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Three new species of Entoloma subgenus Pouzarella from China based on morphological and molecular data
expand article infoXiao-Lan He, Egon Horak§, Di Wang, Wei-Hong Peng, Bing-Cheng Gan
‡ Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences, Chengdu, China
§ Unaffiliated, Innsbruck, Austria
Open Access

Abstract

In the present paper, three additional species of Entoloma subgenus Pouzarella 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 Entoloma subgen. 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 Entoloma subgen. 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 Entoloma subgen. Pouzarella probably has to be fundamentally re-adjusted based on additional data.

Keywords

Entolomataceae, systematics, taxonomy, multi-gene analyses

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, 1992, 2004; Noordeloos 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 multi-loci 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 1980, 1983, 2008; Desjardin and Baroni 1991; Noordeloos 1992, 2004; Noordeloos 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.

Materials and methods

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.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted with Biospin Fungus Genomic DNA Extraction Kit following the manufacturer’s instructions. PCR amplification was performed using DreamTaq™ Green PCR Master qMix (2×), Fermentas. The primers for RPB2 amplification were rpb2-6F and rpb2-7R, rpb2-i6f and rpb2-i7r (Matheny 2005; Co-David et al. 2009). ITS regions were amplified with ITS5 and ITS4; LR0R and LR5, MS1 and MS2 were used for nLSU and mtSSU amplification, respectively (White et al. 1990; http://www.biology.duke.edu/fungi/mycolab/primers.htm).

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 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.

A list of taxa, specimens and GenBank accession numbers of sequences used in this study.

