Research Article
Print
Research Article
Three new Melanogaster species (Boletales, Paxillaceae) from southwestern China based on morphological and molecular evidence
expand article infoTian-Jun Yuan‡§, Hong-Mei Luo§, Kai-Mei Su§, Shu-Hong Li§, Olivier Raspé‡
‡ Mae Fah Luang University, Chiang Rai, Thailand
§ Institute of Biotechnology and Germplasm Resources, Yunnan Academy of Agricultural Sciences, Kunming, China

Corresponding author: Shu-Hong Li ( shuhongfungi@126.com )

Corresponding author: Olivier Raspé ( Olivier.Ras@mfu.ac.th )

Academic editor: Jennifer Luangsa-ard
© 2024 Tian-Jun Yuan, Hong-Mei Luo, Kai-Mei Su, Shu-Hong Li, Olivier Raspé.
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: Yuan T-J, Luo H-M, Su K-M, Li S-H, Raspé O (2024) Three new Melanogaster species (Boletales, Paxillaceae) from southwestern China based on morphological and molecular evidence. MycoKeys 107: 141-160. https://doi.org/10.3897/mycokeys.107.123565
Open Access

Abstract

Three newly discovered Melanogaster species, namely M. cyaneus, M. diqingensis, and M. truncatisporus, are introduced and illustrated based on both morphological and molecular data from Sichuan and Yunnan provinces in China. A multigene phylogenetic analysis (nrITS, nrLSU, and rpb2) was performed mainly to verify the placement of the new species in Melanogaster. A second, nrITS-only phylogenetic analysis comprising more Melanogaster species for which only ITS sequences were available, was used to infer the relationship between the new species and as many known Melanogaster species as possible. Specimens of M. cyaneus, M. diqingensis, and M. truncatisporus formed three independent clades in a phylogenetic tree inferred from the ITS data set. The robust support from ITS for these clades and genetic similarity with other species being lower than 93.2% suggest that these three species are indeed distinct from the other Melanogaster species in the phylogeny. Morphologically, M. cyaneus is characterized by its blue or bluish gleba, light brown to yellowish brown peridium, and subglobose to globose basidiospores, 6.2–15 × 4.6–9.0 μm. Melanogaster diqingensis is distinguished from other Melanogaster species by its pale yellow to brown-yellow peridium and obovate to subglobose basidiospores, 3.0–5.1 × 2.0–4.0 μm. Melanogaster truncatisporus is diagnosed by its subglobose to globose or irregularly elongate-pyriform basidiomata, pale yellow to deeply orange-yellow peridium, and subglobose to globose or pyriform, truncate basidiospores. Additionally, infrageneric classification based on the number of peridium layers, the average thickness of the peridium, and the average length and width of basidiospores was tested with M. cyaneus, M. diqingensis, and M. truncatisporus. Orthogonal partial least squares discriminant (OPLS-DA) analysis placed the three new species within the Melanogaster, Rivulares, and Variegati sections, respectively. However, the morphologically circumscribed sections were not monophyletic in the phylogenetic tree. Therefore, the current infrageneric classification should be abandoned.

