Taxonomy and phylogeny of Dichostereum (Russulales), with descriptions of three new species from southern China.

Nine species of Dichostereum were subjected to phylogenetic analyses, based on a combined dataset of ITS1-5.8S-ITS2-nrLSU-tef1 sequences. The morphology of specimens collected from China and Australia were studied. Three species, D.austrosinense, D.boidinii and D.eburneum, collected from southern China, are described and illustrated as new to science, based on the morphological and molecular evidence. Dichostereumaustrosinense is characterised by the relatively large gloeocystidia (80-130 × 8-15 µm) and basidiospores (7.3-8 µm in diam.) with large warts and crests. Dichostereumboidinii is distinguished by its thick basidiomata and relatively small basidiospores (5.5-6.5 µm in diam.) with large warts and crests. Dichostereumeburneum is unique in having pale basidiomata growing on bark of living Castanopsis, abundant crystals in the context and basidiospores with dense and large ornamentations. A key to the 5 species of Dichostereum in China is given.

Introduction

Pilát, typified with (Bourdot & Galzin) Pilát, is a small and well-delimited corticioid genus in . It is characterised by resupinate basidiomata with smooth or grandinioid hymenophore, dimitic hyphal system with dextrinoid dichohyphae and clamped generative hyphae, gloeocystidia and ellipsoid or PageBreaksubglobose, ornamented basidiospores with a strong amyloid reaction in Melzer’s reagent (Hallenberg 1985; Bernicchia and Gorjón 2010). Previously, was placed in , which includes genera with dextrinoid skeletal hyphae (Reid 1965; Parmasto 1968, 1970; Hallenberg 1985). However, recent phylogenetic analyses, based on DNA sequences, showed that formed a monophyletic lineage in the clade, which includes genera with or without dextrinoid hyphae (Larsson and Larsson 2003; Miller et al. 2006; Larsson 2007).

was once treated as a subgenus of P. Karst. (, ) by some mycologists since the two genera are very similar in morphology except that the latter has smooth basidiospores (Boidin 1967; Parmasto 1970; Lanquetin 1973). Boidin and Lanquetin (1977) emended the description of and retained it as a separate genus. Later, Boidin and Lanquetin (1980) monographed the genus and provided a key to its 11 species based on evidence of morphology, distribution and intercompatibility tests of cultures. Based on limited sampling, their results showed that (Cooke & Ellis) Boidin & Lanq. and (Pers.) Boidin & Lanq. were widely distributed, while the other species seemed to be rather endemic (Boidin and Lanquetin 1980). Few studies on the genus have been carried out since then and many regions including East Asia need further collecting and study (Boidin et al. 1987, Boidin and Michel 1998).

Previously, two species, (Pouzar) Ginns & M.N.L. Lefebvre (= ) and (Schwein.) Boidin & Lanq., were reported in temperate China (Dai 2011). The species diversity of the genus in China is still unclear. In the present paper, we provide a morphological and phylogenetic study of the genus based on specimens mostly collected from southern China. This is part of an ongoing study of the corticioid fungi of the in China.

Materials and methods

Morphological studies

Voucher specimens were deposited in the herbaria of Beijing Forestry University, Beijing, China () and in the Centre for Forest Mycology Research, U.S. Forest Service, Madison, USA (). Freehand sections were made from dried basidiomata and mounted in 2% (w/v) potassium hydroxide (KOH), 1% phloxine (w/v) or Melzer’s reagent. Microscopic examinations were carried out with a Nikon Eclipse 80i microscope (Nikon Corporation, Japan) at magnifications up to 1000×. Drawings were made with the aid of a drawing tube. All measurements were carried out with sections mounted in Melzer’s reagent. Ornamentations were excluded from the measurements of basidiospores. Scanning electron micrographs (SEM) were taken with a JEOL JSM-6700F microscope (JEOL, Japan). Dried specimens were mounted directly in gold and platinum and examined and photographed at 10.0 kV. Colour names and codes follow Kornerup and Wanscher (1978). Herbarium code designations are from Index Herbariorum (Thiers, continuously updated).

