1 Introduction
The genus Russula Pers. (Russulaceae, Russulales, Basidiomycota) is one of the most highly diverse genera of mushroom-forming fungi and range of habitats from the tropics to polar ecosystems, which resulted in many complex and multilevel classifications[1,2,3]. It is the largest group of all seven genera in Russulaceae, which includes eight subgenera and at least 2000 members within the genus[3, 4-6]. Some species of Russula can be as food for humans in the world. According to the recent statistics of resource diversity of Chinese macrofungi, there are 78 edible species in China[7,8,9].
Russula subg. Heterphyllidia Romagn. sect. Ingratae (Quél.) Maire is mainly characterized by a fetid odor (sometimes spermatic, waxy, or bitter), pale to dark yellowish brown to dark greyish brown colors, tuberculate striate pileus margin, articulated and branched hair cuticles, acute to subacute lamellae, spores print white to cream, inamyloid supraphilar spot, and short-celled and branched hyphal extremities in the pileipellis[10,11]. Resently, many species belonged to sect. Ingratae have been reported from China[12,13,14], indicating that China has been a hot spot for the exploration of unknown Russula species. During a survey on the habitat diversity and geographic distribution of Chinese Russula, some interesting specimens were made. Then they were identified as R. indocatillus A. Ghosh, K. Das & R.P. Bhatt, a species originally described from India, based on morphological features and phylogenetic analysis, which is a taxon new to China.
2 Materials and methods
2.1 Morphological study
The basidiomata were photographed under daylight in the field. Specimens were dried at 45-55 ℃ and deposited in the Herbarium of Research Institute of Tropical Forestry, Chinese Academy of Forestry (RITF). Terminology for descriptive terms follows Vellinga and Noordeloos (1988)[15]. Color designations were from Kornerup and Wanscher (1981)[16]. Macroscopic characteristics and ecological aspects were based on detailed notes and the photographs of fresh materials. Spores were observed and measured in Melzer’s reagent. After pretreatment of dried materials in 5% KOH, the observations and measurements of other microscopic features were made in Congo red with ZEISS Imager M2. Pileipellis was examined in Cresyl blue to verify the presence of ortho- or metachromatic reactions[17]. Cystidia were examined in Sulfovanillin (SV) solution. Structure and ornamentation of basidiospore were illustrated using scanning electron microscope (SEM-JEOL JSM-6510). The abbreviation (n/m/p) indicates measurements were made on n basidiospores from m fruit bodies of p specimens. In the dimension notation of basidiospores (a) b-c (d), b-c is the range containing 95% of the measured values for length or width with the extremes (a and d) of all measurements. Q value indicates length/width ratio of the basidiospores excluding ornamentation with bold Q denoting the average Q of all basidiospores ± standard deviation.
2.2 Molecular study
The total genomic DNA was isolated by an improved CTAB protocol[18] from dry herbarium. We amplified and sequenced the three following loci: 600 base pairs of the ITS region of rDNA using the primers ITS1 and ITS4[19], 900-1400 base pairs of the nuclear ribosome large subunit (nrLSU) using the primers LROR and LR5[20]; 700 base pairs of the second largest subunit of the RNA polymerase II (RPB2) using the primers bRPB2-6f and fRPB2-7cr[21,22]. The PCR products were subjected to automated DNA sequencing on an ABI 3730 DNA analyzer and an ABI BigDye 3.1 terminator cycle sequencing kit (Shanghai Sangon Biological Engineering Technology and Services CO., Ltd, Shanghai, China). The sequences newly generated were submitted to GenBank database (Tab. 1).