Key words

False truffles, gasteroid Boletales, phylogeny, taxonomy, three new species

Introduction

Melanogaster Corda, belonging to Paxillaceae within the Boletales (Basidiomycota), stands out as one of the ecologically significant groups of hypogeous fungi. According to He et al. (2019), Melanogaster encompasses approximately 26 species. However, recent discoveries by taxonomists worldwide have identified new species, expanding the count to 34 as listed in Index Fungorum (https://www.indexfungorum.org/, accessed on November 10, 2023). While Melanogaster species are predominantly distributed in the Northern Hemisphere, an exception is noted with Melanogaster quercus L., reported by Trappe et al. (2009) in the Southern Hemisphere. Melanogaster species typically establish ectomycorrhizal associations (EcM) with various plant families, including Betulaceae, Cistaceae, Fagaceae, Pinaceae, and Salicaceae (Comandini et al. 2006; Krisztián 2008; Perič and Moreau 2010; Türkoğlu and Castellano 2013; Lacheva 2015). These fungi are recognized by their peridium, varying in color from brownish to yellowish, occasionally featuring mycelial strands at the base or on the surface. The sequestrate hymenophore is composed of rounded to irregular locules of varying sizes, filled with black or dark brown basidiospores embedded in a gel and separated by whitish to yellowish veins or walls. Melanogaster basidiospores exhibit a range of shapes, including globose, ellipsoid, pyriform, or cirriform. Most species in the genus emit distinctive odors, ranging from sweet and pleasant to garlic-like or nauseating (Castellano et al. 1986; Montecchi and Sarasini 2000; Cázares et al. 2008; Trappe et al. 2009; de la Fuente et al. 2021; Alvarado et al. 2021; Xu et al. 2022). Melanogaster can be differentiated from related sequestrate Paxillaceae such as Alpova C.W. Dodge, Neoalpova Vizzini, and Paralpova Cabero & P. Alvarado based on differences in peridium structure. In Melanogaster, the peridium exhibits prostrated or interwoven hyphae, contrasting with the pseudoparenchymatous structure with inflated hyphae found in Alpova, Neoalpova, and Paralpova. Another false-truffle genus, Rhizopogon Fr. (Rhizopogonaceae), differs from Melanogaster by the absence of gel in the locules and the hyaline basidiospores (yellow to dark brown in Melanogaster). The relationships among these genera have been extensively discussed and Melanogaster has been placed in the Paxillinae, along with Gyrodon Opat., Paxillus Fr., and Alpova by Trappe (1975), Grubisha et al. (2001), Binder and Hibbett (2006), Vizzini et al. (2010), Moreau et al. (2011, 2013), and Alvarado et al. (2021).

Knapp (1954) proposed a subdivision of the genus Melanogaster into three groups based on the spore length (L), namely the ambiguous group (L > 10 μm), the variegatus group (7 < L < 10 μm), and the microsporus group (L < 7 μm). Then, Svrček (1958) proposed the subdivision into three sections, namely sect. Melanogaster (L> 10 μm), sect. Variegati (6 < L < 10 μm), and sect. Rivulares (L < 6 μm). Those sections were later validated by Moreau et al. (2011).

In China, the first specimen of Melanogaster was gathered from the Jinshajiang Valley in Yunnan Province, southwestern China, in 1915. Initially identified as M. variegatus (Vittad.) Tul. & C. Tul. by Keissler and Lohwag in 1937, it was later erected as a distinct species named M. ovoidisporus Y. Wang (Wang et al. 1995). Up to now, twelve species have been reported from China: M. fusisporus Y. Wang, M. natsii Y. Wang, K. Tao & B. Liu, M. obovatisporus B. Liu, K. Tao & Ming C. Chang, M. ovoidisporus Y. Wang, M. shanxiensis B. Liu, K. Tao & Ming C. Chang, M. spinisporus Y. Wang, M. subglobisporus K. Tao, Ming C. Chang & B. Liu, and M. utriculatus Y. Wang, Castellano & Trappe, M. minobovatus, M. panzhihuaensis, M. quercicola, M. tomentellus L. Fan, X. Y. Yan & Y. Y. Xu (Liu et al. 1989; Wang et al. 1995; Wang et al. 2005; Xu et al. 2022). China seems to have a rich variety of Melanogaster species; however, most of the previous records relied primarily on morphological evidence (Xu et al. 2022). During our survey of hypogeous fungi in Yunnan and Sichuan provinces, located in southwest China, from 2019 to 2021, we uncovered three novel species of Melanogaster under Castanea mollissima Bl. and Quercus aquifolioides Rehd. et Wils. Through a comprehensive analysis encompassing both morphology and phylogenetic considerations (including Alpova, Neoalpova, Paralpova, and Melanogaster), we introduce three new species: Melanogaster cyaneus, M. diqingensis, and M. truncatisporus.