Taxa Collection No. Origin GenBank accessions Remarks
ITS LSU RPB2 mtSSU
Entoloma albostrigosum DL Largent 9641 Australia: Queensland HQ876535 HQ876513 HQ876557 GenBank ID: Pouzarella albostrigosa
DL Largent 9663 Australia: Queensland HQ876536 HQ876514 HQ876558 GenBank ID: P. albostrigosa
E. araneosum ME Noordeloos 200314 China: Jilin KC710056 GQ289153 GQ289225 GQ289293
E. barringtonense DL Largent 9901 (Holotype) Australia: Queensland HQ876524 HQ876543 HQ876546 GenBank ID: P. parvula
E. changchunense HMJAU 3886 (Holotype) China: Jilin JQ993095 JQ993061
E. crassicystidiatum GDGM 28821 China: Guangdong KC678997 JQ291567 JQ993085 JQ993058
GDGM 27357 (Holotype) China: Guangdong KC678996 JQ291569 JQ993083 JQ993056
E. debile DLL 9784 Australia: Queensland HQ876528 HQ876506 HQ876550 GenBank ID: P. debilis
E. dindenense DL Largent 9623 Australia: Queensland HQ876527 HQ876505 HQ876549 GenBank ID: P. fusca
E. erectoides SAAS 1232 (Holotype) China: Sichuan MH020746 KU534255 KU534496
SAAS 945 China: Sichuan MH020769 KU534239 KU534498
SAAS 1361 China: Jilin MH020755 KU534484
E. farinosum DL Largent 9934 (Holotype) Australia: Queensland HQ876516 HQ876495 HQ876538 GenBank ID: P. farinosa
DL Largent 9900 Australia: Queensland HQ876515 HQ876494 HQ876537 GenBank ID: P. farinosa
E. furfuraceum GDGM 28818 (Holotype) China: Jinlin JX975293 JQ993094 JQ993084 JQ993062
SAAS 104 China: Jilin KU534240
E. griseocarpum SAAS 1230 China: Tibet MH020753 KU534253 KU534500 KU534438
SAAS 1328 (Holotype) China: Tibet MH020766 KU534256 KU534501 KU534455
SAAS 951 China: Sichuan MH020770 KU534242 KU534499 KU534457
E. lageniforme DL Largent 9895 Australia: Queensland HQ876523 HQ876502 HQ876545 GenBank ID: P. lageniformis
E. lasium DL Largent 9662 Australia: Queensland HQ876529 HQ876507 HQ876551 GenBank ID: P. lasia
DL Largent 9670 Australia: Queensland HQ876530 HQ876508 HQ876552 GenBank ID: P. lasia
DL Largent 9807 Australia: Queensland HQ876533 HQ876511 HQ876555 GenBank ID: P. lasia
DL Largent 9811 Australia: Queensland HQ876534 HQ876512 HQ876556 GenBank ID: P. lasia
DL Largent 9729 Australia: Queensland HQ876531 HQ876509 HQ876553 GenBank ID: P. lasia
DL Largent 9778 Australia: Queensland HQ876532 HQ876510 HQ876554 GenBank ID: P. lasia
E. nodosporum TENN:068582 USA: Tennessee KY744163 MF797654 GenBank ID: P. nodospora
E. pamiae DL Largent 9794 Australia: Queensland HQ876517 HQ876496 HQ876539 GenBank ID: P. pamiae
DL Largent 9834 Australia: Queensland HQ876519 HQ876498 HQ876541 GenBank ID: P. pamiae
DL Largent 9808 Australia: Queensland HQ876518 HQ876497 HQ876540 GenBank ID: P. pamiae
E. perbloxamii MEN 2004071 (Holotype) Australia: Tasmania KC710117 GQ289178 GQ289249 GQ289318
E. pilocystidiatum DL Largent 9848 Australia: Queensland HQ876520 HQ876499 HQ876542 GenBank ID: P. pilocystidiata
E. pilocystidiatum DL Largent 9932 (Holotype) Australia: Queensland HQ876521 HQ876500 HQ876543 GenBank ID: P. pilocystidiata
DL Largent 9949 Australia: Queensland HQ876522 HQ876501 HQ876544 GenBank ID: P. pilocystidiata
E. prunuloides MEN 200340 Slovakia KC710073 GQ289184 GQ289255 GQ289324
E. rubropilosum SAAS 406 (Holotype) China: Sichuan MH020761 KU534218 KU534488 KU534439
SAAS 1112 China: Tibet MH020767 KU534252 KU534502 KU534454
E. setiforme DL Largent 9809 (Holotype) Australia: Queensland HQ876525 HQ876503 HQ876547 GenBank ID: P. setiformis
DL Largent 9810 Australia: Queensland HQ876526 HQ876504 HQ876548 GenBank ID: P. setiformis
E. silvanum K(M) 191739 (Holotype) India: Kerala KY643747 KY643724
E. sp. 1 SAAS 894 China: Sichuan MH020765 KU534245 KU534491 KU534447
E. sp. 2 SAAS 1088 China: Jilin MH020749 KU534246 KU534441
SAAS 1210 China: Jilin MH020752 KU534248 KU534449
E. sp. 3 SAAS 249 China: Sichuan MH020759 KU534243
E. sp. 4 SAAS 1209 China: Jilin MH020751 KU534492 KU534448
SAAS 291 China: Jilin MH020760 KU534486 KU534444
E. sp. 5 SAAS 1360 China: Jilin MH020754 KU534249 KU534456
E. sp. 6 SAAS 1464 China: Sichuan MH020756 KU534258 KU534493 KU534450
E. sp. 7 SAAS 100 China: Sichuan MH020747 KU534442
E. sp. 8 080301 China: Sichuan MH020745 KU534254
SAAS 102 China: Sichuan MH020748 KU534489 KU534443
E. sp. 9 SAAS 1527 China: Shaanxi MH020758 KU534251 KU534495 KU534452
E. sp. 10 SAAS 529 China: Sichuan MH020763 KU534244 KU534497 KU534446
SAAS 772 China: Sichuan MH020764 KU534241 KU534487 KU534458
E. sp. 11 SAAS 526 China: Shaanxi MH020762 KU534257 KU534490 KU534445
E. strigosissimum 152 Italy JF908004
E. subaraneosum GDGM 28823 (Holotype) China: Jilin JQ320113 JQ410329
KA 12-1534 South Korea KJ523135
E. tenuissimum GDGM 28813 China: Jilin JX975295 JQ993097 JQ993086 JQ993059
GDGM 28814 (Holotype) China: Heilongjiang JX975294 JQ993096 JQ993087 JQ993060
E. violaceovillosum P. Manomohan 645 (Holotype) India: Kerala GQ289205 GQ289273 GQ289345
E. yunnanense GDGM 28815 China: Yunnan JQ320108 JQ320128 JQ993057

Taxonomy

Entoloma erectoides Xiao L. He & E. Horak, sp. nov.

MycoBank No: 828692
Figs 1a, b, 2

Diagnosis

E. erectoides is distinguished by the greyish brown pileus covered with silvery fibrils, large basidiospores (13.5–17.5 × 8–9.5 µm) and presence of ovoid to subutriform cheilocystidia.

Type

CHINA. SICHUAN PROV.: Yajiang County, Gexigou National Nature Reserve, 29°33'N, 100°50'E, elevation ca. 2980 m, August 2014, He XL (SAAS 1232, holotype; ZT 14180, isotype).

