Short communication
Rhodocybe subasyae, a new species of Rhodocybe sect. Rufobrunnea (Entolomataceae, Agaricales) from northeast China
2023 Volume 64 Issue 3 Pages 96-100
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2023 Volume 64 Issue 3 Pages 96-100
Rhodocybe subasyae is described from northeast China as a new species with both morphological and molecular characteristics, which is morphologically characterized by its tricholomatoid basidiomata, orange white to beige red pileus, adnexed to sinuate lamellae and long clavate branched cheilocystidia within section Rufobrunnea. The phylogenetic tree based on internal transcribed spacers of rDNA (nrITS) sequences using Bayesian methods showed that new taxon was distinguished from other species of Rhodocybe.
The genus Rhodocybe Maire was proposed by Maire (1926) with the type species Rhodocybe caelata (Fr.) Maire and the majority of Rhodocybe species are characterized by variable shape and color of basidiomata, attached and pinkish to vinaceous cinnamon lamellae, basidiospores usually with randomly bumpy to pustulate ornamentation and minutely angular in polar view with 6-12 facets, clamp connections absent in all parts (Baroni, 1981; Kluting, Baroni, & Bergemann, 2014).
Rhodocybe sect. Rufobrunnea Singer ex T.J. Baroni was published by Baroni (1981) to retain the clampless species with pinkish or reddish brown pigments. The section was characterized by a central stipe; adnexed to adnate or decurrent lamellae; reddish, reddish brown, pinkish, ochraceous brown, rusty orange or dark brown pileus; cheilocystidia present or not and absent of clamp-connections (Baroni, 1981).
According to He et al. (2019), approximately 50 taxa of Rhodocybe have been described around the world, but only 2 species were reported in China viz. Rhodocybe cf. lignicola Singer and R. truncata (Schaeff.) Singer (Li, 2009; Zhong, 2019), due to some species reallocated to closely related genera (Kluting et al., 2014). During the field investigations in northeastern China, a new species of Rhodocybe was identified and described here based on morphological and phylogenetic analyses.
In this study, basidiomata of the type were photographed and collected from Tianfozhishan National Nature Reserve, Jilin Province, China. Fresh basidiomata were dried and preserved in the Herbarium of Mycology of Jilin Agricultural University (HMJAU). Macroscopic description was based on detailed field notes on fresh basidiomata. Color codes are according to Kornerup and Wanscher (1978). Microscopic features were observed with dried specimens, rehydrated in distilled water or 5% KOH and then stained with Congo Red as universal, using a Nikon 80i light microscope (Nikon, Tokyo, Japan). Melzer's reagent was used to check amyloidity of basiodiospore walls. At least 20 mature spores from each specimen were measured. The sterigmata of the basidia and the ornamentation of basidiospores were not included in the spore measurements based on the recent related studies (Liu & Bau, 2019; Sesli & Vizzini, 2017). The following abbreviations are used: L = number of lamellae reaching the stipe; l = number of lamellulae between each pair of lamellae; Q = the length/breadth ratio of basidiospores; Qav = average quotient.
Genomic DNA was extracted from 0.1-0.2 mg of dried specimens using a NuClean Plant Genomic DNA kit (CWBIO, Beijing, China). ITS1/ITS4 were used as primer sequences (White, Bruns, Lee, & Taylor, 1990). PCR cycling conditions were identical to the touchdown cycling program (Don, Cox, Wainwright, Baker, & Mattick, 1991). A total of 51 sequences of Entolomataceae were downloaded for molecular phylogenetic analysis based on BLAST results from NCBI GenBank database. Rugosomyces carneus (Bull.) Bon (AF357028) and Lyophyllum leucophaeatum (P. Karst.) P. Karst (AF357032) were selected as outgroups (Sesli & Vizzini, 2017; Vizzini, Ferrari, Ercole, & Fellin, 2018). Sequences were aligned using BioEdit 7.2.5 (Hall, 1999). Alignments were then imported into MEGA 7.0.26 (Kumar, Stecher, & Tamura, 2016) for manual adjustment. MrModeltest 2.3 was used to estimate optimal model to use with the allied sequences (Nylander, 2004). The alignment was deposited in TreeBASE (https://www.treebase.org/) with the accession number 27480. Bayesian Inference (BI) analysis was performed with MrBayes 3.2.6 (Ronquist & Huelsenbeck, 2003), and selected model for ITS datasets were HKY+I+G. Markov Chains (MCMC) were run for 2.5 million generations with sampling every 100th generation until the standard deviation of the split frequencies fell below 0.01 to obtain estimates for Bayesian posterior probabilities (BPP). The Maximum Likelihood (ML) estimation was performed in RAxML GUI2.0 (Edler, Klein, Antonelli, & Silvestro, 2019) with rapid bootstrapping algorithm involing 1000 replicates under GTRGAMMAI model, the other parameters used the default settings. The phylogenetic tree was displayed by FigTree vl.4.4. Detailed information on nucleotide sequences is shown in Table 1.