Materials and methods

Fungal materials

The Melanogaster specimens were collected from Yunnan and Sichuan Provinces in China. They were photographed in the field, placed in sterilized plastic tubes and boxes, returned to the laboratory, and stored at 4 °C. Macroscopic and microscopic descriptions were based on fresh basidiomes following the methods of Wan et al. (2016). Hand-cut sections were mounted in 5% (w/v) aqueous KOH solution, Cotton blue, or Congo red solutions and examined with an OLYMPUS BH-2 compound microscope. At least 50 basidiospores were measured of selected specimens, and the measurements are presented in the following format: L, W, Q, representing the extreme values of length, width, length to width ratio, Lm = L ± S.D., Wm = W ± S.D. and Qm = Q ± S.D. For scanning electron microscopy (SEM), spores were scraped from the gleba of dried specimens onto double-sided tape, which was mounted directly on a SEM stub, coated with gold-palladium, examined and photographed with an SEM JSM-5600LV (JEOL, Tokyo, Japan).

The dried specimens were deposited in the Herbarium of Biotechnology and Germplasm Resources Institute of the Yunnan Academy of Agricultural Sciences (YAAS), and the Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS), Yunnan, China.

DNA extraction, PCR, and sequencing

About 10–20 mg of dried gleba were placed in a 1.5 mL tube together with one 3 mm in diameter tungsten carbide bead, and crushed by shaking two to four times for 50 s at 30Hz with a Mixer Mill MM301 (Haan, Germany). Total DNA was extracted using the CTAB method described by Hofstetter et al. (2002). The following primer pairs were used for PCR amplification: the primer pair ITS4/ITS5 was used to amplify the nuclear ribosomal internal transcribed spacer region (White et al. 1990), the primers bRPB2-5F/bRPB2-7.1R for the second largest subunit of RNA polymerase II gene (RPB2) (Matheny 2005; Matheny et al. 2007), and the LR0R/LR5 primers (Vilgalys and Hester 1990; Cubeta et al. 1991) for the 28S nrDNA region (nrLSU). Amplifications were carried out in 25 μL reaction containing 12.5 μL 2×Taq Plus Master Mix II (Vazyme Biotech Co. Ltd, China), 9.5 μL ddH2O, 1 μL 10 μM of forward and reverse primers, and 1 μL template DNA. Standard cycles of denaturation at 94 °C for 45 seconds, annealing for 45 seconds at different temperatures depending on the primer set (50 °C for RPB2, 54 °C for nrLSU, and 55 °C for ITS), and elongation at 72 °C for 1.5 min, followed by a final elongation step at 72 °C for 10 min. Post-cycling, samples were held at 4 °C. PCR products were sent to Shanghai Sangon Biotechnology (Shanghai, China) for purification and sequencing.

Phylogenetic analyses

The newly generated sequences were edited and assembled using SeqMan II (SeqMan Pro, DNAStar) with generic-level identifications for sequences corroborated via BLAST queries of GenBank. A total of 89 sequences (including ITS1-2, 5.8S, nrLSU, and RPB2) were used in the molecular phylogenetic analyses (Table 1), including 20 sequences newly generated in this study and 82 downloaded from GenBank. Paragyrodon sphaerosporus and Paxillus involutus were selected as outgroup for the multilocus phylogenetic analyses while Alpova alpestris, Alpova concolor, and Alpova cinnamomeus were selected as outgroup for the ITS analysis. A sequence (AJ555527) of M. tuberiformis served as the reference for delineating 5.8S, ITS1, and ITS2. The ITS, 5.8S, nrLSU and RPB2 sequences were separately aligned using MAFFT ver. 7 (Katoh and Standley 2013) on the online server accessed at https://mafft.cbrc.jp/alignment/server/, with the G-INS-I algorithm. The obtained alignment was manually refined in BioEdit, and ambiguously aligned sites were pinpointed using Gblocks 0.91b (Castresana 2000), using default options, except “Allowed Gap Positions” = half. After Gblocks, 90%, 99%, 99.5%, and 99% of the positions were kept for ITS1-2, 5.8S, nrLSU, and RPB2, respectively. The ITS1-2, 5.8S, nrLSU, and RPB2 alignments were 537, 157, 886, 715 bp long, respectively (including gaps). The alignments were deposited in Figshare (doi: 10.6084/m9.figshare.25440544). Phylogenetic analyses were performed using maximum likelihood (ML) and Bayesian inference (BI) to validate the affiliation of our specimens with Melanogaster, relying on multi-gene sequences, which included Alpova, Paralpova, and Neoalpova (Fig. 1). Additionally, an ITS dataset (including 159 bp 5.8S, and 527 bp ITS1+ITS2) was compiled to infer the phylogenetic relationships between Melanogaster species. The ML analyses were performed with RAxML 8.0.14 (Stamatakis et al. 2005; Stamatakis 2014) with all parameters at default settings, except a mixed-model partitioning (with the same character sets as in the BI analyses), GTRGAMMA+I for all character sets, and 1,000 bootstrap pseudoreplicates. Partitioned BI analyses were performed with MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003) based on the following best-fit substitution models estimated by jModelTest2 on Cipres XSEDE (2.1.6, https://www.phylo.org; Miller et al. 2010). For the multi-gene dataset, 5.8S: K80+G; ITS1-2: HKY+I+G; nrLSU and RPB2: GTR+I+G). For the ITS-only dataset, 5.8S: HKY+I+G; and ITS1-2: GTR+I+G. Two independent runs of four chains were conducted for 2.5 106 generations (ITS dataset) and 5 106 generations (multiple-gene dataset), with trees sampled every 100 generations. The average standard deviation of split frequency (ASDSF) values at the conclusion of the runs were 0.009248 for the multi-gene tree and 0.001889 for the ITS tree. After discarding the samples from the burn-in phase (first 25% of trees), a 70% majority-rule consensus tree was constructed and posterior probabilities computed. No outgroup was specified when running the BI analyses, but the obtained tree was rerooted with the outgroups used in the ML analysis.