DNA extraction and sequencing

The CTAB plant genome rapid extraction kit DN14 (Aidlab Biotechnologies Co. Ltd, Beijing) was used for DNA extraction and PCR amplification from dried specimens or cultures. The ITS, partial nrLSU and tef1 markers were amplified with the primer pairs ITS5/ITS4 (White et al. 1990), LR0R/LR7 (Vilgalys and Hester 1990) and 983F/1567R (Rehner and Buckley 2005), respectively. The PCR procedures followed Dai and He (2016). DNA sequencing was performed at Beijing Genomics Institute and the sequences were deposited in GenBank (Benson et al. 2018). The sequence quality control followed Nilsson et al. (2012). BioEdit v.7.0.5.3 (Hall 1999) and Geneious v.11.1.15 (Kearse et al. 2012) were used for chromatogram check and contig assembly.

Phylogenetic analyses

The molecular phylogeny was inferred from a combined dataset of ITS1-5.8S-ITS2-nrLSU-tef1 sequences of representative members of sensuLarsson (2007) (Table 1). (Ellis & Everh.) Ellis & Everh. was selected as the outgroup (Liu et al. 2017a). The sequences of the three markers (ITS, nrLSU and tef1) were aligned separately using MAFFT v.7 (Katoh et al. 2017, http://mafft.cbrc.jp/alignment/server/) with the G-INS-i iterative refinement algorithm and optimised manually in BioEdit v.7.0.5.3. The programme Gblocks v.0.91b (Castresana 2000) was then used to exclude poorly aligned positions of the ITS alignment. The separate alignments were concatenated using Mesquite v.3.5.1 (Maddison and Maddison 2018). The combined alignments were deposited in TreeBase (http://treebase.org/treebase-web/home.html, submission ID: 23332).

Table 1.

Species and sequences used in the phylogenetic analyses. Newly generated sequences are set in bold. Holotypes are marked with *.

Taxa	Voucher	Locality	ITS	nrLSU	tef1
Asterostroma bambusicola	He 4132	Thailand	KY263865	KY263871	MH669240
A. cervicolor	He 4020	China	KY263859	KY263869	–
Baltazaria eurasiaticogalactina	CBS 666.84	France	–	AY293211	–
B. octopodites	FLOR 56449	Brazil	MH260025	MH260047	–
Dichostereum austrosinense	He 4871*	China	MH538317	MH538334	–
	He 4316	China	MH538316	MH538335	–
	He 3551	China	MH538314	–	MH550363
D. boidinii	He 5026*	China	MH538324	MH538330	–
	He 1662	China	MH538309	–	MH550360
	He 4410	China	MH538315	MH538331	MH550361
	He 462	China	MH538311	–	–
	Dai 16117	China	MH538312	MH538327	MH550362
D. durum	Fungi Gallici 1985	France	AF506429	AF506429	–
D. eburneum	He 5374*	China	MH538318	MH538337	MH550366
D. effuscatum	GG 930915	France	AF506390	AF506390	–
	FP 101758 Sp	USA	MH538323	MH538336	MH550367
	CBS 516.80	USA	–	AF323739	–
D. granulosum	NH 7137	Canada	AF506391	AF506391	–
	FP 133479 Sp	USA	MH538321	MH538333	MH550368
	He 1887	China	MH538313	MH538332	–
D. pallescens	Kropp 2	USA	MH538320	MH538326	MH550365
	CBS 717.81	USA	–	AF518614	–
	He 3266	China	MH538310	MH538325	MH550364
D. aff. pallescens	KHL 10258	Puerto Rico	AF506428	AF506428	–
D. rhodosporum	Dai 18625A	Australia	MH538319	MH538329	MH550370
D. sordulentum	FP 11735 Sp	USA	MH538322	MH538328	MH550369
Duportella tristicula	He 4775	China	MH669235	MH669239	MH669245
Echinodontium tinctorium	HHB 12866 Sp	USA	KY172888	KY172903	MH550371
Gloiothele lactescens	EL 8-98	Sweden	AF506453	AF506453	–
G. lamellosa	KHL11031	Venezuela	AF506454	AF506454	–
Lachnocladium cf. brasiliense	CALD 161213-1	Brazil	MH260037	MH260055	–
L. schweinfurthianum	KM 49740	Cameroon	MH260033	MH260051	–
L. sp.	KHL10556	Jamaica	AF506461	AF506461	–
Parapterulicium subarbusculum	FLOR 56456	Brazil	MH260026	MH260026	–
	FLOR 56459	Brazil	MH260027	MH260049	–
Peniophora polygonia	He 3668	China	MH669233	MH669237	MH669243
P. rufa	He 2788	China	MH669234	MH669238	MH669244
Scytinostroma portentosum	EL11-99	Sweden	AF506470	AF506470	–
Vararia investiens	He 2104	USA	–	MH669236	MH669242
	FP 151122	USA	MH971976	MH971977	–
Vesiculomyces citrinus	He 3716	China	KY860369	KY860429	MH669241