| Species | Voucher No. | Genbank accession No. | Locality |
| Clitocella mundula | TJB7599 | DQ494694 | USA |
| Clitocella popinalis | CBS821.87 | FJ770400 | - |
| Clitopilopsis hirneola | CBS 126.46 | FJ770392 | UK |
| Clitopilus amarus | A. d. Haan 98031 | KC885963 | - |
| Clitopilus chrischonensis | TO HG1994 | HM623128 | Switzerland |
| Clitopilus cystidiatus | UBC F-30425 | MF955170 | Canada |
| Clitopilus cystidiatus | E. Arnolds 03-27 | KC885964 | Norway |
| Clitopilus cystidiatus | TO AV131 | HM623130 | France |
| Clitopilus prunulus | CBS 227.93 | FJ770408 | UK |
| Clitopilus prunulus | HMJAU 4521 | MN061302 | Russia |
| Clitopilus scyphoides | 148 | MN945081 | Spain |
| Entocybe nitida | 287 | JF907989 | Italy |
| Entocybe haastii | MEL:2379812 | MF977980 | - |
| Entoloma abortivum | GDGM27313 | JQ291565 | China |
| Entoloma caccabus | 17 | KC710063 | The Netherlands |
| Entoloma conferendum | UBCF23758 | KC581296 | Canada |
| Entoloma myrmecophilum | 231 | KC710120 | Norway |
| Entoloma prunuloides | TJB4765 | DQ206983 | - |
| Entoloma sordidulum | Co-David 2003 | KC710062 | Norway |
| Entoloma sphagneti | OW-E2-14 | KX945366 | Norway |
| Lyophyllum leucophaeatum | Hae251.97 | AF357032 | - |
| Rhodocybe asanii | KATO:Fungi:3659 | NR154442 | Turkey |
| Rhodocybe asanii | KATO:Fungi:3657 | KX834265 | Turkey |
| Rhodocybe asyae | Personal collection:NA131019 | MN840644 | Turkey |
| Rhodocybe asyae | KATO:Fungi:3640 | NR154443 | Turkey |
| Rhodocybe asyae | KATO:Fungi:3653 | KX834268 | Turkey |
| Rhodocybe brunnescens | TENN056140 | HQ222033 | USA |
| Rhodocybe brunnescens | TENN:056140 | NR119914 | USA |
| Rhodocybe caelata | JVG 1070904-2 | KU862855 | Spain |
| Rhodocybe formosa | Herb. B. Picillo 12/208 | KU862858 | Italy |
| Rhodocybe formosa | Herb. B. Picillo 12/198 | KU862857 | Italy |
| Rhodocybe formosa | LIP JVG 1061015 | KU862856 | Spain |
| Rhodocybe fumanellii | MCVE:29550 | NR166243 | Italy |
| Rhdodcybe fusipes | DLK 298 | MN306209 | Brazil |
| Rhdodcybe fusipes | DLK 587 | MN306210 | Brazil |
| Rhodocybe griseoaurantia | CAL:1324 | NR154435 | India |
| Rhodocybe griseoaurantia | CAL 1324 | KX083571 | India |
| Rhodocybe griseonigrella | LIP:JVG 1081204 | NR163283 | Spain |
| Rhodocybe matesina | MCVE:29261 | KY629962 | Italy |
| Rhodocybe matesina | MCVE:29262 | NR154455 | Italy |
| Rhodocybe minutispora | LIP JVG 1071101 | KU862860 | Spain |
| Rhodocybe roseiavellanea | PBM4056 (TENN) | MF686525 | USA |
| Rhodocybe rubrobrunnea | CAL:1387 | NR154445 | India |
| Rhodocybe rubrobrunnea | CAL 1387 | KX951452 | India |
| Rhodocybe subasyae | HMJAU56921-1 | MW298803 | China |
| Rhodocybe subasyae | HMJAU56921-2 | MW298804 | China |
| Rhodocybe subasyae | HMJAU56921-3 | MW298805 | China |
| Rhodocybe truncata | CBS482/50 | EF421110 | - |
| Rhodocybe tugrulii | MSNG3938 | KY945354 | Italy |
| Rhodocybe tugrulii | KATO:Fungi:3340 | NR154436 | Turkey |
| Rugosomyces carneus | CBS552.50 | AF357028 | - |
Rhodocybe subasyae T. Bau & Y. L. Sun, sp. nov. Figs. 1, 2.