The trees were visualized with TreeView32 (Page 2001), exported in PDF format, and edited in Adobe Illustrator CS6. Clades with bootstrap support (BS) ≥ 70% and Bayesian posterior probabilities (PP) ≥ 0.90 were considered significantly supported (Alfaro et al. 2003).

Table 1.
Download as
CSV
XLSX

Specimen information and GenBank accession numbers for sequences used in this study.

Species Isolate/ Strain/ Country Genbank accession numbers Reference
Clone/Voucher ITS nrLSU RPB2
Alpova alpestris S123 France HQ714711 / HQ714846 Moreau et al. 2013
Alpova alpestris S159 France HQ714721 / HQ714853 Moreau et al. 2013
Alpova cf. cinnamomeus PAM09082702 France HQ714779 / HQ714901 Moreau et al. 2013
Alpova cinnamomeus BROWN FP73 USA KF835996 / / Hayward et al. 2014
Alpova cinnamomeus HRL1384 Canada MN594282 MN594298 MN594770 Unpublished
Alpova concolor UBC F14673 USA KF835997 / / Hayward et al. 2014
Alpova concolor OSC 65696 USA NR_154686 / / Unpublished
Alpova corsicus S287 France HQ714769 / HQ714893 Hayward et al. 2014
Alpova corsicus S288 France HQ714770 / HQ714894 Moreau et al. 2011
Alpova komovianus PAM10081201 Montenegro JQ436850 / JQ436862 Moreau et al. 2013
Neoalpova arenicola JC150513NR Spain MN594292 MN594304 MN594775 Unpublished
Neoalpova cf. rubescens JC140920BT Spain MN594294 MN594305 MN594776 Unpublished
Neoalpova montecchii JC181021NR Spain MN594296 MN594306 MN594777 Alvarado et al. 2021
Paralpova artikutzensis AH 49154 Spain NR_173892 MN594307 MN594778 Alvarado et al. 2021
Melanogaster ambiguus B-2220 Hungary AJ555510 / / Unpublished
Melanogaster ambiguus 51745 Hungary AJ555511 / / Unpublished
Melanogaster ambiguus B-1599 Hungary AJ555512 / / Unpublished
Melanogaster ambiguus B-1613 Hungary AJ555513 / / Unpublished
Melanogaster ambiguus B-2409 Hungary AJ555514 / / Unpublished
Melanogaster ambiguus Ch12 Poland KX438335 / / Unpublished
Melanogaster ambiguus JC180719NR Spain MN594286 MN594299 MN594771 Unpublished
Melanogaster ambiguus OSC158337 MES304 Poland MN984308 / / Unpublished
Melanogaster ambiguus MTH1 Germany MN994353 / / Unpublished
Melanogaster broomeanus JC091213NR Spain MN594287 MN594300 MN594772 Unpublished
Melanogaster broomeanus OTU_718s United Kingdom MT095837 / / Arraiano-Castilho et al. 2021
Melanogaster broomeanus OTU_719s United Kingdom MT095838 / / Arraiano-Castilho et al. 2021
Melanogaster cyaneus TJ75_1 (TYPE) China ON427476 ON427489 ON533869 This study
Melanogaster cyaneus TJ75_2 China ON427477 ON427490 ON533870 This study
Melanogaster diqingensis WXH_9068 (TYPE) China ON427482 ON427495 ON533874 This study
Melanogaster euryspermus OSC158352 DS1257 USA MN984309 / / Unpublished