Tab. 1 Samples used and their GenBank accession numbers |
| Taxon | Voucher | Location | ITS | nrLSU | RPB2 | Reference |
|---|---|---|---|---|---|---|
| R. amerorecondita | F (PGA17-017) | USA | MN130066 | - | MN380515 | [5] |
| R. amerorecondita | SH120828 | USA | MN130067 | - | - | [5] |
| R. amoenolens | 46372 (AH) | Spain | MK105626 | MK105707 | MK102750 | [24] |
| R. amoenolens | 46371 (AH) | Spain | MK105625 | - | MK102749 | [24] |
| R. benghalensis | CUH AM654 | India | MK414583 | - | - | [25] |
| R. catillus | SFC20120725-52 | Korea | KX574685 | - | - | [26] |
| R. catillus | SFC20120827-01 | Korea | KX574686 | - | - | [26] |
| R. dinghuensis | GDGM45244 | China | KU863579 | - | - | [27] |
| R. fluvialis | KUO (JR8666) | Finland | MN130084 | MN130125 | MN380532 | [5] |
| R. fluvialis | KUO (JR8313) | Finland | MN130085 | MN130126 | - | [5] |
| R. foetens | FH12277 | Germany | KT934016 | KT933877 | KT933948 | [28] |
| R. garyensis | F (PGA17-008) | USA | MN130088 | - | MN380519 | [5] |
| R. garyensis | F (PGA15-910) | USA | MN130089 | - | - | [5] |
| R. gelatinosa | K15052626 | China | MH168575 | - | - | [12] |
| R. gelatinosa | K16053119 | China | MH168574 | - | - | [12] |
| R. granulata | BPL272 | USA | KT933971 | KT933832 | KT933903 | [28] |
| R. illota | UE26.07.2002-3 | Sweden | DQ422024 | DQ422024 | DQ421967 | [29] |
| R. indocatillus | AG 17-1571 | India | MN581483 | - | - | [30] |
| R. indocatillus | AG 18-1653 | India | MN581165 | - | - | [30] |
| R. indocatillus | RITF3275 | China | MN317307 | MN325084 | MN329157 | This work |
| R. indocatillus | RITF1320 | China | MN317306 | MN325083 | MN329156 | This work |
| R. indocatillus | RITF1369 | China | MN317305 | MN325082 | MN329155 | This work |
| R. laurocerasi | FH12178 | Germany | KT933988 | KT933849 | KT933920 | [28] |
| R. livescens | F0177 | China | GU371295 | - | - | [31] |
| R. mutabilis | DPL10654 | USA | KF810137 | - | - | [32] |
| R. mutabilis | BHI-F384a | USA | MF161239 | - | - | [33] |
| R. pectinata | Buyck 2304 | Europe | AY061706 | - | - | [34] |
| R. pectinatoides | BPL276 | USA | KT933975 | KT933836 | KT933907 | [28] |
| R. pila | JMV800654 (BCN) | Spain | MK105691 | MK105736 | MK102768 | [5] |
| R. pseudocatillus | K16042406 | China | MK049974 | - | - | [13] |
| R. pseudocatillus | K15060706 | China | MK049975 | - | - | [13] |
| R. pseudopectinatoides | HMAS 265020 | China | KM269079 | - | - | [35] |
| R. punctipes | K17052318 | China | MH168576 | - | - | [12] |
| R. putida | IB0791 | Italy | HG798527 | HG798526 | - | Unpublished |
| R. rufobasalis | H15060622 | China | MH168567 | - | - | [12] |
| R. subpallidirosea | RITF4083 | China | MK860697 | MK860702 | - | [36] |
| R. subpunctipes | RITF2616 | China | MK860692 | MK860695 | - | [14] |
| R. subpunctipes | RITF2617 | China | MK860693 | MK860696 | - | [14] |
Note: Newly generated sequences in bold. |
2.3 Phylogenetic analysis
Reference sequences were selected for phylogenetic analysis based on NCBI blast search results and published molecular analyses, and then combined with the newly generated sequence. NCBI accession numbers for the sequences used in the phylogenetic analysis are listed in Tab. 1. A preliminary multiple sequence alignment was performed using MAFFT 7.0 (http://mafft.cbrc.jp/alignment/server/), with manually checking and adjusting when necessary in order to obtain reliable results in BioEdit[23]. The final sequence alignment was submitted to TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S27534).
Both maximum likelihood (ML) and Bayesian analysis (BA) were employed. Maximum likelihood of the phylogenetic relationships were conducted with RAxML-HPC2 on XSEDE (8.2.12) through the Cipres Science Gateway (www.phylo.org). Bootstrap analyses were done to affirm consistency of results with 1000 replicates. The GTR model of nucleotide substitution and GAMMA rate heterogeneity parameters were implemented. Bootstrap support (BS)≥70% was regarded as significance. BA analysis were performed with XSEDE (3.2.7a) through the Cipres Science Gateway (www.phylo.org) under the GTR model. Analyses were run with 4 chains for a total of 50000000 generations and trees were sampled every 1000 generations, and first 25% of the trees were discarded as the burn-in phase of each analysis. Bayesian Posterior Probabilities (PP) values were obtained from the 50% majority-rule consensus trees and nodes with PP≥0.95 were considered as significantly supported.