MycoBank no.: MB 838088.
Diagnosis: R. subasyae differs from R. asyae Sesli & Vizzini by the tricholomatoid basidiomata, orange white (6A2) to beige red (7B4) pileus and sinuate to adnate lamellae. Basidiospores inamyloid, ellipsoid to broadly ellipsoid. Cheilocystidia slenderly clavate, sometimes with short branch.
Holotype: CHINA, Jilin Province, Longjing City, Yanbian Korean Autonomous Prefecture, Tianfozhishan National Nature Reserve, Jul 10, 2020, leg. Ya-li Sun, (HMJAU56921).
Gene sequences ex-holotype (ITS): MW298803-MW298805.
Etymology: sub = somewhat, asyae- refers to the closest phylogenetic relationship with the species R. asyae.
Pileus 19-25 mm diam, convex to plane, sometimes irregular, beige red (7B4) over most of the surface, margin ivory to beige (6A2-6A3); surface dry, smooth, with off-white (7B1) slightly tomentose at the centre. Context thin, pale white (1A1). Lamellae adnexed to sinuate, concolorous with the pileus or paler, crowded, fragile, edge regular, L = 40-50, l = 2-4. Stipe 22-37 × 5-7 mm, cylindrical, sometimes slightly flattened, fibrillose, orange white (6A2) with white (1A1) basal mycelium. Odor and taste not distinctive.
Basidiospores (4.9-) 5.4-6.8 (-7.8) × (3.4-) 3.9-4.9 (-5.8) μm, 6.0 × 4.3 μm on average, Q = 1.2-1.6, Qav = 1.4, ellipsoid to broadly ellipsoid in profile view, colorless, angular in polar view with 6-8 facets, walls cyanophilic, inamyloid. Basidia 18.5-28.2 × 5.8-6.8 μm, 2 to 4-spored (mostly 2-spored), sterigmata 2-3 μm, clavate, colorless, smooth, thin-walled, inamyloid, sometimes with oil drops. Basidioles 9.7-23.4 × 3.9-6.8 μm, similar to basidia, clavate, colorless, smooth, thin-walled. Cheilocystidia rare, 22.4-28.2 × 3.9-6.8 μm, slenderly clavate, flexuous, occasionally with short branch, colorless, smooth, thin-walled. Pleurocystidia absent. Lamellar trama regular, cylindrical, thin-walled, 19-25 mm diam, colorless, thin-walled. Pileipellis two layered, a layer of short cylindrical colorless hyphae, 4-7 μm diam and thin-walled; a layer of enlarged cylindrical colorless hyphae without encrusted, 7-12 μm diam. Pileus context consisting of short cylindrical, enlarged, colorless, smooth, thin-walled, up to 11 μm wide hyphae. Stipitipellis a cuits made up of cylindrical, parallel, colorless, smoooth, thin-walled, up to 10 μm wide hyphae. Caulocystidia absent. Clamp connections absent in all tissues.
Habitat and distribution: Scattered in the sandy soil under mixed forests (Pinus L., Quercus L., Larix Mill.), currently known from northeast China (Tianfozhishan National Nature Reserve, Jilin).
Specimen examined: CHINA. Jilin Province: Tianfozhishan National Nature Reserve, Longjing City, Yanbian Korean Autonomous Prefecture, 42°57′13″N , 129°67′17″E, elev. 428 m, Ya-li Sun, 10 Jul 2020, HMJAU56921 (Holotype!).
The Bayesian analyses based on the nrITS of Entolomataceae shows that species in each genus located in separate clades with high node support which agrees with the previous studies (Sesli & Vizzini, 2017; Vizzini et al., 2018). The ML tree shows the similar topologies with the BI tree. The species of genus Rhodocybe fell in one clade and the sect. Rufobrunnea form a well-supported clade. The new species, R. subasyae occupied a distinct position together with another species R. asyae of sect. Rufobrunnea by a high node support (BPP=1.00/ML=100). Based on the results of phylogeny study, R. subasyae is independent to other Rhodocybe taxa (Fig. 3).