Etymology

Erectoides, refers to the suberect to erect fibrils on the pileus.

Figure 1. 

Basidiomata of the new species. a, b E. erectoides (SAAS 1361, SAAS 1232) c, d E. griseocarpum (SAAS 951, SAAS 1328) e, f E. rubropilosum (SAAS 406, SAAS 1112).

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.

Basidiospores (13–) 13.5–17.5 × 8–9.5 (–10.5) µm (x = 15.5 ± 0.5 × 8.8 ± 0.3 µm), Q = 1.50–1.94 (Q = 1.72 ± 0.03), heterodiametric, strongly angled in profile and face views with 6–9 facets, appearing nodulose, pale yellow brownish, thick-walled. Basidia 39–48 × 13–18 µm, subclavate or clavate, 4-spored. Aborted basidia scattered in the hymenium, often filled with dark brown amorphous cytoplasmic pigment. Lamellar trama dark brown, composed of parallel, cylindrical, heavily encrusted and thin-walled cells, 6–15 µm wide. Lamellar edge sterile. Cheilocystidia 33–90 × 12–33 µm, broadly ovoid to utriform (32–65 × 22–30 µm) or lageniform (70–90 × 16–20 µm), with pale brownish, intracellular pigment, slightly thick-walled. Pileipellis a trichoderm composed of clustered and suberect hyphae, walls externally encrusted with brown pigment; terminal cells 30–50 (–90) × 8–15 µm, cylindrical to slightly fusoid; subpellis composed of cylindrical, encrusted hyphae, up to 20 µm broad. Stipitipellis composed of thin-walled and pale yellowish-brown encrusted hyphae; terminal cells 40–80 × 9–15 µm, cylindrical to slender fusoid, walls encrusted with pale yellow-brown pigment. Oleiferous hyphae absent. Clamp connections absent.

Habitat

Scattered or gregarious on soil and amongst leaf litter in broadleaf forest dominated by Quercus or on soil amongst decaying leaves of Betula, Pandus and Abies.

Additional collections examined

CHINA. SICHUAN PROVINCE: CHINA: SICHUAN PROV. Yajiang County, Gexigou National Nature Reserve, 29°33'N, 100°50'E, elevation ca. 2980 m, 24 July 2013, He X.L. (SAAS 945). JILIN PROV.: Antu County, Changbai Mountains, 42°10'N, 127°55'E, elevation ca. 750 m, 25 August 2014, He X.L. (SAAS 1361).

Comments

Morphologically, Entoloma erectoides is a member of section Dysthales. In literature, a few species in section Dysthales are described having silvery fibrils or squamules on pileus and stipe. Accordingly, E. erectoides can be confused with the Argentinean E. calileguense Blanco-Dios (as Pouzarella variabilis T.J. Baroni, Albertó, Niveiro & B.E. Lechner in Baroni et al. 2012). Both species have silvery greyish-brown erect fibrils or squamules on the pileus and stipe. However, the latter species is easily separated by the much larger basidiospores (16–23.5 × 10–12 μm, Baroni et al. 2012). E. farinosum (Largent & Skye Moore) Noordel. & G.M. Gates, reported from Australia, differs by globose or nearly napiform cheilocystidia (Largent et al. 2011). In addition, this taxon is separated from E. erectoides by molecular evidence. E. tenuissimum T.H. Li & Xiao-Lan He, also recorded from China, is distinguished by the smaller and slimmer basidiomes and taxonomically is also distinctly different based on molecular analysis (He et al. 2013). E. argenteolanatum (T.J. Baroni, Perd.-Sánch. & S.A. Cantrell) Noordel. & Co-David was found on decaying leaves of tropical trees and shrubs in the Dominican Republic and is characterised by denser and longer silvery fibrils and the place of discovery in the Caribbean on the island of Hispaniola (Baroni et al. 2008). The other grey-brown species with silvery fibrils in section Versatile could be distinguished by the innately fibrillose pileus and stipe and colourless hymenial cystidia.

Figure 2. 

Microscopic structures of E. erectoides (drawn from the holotype). a Cheilocystidia b Basidiospores c Pileipellis.

Entoloma griseocarpum Xiao L. He & E. Horak, sp. nov.

MycoBank No: 828701
Figs 1c, d, 3

Diagnosis

E. griseocarpum is characterised by the greyish-brown pileus, large basidiospores (12.5–15.5 × 7.5–9 µm) and broadly clavate, ovoid to lageniform cheilocystidia.