For both Maximum Likelihood (ML) and Bayesian Inference (BI), a partitioned analysis was performed with the following five partitions: ITS1, 5.8S, ITS2, nrLSU and tef1. The ML analysis was performed using RAxML v.8.2.10 (Stamatakis 2014) with the bootstrap values (ML-BS) obtained from 1,000 replicates and the GTRGAMMA model of nucleotide evolution. The maximum parsimony (MP) analysis was performed using PAUP* 4.0a162 (Swofford 2003). Trees were generated using 100 replicates of random stepwise addition of sequence and tree-bisection reconnection (TBR) branch-swapping algorithm with all characters given equal weight. Branch supports (MP-BS) for all parsimony analyses were estimated by performing 1,000 bootstrap replicates with a heuristic search of 10 random-addition replicates for each bootstrap replicate. The BI was performed using MrBayes 3.2.6 (Ronquist et al. 2012). The best-fit substitution model for each partitioned locus was estimated separately with jModeltest v.2.17 (Darriba et al. 2012). Four Markov chains were run for 6,000,000 generations until the split deviation frequency value was lower than 0.01. The convergence of the runs was checked using Tracer v.1.7 (Rambaut et al. 2018). Trees were sampled every 100th generation. The first quarter of the trees, which represented the burn-in phase of the analyses, was discarded and the remaining trees were used to calculate Bayesian posterior probabilities (BPP) in the majority rule consensus tree. All trees were visualised in FigTree 1.4.2 (Rambaut 2014).

Results

Phylogenetic inference

The ITS-nrLSU-tef1 sequence dataset contained 37 ITS, 38 nrLSU and 18 tef1 sequences from 40 samples representing 26 ingroup taxa and the outgroup (Table 1). Twenty ITS, 18 nrLSU and 18 tef1 sequences were generated for this study. The dataset had an aligned length of 2239 characters, of which 1596 were constant, 163 variable characters were parsimony-uninformative and 480 were parsimony-informative. MP analysis yielded six most parsimonious trees. jModelTest suggested TIM2ef+G, K80+G, TPM1uf+G, TIM2+I+G and TrN+I+G to be the best-fit models of nucleotide evolution for ITS1, 5.8S, ITS2, nrLSU and tef1 markers, respectively, for the Bayesian analysis. The average standard deviation of split frequencies of BI was 0.004704 at the end of the run. MP and BI analyses resulted in an almost identical tree topology compared to the ML analysis. Only the ML tree is shown in Fig. 1 with ML and MP bootstrap values ≥50% and Bayesian posterior probabilities ≥0.95 labelled along the branches.

Figure 1.

Phylogeny of and representatives of inferred from ITS-nrLSU-tef1 sequences. Topology is from ML analysis with maximum likelihood bootstrap support values (≥50, former), parsimony bootstrap support values (≥50, middle) and Bayesian posterior probability values (≥0.95, latter) shown along the branches. Different species of are indicated as coloured blocks. The new species are set in bold. Scale bar: 0.05 nucleotide substitutions per site.