3 Results
3.1 Phylogeny
The final ITS-nrLSU-RPB2 sequences dataset was analysed by ML and BA. Both the ML and BA analyses result in similar tree topologies and only the best ML tree is shown in Fig. 1. BA analysis posterior probabilities are also showed along the branches.
The phylogenetic analysis showed that sect. Ingratae was a strongly supported monophyletic group by BS (100%) and PP (1) (Fig. 1). Our phylogeny indicates the Chinese collections and the holotype of Indian R. indocatillus formed an independent lineage within sect. Ingratae with high statistical support (BS=100%, PP=1) (Fig. 1), which is sister to the lineage consisting of South Korean species R. catillus Lee with 100% bootstrap support and 1.00 posterior probabilities (Fig. 1).
3.2 Taxonomy
Russula indocatillus A. Ghosh, K. Das & R.P. Bhatt Figs. 2, 3 Basidiomata small to medium-size. Pileus 40-52 mm in diam., first hemispheric, then planoconvex, applanate with a slightly depressed center when mature; margin incurved when young, relatively flat at maturity, stripes 5-10 mm from edge inward; surface smooth, glabrous, viscid when wet, not peeling readily; brownish orange (6C7) to yellow ochre (6D7) at the center, margin first butter yellow (4A5) to apricot (5B6), turning pale (2A2) to pale yellow (2A3) after maturation, unchanging in color when bruised. Lamellae adnate, 3-4 mm in height, 15-20 pieces per centimeter at edge, not forked, slightly interveined, satin white (2A1) to yellowish white (3A2), unchanging after bruising, not brittle when toughing, lamellulae absent. Stipe 22-36 mm×8-12 mm, central to subcentral, subcylindrical to cylindrical, slightly curved towards center, slightly attenuate towards the base, surface dry, rugulose longitudinally, pithy and hollow when mature, pale yellow (3A3) to grayish yellow (5B5), unchanging when bruised. Context 2-3 mm thick at pileus center, white to cream, without color changing when bruised. Taste slightly bitter or slightly spicy. Oder indistinct. Spore print white to cream.
Fig. 2 Basidiomata and basidiospores of R. indocatillus under SEMA, B: Basidiomata of Russula indocatillus (RITF 1369, holotype), bar = 15mm; C, D: Basidiospores of R. indocatillus |
Basidiospores [60/3/3] (5) 5.4-6.8 (7.5)×(4) 4.5-5.2 (5.6) μm, [Q=1.10-1.32, Q=1.18±0.11], subglobose to broadly ellipsoid to ellipsoid; ornamentation amyloid, warts bluntly conical to subcylindrical, isolated or connected with irregular lines or ridge, not forming a reticulum, measuring 0.3- 0.55 (1.0) μm high; suprahilar spot large. Basidia (27) 32.8-44.8 (47)×(6.2) 7.4-9.6 (11) μm, mostly 4-spored, sometimes 2- and 3-spored, subclavate to clavate, thin-walled, sterigmata 3-6×1.5-2.3 μm. Lamellar trama mainly composed of numerous nested spherocytes (20-50×17-43 μm) surrounded by connective hyphae (1.5-4.5 μm wide). Pleurocystidia (46) 51-64 (69)×(7) 7.6-9.4 (11) μm, clavate to subclavate to subcylindrical, apically obtuse or mucronate to moniliform, granular or crystalline contents turning to blackish grey in SV. Cheilocystidia (42) 45.8-66 (73)×6-10 μm, mostly similar to pleurocystidia, clavate to subcylindrical, apically obtuse to mucronate, granular or crystalline contents blackish grey in SV. Pileipellis orthochromatic in Cresyl blue, 120-160 μm thick, distinctly two layered, not sharply delimited from the underlying sphaerocysts of the context, composed of suprapellis and subpellis; suprapellis 90-120 μm thick, consisting of thin- walled, often ramifying, septate, erect or repent hyphae and pileocystidia, terminal elements 12-36×2.4-6 μm, apically obtuse or attenuate; subpellis 30-40 μm thick, consisting of horizontally oriented hyphae (2-6 μm diam.); pileocystidia 25-45×4-7 μm, always one-celled, cylindrical to narrowly clavate, apically mucronate or obtuse, usually with an appendage, granular or crystalline contents turning to blackish grey in SV.