Type

CHINA. TIBET: Linzhi, Lulang, 29°94'N, 94°79'E, elevation ca. 3800 m, 18 September 2014, He X.L. (SAAS 1328, holotype).

Etymology

griseocarpum, refers to the greyish-brown coloured basidiomes.

Description

Pileus 5–20 mm broad, hemispherical, convex, bluntly conic to broadly campanulate, dry, not hygrophanous, greyish-brown to brown (4D3–4E3), densely covered by suberect hispid or minutely squamulose overall, denser in centre; fibrils dark grey, pale grey brownish or concolorous with pileal surface (4D2–4D3), striate from entire margin to near centre. Lamellae sinuate with short decurrent tooth, ventricose, distant, moderately thick, up to 3 mm broad, with two tiers of lamellulae, dark grey to brownish-grey, with entire and concolorous edges. Stipe 20–50 × 0.7–1.5 mm, cylindrical, equal, dry, concolorous with pileus, densely covered with pale yellow brownish flocculose hairs, hollow, with a dirty yellowish to pale yellow brownish strigose base. Context thin, concolorous with pileus. Odour and taste not distinctive.

Basidiospores 12.5–15.5 (–17) × (6.5–) 7.5–9 (–9.5) µm (x = 13.8 ± 0.3 × 8.3 ± 0.3 µm), Q = 1.60–1.94 (Q = 1.71 ± 0.02), heterodiametric, strongly angled in profile and face view with 6–10 facets, appearing nodulose, pale yellow brownish, thick-walled. Basidia 35–55 × 11–13 (–15) µm, subclavate to clavate, 4-spored. Aborted basidia scattered in the hymenium, filled with dark brown amorphous cytoplasmic pigment. Lamellar trama dark brown, composed of parallel, cylindrical, heavily encrusted and thin-walled elements. Lamellar edges sterile. Cheilocystidia 23–50 × (10–) 12 20 µm, broadly clavate, ovoid to lageniform; with brownish, intracellular pigment, slightly thick-walled. Pileipellis a trichoderm composed of yellow brown, suberect and multiseptate hyphae, walls heavily encrusted with brown pigment; terminal cells 35–105 × 8–27 µm, cylindrical, subclavate or bullet-shaped, thin to moderately thick-walled; subpellis composed of cylindrical encrusted hyphae, up to 25 µm diam. Stipitipellis composed of yellow-brown encrusted hyphae; terminal cells 40–80 × 4–10 µm, slender cylindrical with obtuse apex, thin-walled, sparsely encrusted with pale yellowish-brown pigment. Oleiferous hyphae absent. Clamp connections absent.

Habitat

Scattered on soil amongst decaying litter in mixed conifer-broadleaf forest dominated by Quercus, Betula, Rhododendron and Abies.

Additional collections examined

CHINA. TIBET: Linzhi, Lulang, 29°94'N, 94°79'E, elevation ca. 3800 m, 18 September 2014, He X.L. (SAAS 1230, SAAS 1657, SAAS 1751, SAAS 1871). SICHUAN PROV.: Jiuzhaigou, 33°28'N, 103°59'E, elevation ca. 3000 m, 20 July 2013, He X.L. (SAAS 951).

