Research Article |
Corresponding author: Xiao-Lan He ( xl_he@qq.com ) Academic editor: Olivier Raspé
© 2018 Xiao-Lan He, Egon Horak, Di Wang, Wei-Hong Peng, Bing-Cheng Gan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
He X-L, Horak E, Wang D, Peng W-H, Gan B-C (2018) Three new species of Entoloma subgenus Pouzarella from China based on morphological and molecular data. MycoKeys 44: 1-18. https://doi.org/10.3897/mycokeys.44.24998
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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.
Entolomataceae , systematics, taxonomy, multi-gene analyses
Pouzarella Mazzer is a distinctive group of entolomatoid species that was accepted as a genus by some researchers (
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 (
Fresh basidiomes were photographed in the field and described macroscopically. Colour notations follow
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 (
Sequences used in phylogenetic analyses are listed in Table
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 (
Maximum parsimony analysis – MP analysis was performed using PAUP* version 4.0b10 (
Bayesian analysis – Bayesian analysis was performed using MrBayes 3.2.6 (
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 |
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.
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).
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.
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.
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).
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
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.
CHINA. TIBET: Linzhi, Lulang, 29°94'N, 94°79'E, elevation ca. 3800 m, 18 September 2014, He X.L. (SAAS 1328, holotype).
griseocarpum, refers to the greyish-brown coloured basidiomes.
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.
Scattered on soil amongst decaying litter in mixed conifer-broadleaf forest dominated by Quercus, Betula, Rhododendron and Abies.
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).
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 (
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.
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).
Rubropilosum, refers to the reddish coloured fibrils on the pileus.
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.
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.
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).
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 (
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 |
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.
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.
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 (
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,
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.
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).