In the tree (Fig. 1), the nine sampled species of formed a strongly supported clade in (ML-BS = 91, MP-BS = 97, BPP = 1.00). (Schwein.) P. Karst., the generic type of , formed a sister lineage to , but this close relationship did not receive significant support. Of the three new species, samples of and formed two strongly supported lineages, whilst the single specimen of formed the sister taxon to and . from France and USA and both and from north America and China, formed three strongly supported lineages. Single samples of from France, from Australia and from USA formed their own distinct lineages.

Taxonomy

S.H. He & S.L. Liu sp. nov.

MB826931

Figs 2a , 3 , 6a

Figure 2.

Basidiomata of species. a (holotype, He 4871) b (holotype, He 5026) c (holotype, He 5374) d (He 1887) e (He 3266) f (Dai 18625A). Scale bar: 1 cm.

Figure 3.

Microscopic structures of (drawn from the holotype). a Basidiospores b Basidia c Gloeocystidia d Dichohyphae from hymenium e Dichohyphae from subiculum. Scale bar: 10 µm.

Figure 6.

Scanning electron micrographs (SEM) of basidiospores of . a (holotype, He 4871) b (holotype, He 5026) c (holotype, He 5374) d (He 1887) e (He 3266) f (Dai 18625A). Scale bar: 1 µm.

Typification.

CHINA. Guangxi Autonomous Region, Jinxiu County, Dayaoshan Nature Reserve, Shengtangshan, on fallen angiosperm trunk, 15 Jul 2017, He 4871 (holotype, BJFC 024390, ITS GenBank accession number: MH538317; isotype in CFMR).

Etymology.

“” referring to the distribution in southern China.

Basidiomata.

Perennial, resupinate, effused, closely adnate, inseparable from substrates, coriaceous to soft corky, at first as irregular small patches, later confluent up to 15 cm long, 4.5 cm wide, up to 1 mm thick. Hymenophore surface smooth, greyish-orange [5B(4–5)], brownish-yellow [5C(7–8)] to light brown [6D(4–8)], not cracking; margin abrupt, concolorous or darker than hymenophore surface.

Microscopic structures.

Hyphal system dimitic. Context thickening, compact, composed of generative hyphae, dichohyphae, embedded basidiospores and scattered crystals. Generative hyphae rare, with clamp connections, hyaline, thin- to slightly thick-walled, 2–3 µm in diam. Dichohyphae dominant, hyaline to yellow, distinctly thick-walled, dichotomously branched with acute tips, weakly dextriniod. Catahymenium composed of dichohyphae, gloeocystidia, basidia and basidioles. Dichohyphae in this layer abundant, similar to those in the context, but strongly dextrinoid, more slender and more frequently branched, 20–50 μm across, 2–4 µm wide at lowest part. Gloeocystidia abundant, subcylindrical to subfusiform, hyaline, slightly thick-walled, PageBreakwith or without solidified contents, 80–130 × 8–15 µm. Basidia narrowly cylindrical, usually slightly sinuous, hyaline, thin-walled, with 4 sterigmata and a basal clamp connection, 50–80 × 5–8 µm; basidioles in shape similar to basidia, but slightly smaller. Basidiospores abundant, subglobose with a distinct apiculus, hyaline to pale yellowish-brown in KOH, thick-walled, strongly amyloid, (7–) 7.3–8 (–9) µm in diam.; walls ornamented with large warts and crests.

Additional specimens examined.

CHINA. Hainan Province, Lingshui County, Diaoluoshan Nature Reserve, on fallen angiosperm trunk, 17 Mar 2016, He 3551 (BJFC 022052); Jiangxi Province, Lianping County, Jiulianshan Nature Reserve, on fallen angiosperm branch, 13 Aug 2016, He 4316 (BJFC 023758).

Remarks.

is overall characterised by the relatively large gloeocystidia and basidiospores with large warts and crests. (Burt) Boidin & Lanq. is similar to but differs in having wider gloeocystidia (7–22 µm), slightly larger basidiospores (7–9 µm) with larger ornamentations and a distribution in Caribbean regions (Lanquetin 1973; Boidin and Lanquetin 1980). is also similar to (Wakef.) Boidin & Lanq. which differs in having paler basidiomata, smaller ornamentations of basidiospores and a distribution in Australia and New Zealand (Boidin and Lanquetin 1980, Figs 2 and 6).