Comments

The greyish-brown pileus covered by suberect hispid or minutely squamulose, the brown external encrustations on pileipellis and stipitipellis and the cylindrical terminal cells of pileipellis and stipitipellis indicate E. griseocarpum belongs to the sect. Dysthales. It is very similar to E. albostrigosum (Largent & Abell-Davis) Blanco-Dios and E. lasium (Berk. & Broome) Noordel. & Co-David (Largent et al. 2011). However, E. albostrigosum is distinguished by the white strigose base and E. lasium differs by the smaller basidiospores (8.9–14.5 × 5.1–8.7 μm, Largent et al. 2011). In addition, the two species are distant from E. griseocarpum following phylogenetic analysis. E. puertoricense Blanco-Dios (as P. caribaea T.J. Baroni & B. Ortiz in Baroni and Ortiz 2002) resembles E. griseocarpum by the brownish-grey coloured basidiomes but is separated by its broader basidiospores (12.5–16.5 × 8.3–11.3 μm, Q = 1.26–1.65, Baroni and Ortiz 2002). Moreover, E. puertoricense was discovered in a tropical habitat in Puerto Rico (Baroni and Ortiz 2002). The similar E. japonicum (Hongo) Hongo, described from Japan, is also reminiscent of E. griseocarpum in the brownish pileus but is distinguished by the much larger basidiospores (15–18.5 × 9–10.5 μm, Hongo 1959). The well-known E. dysthales (Peck) Sacc. also differs by the larger basidiospores (14–20 × 7.5–10 µm, Mazzer 1976). E. fulvolanatum (Berk. & Broome) Blanco-Dios from Sri Lanka is not only separated by its type locality but also by the narrower basidiospores measuring 12–16 × 7–8 μm (Mazzer 1976). Two species in subgen. Pouzarella, recently described from geographically neighbouring India, viz. E. peechiense K. N. A. Raj & Manim. and E. silvanum K. N. A. Raj & Manim., have somewhat similar basidiomes as compared to E. griseocarpum; however, their ITS and LSU sequences are distinctly different (Raj and Manimohan 2017). The third Indian species E. lomapadum Manim., Joseph & Leelav. is readily recognised by the much smaller basidiospores measuring 11–13 × 6–9 μm (Manimohan et al. 1995). There were four other species in subgen. Pouzarella which showed some similarities to E. griseocarpum. E. fibrillosipes (Murrill) Noordel. & Co-David is distinguished by the much larger basidiospores (17–22 × 7.5–10 μm, Mazzer 1976). E. subdeceptivum Courtec. and E. rotula (Romagn.) Noordel. & Co-David are lignicolous (Mazzer 1976). E. homomorphum (Romagn.) Singer differs by the larger basidiospores (15–19 × 9–11.5 μm, Mazzer 1976).

Figure 3. 

Microscopic structures of E. griseocarpum (drawn from the holotype). a Basidiospores b Cheilocystidia c Pileipellis.

Entoloma rubropilosum Xiao L. He & E. Horak, sp. nov.

MycoBank No: 828699
Figs 1e, f, 4

Diagnosis

E. rubropilosum is distinct due to its reddish-brown coloured pileus and stipe, large basidiospores (13–17 × 7.5–9.5 µm), broadly clavate cheilocystidia, distinctive thick-walled setiform caulocystidia and terminal cells of the pileipellis hyphae.

Type

CHINA: SICHUAN PROV.: Yajiang County, Gexigou National Nature Reserve, 29°33'N, 100°50'E, elevation ca. 2950 m, 24 July 2013, He X.L. (SAAS 406, holotype).

Etymology

Rubropilosum, refers to the reddish coloured fibrils on the pileus.

Description

Pileus 7–20 mm broad, conical-convex, truncate conical to broadly campanulate, dark reddish-brown (8D2–8D3) at first, becoming greyish-orange to pale beige brownish (5B2–5C2), dry, slightly hygrophanous, densely covered by reddish-brown erect or suberect squamules and fibrils; fibrils much denser at disc, margin not striate or very slightly striate only. Lamellae adnate to sinuate, ventricose, up to 2.5 mm wide, relatively thick, with two tiers of lamellulae, brownish-pink when mature, with concolorous and entire edges. Stipe 40–73 × 0.8–2 mm, central, cylindrical, hollow, densely covered with rust reddish hairs or fibrils, very dark brown strigose at base. Odour and taste not distinctive.

Basidiospores (12.5–) 13–17 × 7.5–9.5 µm (x = 15.2 ± 0.5 × 8.5 ± 0.3 µm), Q = 1.53–1.98 (Q = 1.76 ± 0.02), heterodiametrical, with 6–8 facets in profile and face views, sometimes multi-angled to nodulose, pale brownish, thick-walled. Basidia (32–) 38–45 (–50) × 12–16 µm, clavate, 4-spored. Aborted basidia inconspicuous. Lamellar edges sterile. Cheilocystidia 25–50 × 12–18 µm, broadly clavate, with faintly pale brownish, intracellular pigment, slightly thick-walled. Pleurocystidia absent. Pileipellis a trichoderm composed of brown hyphae; terminal cells 23–110 × 6–18 µm (diameter was measured at the base), slender setiform, gradually tapering towards subacute apex, sometimes subfusoid to somewhat bullet-shaped, thick-walled, with intraparietal and intracellular brown pigment; subpellis composed of cylindrical, relatively thin-walled hyphae, encrusted with yellow-brown pigment. Stipitipellis composed of loosely entangled, rather slender hyphae; terminal cells 45–120 × 5–11 µm (diameter was measured at the base), distinctly setiform with obtuse or subacute apex, thick-walled, with intraparietal and intracellular brown pigment. Oleiferous hyphae absent. Clamp connections absent.