The tricholomatoid basidiomata, orange white to beige red pileus and clamp connections absent in all tissues indicate that R. subasyae belongs to sect. Rufobrunnea according to Baroni (1981), and it was proved to be an independent and distinct species within this section based on phylogenetic analysis (Fig. 3). Several species of Rhodocybe sect. Rufobrunnea related to R. subasyae were compared in Table 2.
| Characters/species | R. subasyae | R. asyae | R. fusipes | R. fumanellii | R. asanii |
| Pileus diam (mm) | 19-25 | 10-30 | 13-47 | 35-100 | 20-45 |
| Pileus surface | smooth, central slightly tomentose | smooth, color changed when old or injured | smooth to slightly pruninose, deep orange, orange, reddish orange, hiah red | smooth, dry, greasy when wet | dry, minutely felty-tomentose, color changed when old or injured |
| Stipe (mm) | 22-37 × 5-7 | 25-30 × 2-5 | 43-63 × 5-10 | 40-70 × 5-15 | 25-40 × 5-15 |
| Lamellae colour | orange white to beige red | whitish, light ivory to beige red or slightly reddish beige | white becoming reddish with age | at first whitish-cream, finally pinkish, with an irregular-eroded concolorous edge | light ivory to beige red, wood color or paler |
| L, l | L = 40-50, l = 2-4 | L = 35-55, l = 1-3 | - | L = 60-80, l = 1-3(-4) | L = 40-50, l = 2-5 |
| Basidiospores (Size μm) | 5.4-6.8 × 3.9-4.9 | 5-7 × 4-5 | 5-5.5 × 4-5 | 5.68-6.83 × 3.93-4.58 | 5.3-6.3 × 3.5-4.5 |
| Q | 1.2-1.6 | 1.1-1.4 | 1-1.25 | 1.34-1.6 | 1.2-1.5 |
| Basidiospores Shape | ellipsoid to broadly ellipsoid | ellipsoid to broadly ellipsoid | globose to short ellipsoid | ellipsoid | ellipsoid to broadly ellipsoid |
| Basidia (μm) | 18.5-28.2 × 5.8-6.8 | 17-22 × 5-6 | 27-31.5 × 5-7 | 30-40 × 6.5-7 | 14-29 × 8-13 |
| Cheilocystidia (μm) | 22.4-28.2 × 3.9-6.8 | 20-30 × 4-6 | absent | 35-95 × 3-6.5 | absent |
| Caulocystidia (μm) | Absent | Absent | absent | 30-50 × 3-4 | absent |
The closest phylogenetical species is R. asyae which from Turkey can be distinguished from R. subasyae by a clitocyboid, smaller (10-30 mm), salmon pink pileus, decurrent lamellae, a pruinose, 25-30 × 2-5 mm stipe; microscopically, R. asyae differs in the versiform, flexuous cheilocystidia (20-30 × 4-6 μm) without branching (Sesli & Vizzini, 2017). Rhodocybe fusipes Silva-Filho, D.L. Komura & Wartchow from Brazil is closely related to R. subasyae but it has orange to reddish and slightly umbonated pileus and it is absent of cystidia (Silva-Filho et al., 2020). The other allied species R. truncata has 15-80 mm irregular pileus, subdecurrent lamellae and weakly farinaceous odor; microscopically R. truncata has smaller basidiospores (5-6.5 × 4-5 μm) and longer cheilocystidia (20-60 × 2-6.5 μm) (Baroni, 1981).
Morphologically, it is familiar to R. asanii E. Sesli & Vizzini by the tricholomatoid basidiomata, beige red pileus and adnexed to sinuate lamellae. However, R. asanii from Turkey differs in the discolored pileus when injured or old. Furthermore, microscopically R. asanii is distinguished due to the absence of the cheilocystidia and the multicelled layer of interwoven hyphae with angular and spirally pigment encrusted hyphae (Sesli & Vizzini, 2017).
Rhodocybe fumanellii Ferrari, Vizzini & Fellin described from Italy, is circumscribed by a larger (35-100 mm), reddish-brown to ochre pileus, a 40-70 × 5-15 mm stipe, 5.68-6.83 × 3.93-4.58 μm ellipsoid basidiospores and slender cylindrical caulocystidia (Vizzini et al., 2018). Rhodocybe matesina Picillo & Vizzini from Italy differs in a collybioid basidiomata, broadly adnate to shortly decurrent lamellae, a smell similar to Hygrophorus penarioides Jacobsson & E. Larss, an olive-green reaction on the pileus surface with KOH and shorter cheilocystidia (16.5-23 × 3-6.5 μm) (Crous et al., 2017).
The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.
This work was supported by the Program for Ministry of Education Innovation (No. IRT1134, IRT-15R25). We sincerely thank the staff of Longjing Forestry Bureau and Tianfozhishan National Nature Reserve Administration for providing help with field work. We also thank the reviewers and the conscientious editor for their corrections and suggestions to improve our work.