S.H. He & S.L. Liu sp. nov.

MB826932

Figs 2b , 4 , 6b

Figure 4.

Microscopic structures of (drawn from the holotype). a Basidiospores b Basidia c Gloeocystidia d Dichohyphae from hymenium e Dichohyphae from subiculum. Scale bar: 10 µm.

CHINA. Hubei Province, Wufeng County, Breeding base of , on angiosperm stump, 14 Aug 2017, He 5026 (holotype, BJFC 024544, ITS GenBank accession number: MH538324; isotype in CFMR).

“” (Lat.), named to honour Dr. Jacques Boidin (Lyon, France) for his contribution to the taxonomy of .

Perennial, resupinate to effused-reflexed with slightly elevated margin, closely adnate, inseparable from substrates, coriaceous to soft corky, up to 8 cm long, 4 cm wide, 1.5 mm thick. Hymenophore surface smooth, greyish-orange [6B(3–4)], brownish-orange [6C(4–6)] to light brown [6D(4–6)], not cracking; margin abrupt, concolorous or darker than hymenophore surface.

Hyphal system dimitic. Context thickening, compact, composed of generative hyphae, dichohyphae, embedded basidiospores and scattered crystals. Generative hyphae rare, with clamp connections, hyaline, thin-walled, 2–3 µm in diam. Dichohyphae dominant, hyaline to yellow, distinctly thick-walled, dextriniod. Catahymenium composed of dichohyphae, gloeocystidia, basidia and basidioles. Dichohyphae in this layer abundant, similar to those in the context, but strongly dextrinoid, more frequently branched with short terminal branches, 20–40 μm across, 2–4 µm wide at lowest part. Gloeocystidia abundant, fusiform to subulate, hyaline, slightly thick-walled, with solidified contents, 20–60 × 7–12 µm. Basidia subclavate to subcylindrical, hyaline, thin-walled, with 4 sterigmata and a basal clamp connection, 25–40 × 5–7 µm; basidioles in shape similar to basidia, but slightly smaller. Basidiospores subglobose with a distinct apiculus, hyaline to pale yellowish-brown in KOH, thick-walled, strongly amyloid, (5–) 5.5–6.5 (–7) µm in diam.; walls ornamented with warts and crests.

CHINA. Anhui Province, Huangshan County, Huangshan Nature Reserve, on fallen angiosperm trunk, 21 Oct 2011, He 462 (BJFC 012101); Hainan Province, Lingshui County, Diaoluoshan Nature Reserve, on rotten wood of , 13 Nov 2015, Dai 16117 (BJFC 020210); Jiangxi Province, Anyuan County, Sanbaishan Forest Park, on fallen angiosperm trunk, 15 Aug 2016, He 4410 (BJFC 023851); Yunnan Province, Kunming, Xishan Park, on angiosperm stump, 17 Jul 2013, He 1662 (BJFC 016129).

is widely distributed in southern China and mainly characterised by the thick, brownish basidiomata and relatively small basidiospores with large warts and crests. is similar to but differs in PageBreakhaving slender dichohyphae and smaller and sparser ornamentations of basidiospores (Boidin and Lanquetin 1980, Fig. 6). Boidin & Lanq. resembles by sharing short terminal branches of dichohyphae, but differs in having smaller basidiospores (5–5.5 µm in diam.) and a distribution in Africa (Boidin and Lanquetin 1980). The ornamentation of basidiospores of is similar to , but the latter species has larger gloeocystidia, basidia and basidiospores.

S.H. He & S.L. Liu sp. nov.

MB826933

Figs 2c , 5 , 6c

Figure 5.

Microscopic structures of (drawn from the holotype). a Basidiospores; b Basidia c Gloeocystidia d Dichohyphae from hymenium e Dichohyphae from subiculum. Scale bar: 10 µm.