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.

Additional collections examined

CHINA. SICHUAN PROV.: Yajiang County, Gexigou National Nature Reserve, 29°33'N, 100°50'E, elevation ca. 2950 m, 24 July 2013, He X.L. (SAAS 765); 24 July 2013, He X.L. (SAAS 706); 3 August 2014, He X.L. (SAAS 1488, SAAS 1112, ZT 14179). TIBET: Linzhi, Lulang, 29°94'N, 94°79'E, elevation ca. 3800 m, 18 September 2014, He X.L. (SAAS 1618, SAAS 1087); Linzhi, Kadinggou, 29°50'N, 93°26'E, elevation ca. 2950 m, 24 September 2014, He X.L. (SAAS 1456).

Comments

The setiform terminal cells of pileipellis and stipitipellis place E. rubropilosum in sect. Pouzarella. It is readily recognised in the field. A few species of Entoloma subgen. Pouzarella with reddish-brown fibrils or squamules have been reported in literature (Mazzer 1976; Baroni et al. 2008). E. ferreri (T.J. Baroni, Perd.-Sánch. & S.A. Cantrell) Noordel. & Co-David is distinguished by dark blackish stains on the pileus caused from handling and non-setiform pileocystidia and caulocystidia (Baroni et al. 2008). E. strigosissimum (Rea) Noordel. is separated by the larger basidiospores [15–19 (23) × 8.5–10.5 (11.5) µm, Mazzer 1976]. E. squamifolium (Murrill) Singer might be confused with E. rubropilosum due to the ferruginous hairs on the stipe (Mazzer 1976). However, E. rubropilosum can be distinguished by the setiform pileocystidia and caulocystidia. Furthermore, type of E. squamifolium was collected in a tropical location. The recently described E. wayanadense K. N. A. Raj & Manim. from India, also discovered in a tropical area, is similar to E. rubropilosum in its greyish-orange pileus with long hairs and the setiform terminal cells of pileipellis, but differs by the absence of cheilocystidia. In addition, the partial ITS sequence (419 bp, KY 643748) of E. wayanadense is quite different from that of E. rubropilosum (Raj and Manimohan 2017).

Figure 4. 

Microscopic structures of E. rubropilosum (drawn from the holotype). a Basidiospores b Cheilocystidia c Pileipellis d Stipitipellis.

Key to the species of Entoloma subgen. Pouzarella described from China

1 Pileus reddish-brown or greenish-brown 2
Pileus greyish-brown 3
2 Pileus covered with reddish-brown suberect fibrils E. rubropilosum
Pileus greenish-brown with reddish tinge, zonate E. changchunense
3 Pileus covered with appressed or suberect silvery fibrils 4
Pileus squamulose or covered with suberect brownish fibrils 5
4 Pileus covered with appressed silvery fibrils E. subaraneosum
Pileus covered with suberect silvery fibrils 6
5 Pileus fibrillo-squamulose or squamulose-tomentose, growing in tropical forest E. crassicystidiatum
Pileus covered with erect or suberect fibrils 7
6 Pileus pale brownish with pinkish tinge, basidiospores larger, average (16.8 ± 0.5) × (10.8 ± 0.3) µm E. tenuissimum
Pileus greyish-brown, basidiospores smaller, average (15.5 ± 0.5) × (8.8 ± 0.3) µm E. erectoides
7 Average spore length less than 13 µm E. furfuraceum
Average spore length more than 13 µm 8
8 Pileus greyish-brown, striate from entire margin to near centre E. griseocarpum
Pileus peach brown, not striate E. yunnanense

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).

Figure 5. 

Phylogenetic relationships of Entoloma subgen. Pouzarella species inferred from the combined ITS, LSU, mtSSU and RPB2 dataset (new species are in bold). Bayesian posterior probability values (BPP > 0.90) and MP BS support values (> 50%) are indicated above branches as BPP/BS; RAxML BS support values (> 50%) are listed below branches.

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 Entoloma 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.

Acknowledgements

We express special thanks to Ms. Bo Zhang (Jilin Agricultural University) and Dr. Hai-Xia Ma (Chinese Academy of Tropical Agricultural Sciences) for assistance during fieldwork. This study is funded by the National Natural Science Foundation of China (Nos. 31400023, 31770020) and the Sichuan Provincial Innovation Ability Promotion Engineering (2016ZYPZ-028).

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