Specimens examined: China, Hubei Province, Shennongjia Forestry District, Hongping Town, 31°40'17.86''N; 110°26'40.53''E, 2560 m asl., in mixed hardwood forests, dominated by Fagus, 14 July 2012, Yuan117 (RITF1369). Hubei Province, Yichang City, Xingshan County, Nangyang Town, Longmenkou Forest Farm, mixed forests, 31°18'39''N; 110°40'20''E, 260 m asl., 11 July 2012, leg. Yuan54 (RITF1320). Yunnan Province, Kunming City, Yeya Lake, mixed forests, dominated by Quercus, 25°07'34''N; 102°51'42''E, 2126m asl., 27 July 2014, leg. LHJ14072713 (RITF3275).
4 Discussion
Our samples share a remarkable resemblance with the description of Ghosh et al (2020) [29]. Meanwhile, the Chinese collections clustered (BS= 100%, PP=1) with the holotype of Indian R. indocatillus and share 100% ITS sequence identity, suggesting that they are conspecific. Based on the macro-and micro-morphological character, as well as the significant support for its phylogenetic placement (Fig. 1), R. indocatillus was placed in Russula subg. Heterphyllidia sect. Ingratae.
The phylogenetic result indicated R. indocatillus is sister to South Korean species R. catillus, however, they can be easily distinguished for R. catillus has brittle and less lamellae, whitish stipe slightly inflate towards to the base, larger basidia (41.6-48.9×9.3-11.7 μm), smaller cheilocystidia (43.2-59.7×6.9-11.2 μm) and thicker pileipellis (110-210 μm), furthermore, R. catillus grows in the areas with higher latitudes and greater longitudes and is symbiosis with oaks[26].
In addition, the recently reported four new species (R. gelatinosa Y. Song & L.H. Qiu, R. rufobasalis Y. Song & L.H. Qiu, R. pseudocatillus F. Yuan & Y. Song and R. subpunctipes J. Song) and a new recorded species (R. punctipes Singer) of subg. Heterophyllidia sect. Ingratae from China are worthy of attention[12,13,14]. Comparing to R. indocatillus, R. gelatinosa has a surface gelatinized, a reddish brown to ochre to dark brown pileus, whitish lamellae with reddish hue, a longer stipe (7-9.5× 1.2-1.7 μm), larger basidiospores [(7.6) 8.2-9.0- 9.8×(7) 7.5-8.4-9.1 (9.5) μm] with ornamentation composing of high wings and short warts, and large basidia [(44) 48-67.5 (69)×(10) 12-19 (20.5) μm][12]; R. rufobasalis possess whitish to rusty tinged lamellae, a reddish stipe base, and a thicker pileipellis (360-500 μm)[12]; R. pseudocatillus has larger basidiospores [7.0-7.9-8.6 (9.2)×(5.1) 5.5-6.1-6.6 (6.7) μm] with conical to cylindrical isolated warts never forming a reticulum, shorter pleurocystidia (32-37.5×9.5-11.5 μm) and pileocystidia (13-33× 3-6 μm) [13]; R. subpunctipes possess sulfur yellow to honey yellow pileus with a margin cracking into patches, henna lamellar edge, henna warts on the hollow stipe, faint fragrant odor, larger basidiospores [8.7-10.5 (-10.7)×8.3-9.5 μm] with ornamentation composed of large wings and short isolated warts, larger basidia (65-72×11-14 μm) and pleurocystidia (110-132×9-25 μm) [14]; R. punctipes has a dull yellow to brownish stipe with ochraceous brown to dark brown punctuations, strongly fetid odour, larger basidiospores [(7.1) 7.9-9.3-10.3 (11)×(6.0) 7.2-8.7-10.1 (10.3) μm] with ornamentation composed of wings (up to 3 μm high) and short warts, wider basidia [(32)33.5-48.5 (54) × (10.5) 11-18.5 μm] and cheilocystidia (30.5-62.5× 9-18.5) [12].