and Abies, also on soil in bamboo forest.

Molecular analysis
A total 76 sequences were generated in this study and they were deposited in GenBank. The combined dataset in the molecular analyses is composed of 62 specimens and 2925 aligned sites. MP, ML and Bayesian analyses produced almost the same topologies except for the unsupported branches and the MP tree is shown (Fig. 5).
For the ITS sequences used in the analyses, both the size of the entire ITS1-5.8-ITS2 region as well as for ITS1 and ITS2 were separately compared. It is remarkable that the sizes of the entire ITS region and ITS1 were significantly divergent. In general, the total length of ITS sequences in subgen. Pouzarella ranged from 591 bp to 1086 bp. ITS1 was highly variable in length, while the length of ITS2 is relatively conserved within subgen. Pouzarella. ITS1 and ITS2 spacer varied from 229 to 690 bp and from 202 to 255 bp, respectively. However, it is noteworthy that two groups of the whole ITS region and ITS1 spacer could be partitioned in length. One group was varying from 591 bp to 709 bp and the other from 967 bp to 1086 bp. For the 5.8S region, 159-162 bp were yielded. Despite 5.8S is highly conserved, 4 indels and 11 nucleotide substitutes were found in this region. Regarding RPB2 sequences, their length was considerably conserved.
The three new species in this study were placed in different clades, showing they are quite different from each other. E. rubropilosum in relatively close to E. strigosissimum in the analyses, but similarity of their ITS sequences is only 84%. E. griseocarpum is grouped with E. yunnanense J.Z. Ying, but more than 150 different bases were observed in their ITS sequences. E. erectoides and E. furfuraceum nested in the same clade; however, more than 100 different bases were detected amongst their ITS sequences.

Results and discussion
In the present study, three new species of Entoloma subgen. Pouzarella viz. E. griseocarpum, E. erectoides and E. rubropilosum, are reported from southwest China. The description is based on morphological and molecular characters. Together with the seven afore-mentioned species, ten taxa of Entoloma subgen. Pouzarella are now recorded for China. Five of those have been discovered in the northeast of China, four in southwest China and only one was reported from southern China (Ying 1995;He et al. 2013).
In the phylogenetic analyses, 35 taxa in subgen. Pouzarella were included and twenty phylogenetic species from China were recovered, suggesting a high species diversity in this geographical region. Five distinct clades (Clades I-V) were observed and the three new species are phylogenetically separated from each other. Based on morphological characters, Pouzarella (as a genus or subgenus of Entoloma s.l.) was divided into three sections (Dysthales, Pouzarella and Versatiles, Mazzer 1976;Noordeloos 1992). In our phylogenetic tree, Clade I corresponds to sect. Pouzarella while the taxa belonging to Clade II are accommodated in sect. Versatile. Except for several species of uncertain position, most members of Clade III, Clade IV and Clade V belong to the traditional sect. Dysthales morphologically. E. rubropilosum is nested in Clade I, which also includes E. nodosporum (G.F. Atk.) Noordel.and E. strigosissimum, as well as two still unknown species (Entoloma sp. 10 and Entoloma sp. 11) collected in China that morphologically fit the concept of sect. Pouzarella. These four species possess setiform pileocystidia and caulocystidia, somewhat reddish-brown or reddish fibrils on stipe and pileus. E. erectoides and E. griseocarpum are placed in Clade V.
The length of the ITS sequence of the species nested in Clade I, Clade II and Clade IV is relatively short, ranging from 591 bp to 709 bp. Available ITS sequences of Entolo-ma subgen. Pouzarella species in Clade V are recorded from 967 bp to 1086 bp. Unfortunately, no ITS sequences are available for comparison of the taxa belonging to Clade III. Eventual combination, referring both to morphological and molecular evidence, may in the future fundamentally change the classification of Entoloma subgen. Pouzarella.