CHINA. Fujian Province, Wuyishan County, Wuyishan Nature Reserve, on bark of living , 6 Apr 2018, He 5374 (holotype, BJFC, ITS GenBank accession number: MH538318; isotype in CFMR).

“” referring to the white colour of hymenophore.

Perennial, resupinate, effused, closely adnate, inseparable from substrate, coriaceous, at first as irregular small patches, later confluent up to 7 cm long, 2 cm wide, 200–500 µm thick. Hymenophore surface smooth, white (5A1), orange white (5A2) to greyish-orange [5B(3–4)], cracking with age; margin thinning out, concolorous with hymenophore.

Hyphal system dimitic. Context thickening, compact, composed of generative hyphae, dichohyphae, embedded basidiospores and abundant crystals. Generative hyphae rare, with clamp connections, hyaline, thin- to slightly thick-walled, 2–3 µm in diam. Dichohyphae dominant, hyaline to yellow, distinctly thick-walled, dextriniod, frequently branched, aseptate, 1–2 µm in diam. Catahymenium composed of dichohyphae, gloeocystidia, basidia and basidioles. Dichohyphae in this layer abundant, hyaline to pale yellow, distinctly thick-walled, strongly dextriniod, dichotomously branched with acute terminal tips, 15–30 μm across, 2–4 µm wide at lowest part. Gloeocystidia abundant, fusiform to subclavate, hyaline, thin-walled, with solidified contents, 20–50 × 5–10 µm. Basidia subcylindrical with basal part slightly swollen, hyaline, thin-walled, with 4 sterigmata and a basal clamp connection, 30–45 × 6–9 µm; basidioles in shape similar to basidia, but slightly smaller. Basidiospores subglobose with a distinct apiculus, hyaline to pale yellowish-brown in KOH, thick-walled, strongly amyloid, 6–7 (–8) µm in diam.; walls ornamented with dense, large warts and crests.

is characterised by the pale basidiomata on bark of living tree, the presence of abundant crystals in context and basidiospores with dense and large ornamentations. Ecologically and macroscopically, resembles Höhn. & Litsch., but the microscopic features are largely different (Nakasone and Burdsall 2011). Boidin & Lanq. is similar to by sharing the large ornamentations of basidiospores, but differs in having wider span of dichohyphae, slightly larger basidiospores (7–8 µm) and a distribution in Africa (Boidin and Lanquetin 1980).

Discussion

To date, 14 species of have been described worldwide including the three new species in the present paper (Boidin and Lanquetin 1980). Amongst them, 5 species, (S.S. Rattan) Boidin & Lanq. from India, , and (Boidin & Lanq.) Boidin & Lanq. from Africa and from Caribbean regions, were not included in the present analyses. In order to PageBreakresolve the infra-generic phylogenetic relationships of , samples of these species and any additional undescribed taxa should be included.

The family sensuLarsson (2007) formed a strongly supported clade in and included about 15 genera (Larsson and Larsson 2003; Miller et al. 2006; Larsson 2007; Leal-Dutra et al. 2018). Except for the coralloid Lév. and the insect symbiont H.S. Whitney, Bandoni & Oberw., all the other genera in the family are corticioid fungi, such as Massee, Cooke, Donk and . However, recent molecular and morphological studies showed that two species of Corner with coralloid basidiomata belong to in the rather than of the (Leal-Dutra et al. 2018). In the phylogenetic tree, the type species, Corner formed a distinct lineage, while Corner is closely related to (Fr.) Donk and its relatives. More studies on the taxonomy and phylogeny of are needed, since some large genera such as and are still polyphyletic and many species are undescribed.

1	Hymenophore grandinioid; basidiospores ellipsoid	D. granulosum
–	Hymenophore smooth; basidiospores subglobose	2
2	Basidiomata white; on bark of living Castanopsis	D. eburneum
–	Basidiomata brownish; on dead wood	3
3	Gloeocystidia ≥80 μm long	D. austrosinense
–	Gloeocystidia <80 μm long	4
4	Basidiospores 6.5–7.5 µm in diam, ornamentation sparse	D. pallescens
–	Basidiospores 5.5–6.5 µm in diam, ornamentation dense	D. boidinii