Melanogaster euryspermus OSC158364 DS1555 USA MN984310 / / Unpublished
Melanogaster euryspermus OSC158339 JLF1044 USA MN984311 / / Unpublished
Melanogaster euryspermus OSC158325 JLF1129 USA MN984312 / / Unpublished
Melanogaster euryspermus OSC158351 JLF1456 USA MN984313 / / Unpublished
Melanogaster euryspermus OSC158317 JMT22778 USA MN984314 / / Unpublished
Melanogaster euryspermus OSC158333 MES110 USA MN984315 / / Unpublished
Melanogaster intermedius B-1770 Hungary AJ555515 / / Unpublished
Melanogaster intermedius RBG Kew K(M)130202 England EU784372 / / Brock et al. 2009
Melanogaster intermedius MT48 Germany KX168661 / / Unpublished
Melanogaster luteus S328/PAM09082801 France HQ714780 / HQ714902 Moreau et al. 2011
Melanogaster luteus S407/Mon06 Montenegro HQ714794 / / Moreau et al. 2011
Melanogaster macrosporus cI-94 USA AJ555526 / / Unpublished
Melanogaster macrosporus B-2254 USA AJ555528 / / Unpublished
Melanogaster minobovatus BJTC FAN911 China NR_186967 / / Xu et al. 2022
Melanogaster natsii OSC82168 JMT7491 USA MN984331 / / Unpublished
Melanogaster natsii OSC158336 MES297 USA MN984332 / / Unpublished
Melanogaster panzhihuaensis HMAS 81915 China NR_186968 / / Xu et al. 2022
Melanogaster rivularis S190/PAM08090514 France HQ714731 / HQ714862 Moreau et al. 2011
Melanogaster rivularis S285/PAM08090514 France HQ714767 / HQ714891 Moreau et al. 2011
Melanogaster rivularis LIP PAM08090514 (TYPE) France NR_132848 / / Moreau et al. 2011
Melanogaster sp. Melanog002FRA France KU924526 / / Unpublished
Melanogaster sp. Melanog006FRA France KU924529 / / Unpublished
Melanogaster sp. Melanog007FRA France KU924530 / / Unpublished
Melanogaster sp. Melanog008FRA France KU924531 / / Unpublished
Melanogaster sp. Melanog011FRA France KU924533 / / Unpublished
Melanogaster sp. Melanog012FRA France KU924534 / / Unpublished
Melanogaster sp. Melanog018FRA France KU924535 / / Unpublished
Melanogaster sp. Melanog019FRA France KU924536 / / Unpublished
Melanogaster sp. MT15 Germany KX168646 / / Unpublished
Melanogaster sp. MES-1003 Chile KY462394 / / Brock et al. 2009
Melanogaster sp. LMKR1187 United Kingdom MF352733 / / Suz et al. 2017
Melanogaster sp. JC110118BT Spain MN594288 / / Unpublished
Melanogaster sp. OSC AHF420 France MN984333 / / Unpublished
Melanogaster sp. OSC158378 DS1755 USA MN984334 / / Unpublished
Melanogaster sp. OSC LG1042 USA MN984335 / / Unpublished
Melanogaster sp. MVC 753_FLAS-F-65878 Chile MT366708 / / Unpublished
Melanogaster sp. OK-2022a oka331 Turkey OP548647 / / Unpublished
Melanogaster sp. OK-2022a oka332 Turkey OP548648 / / Unpublished
Melanogaster spinisporus BJTC FAN1092 China MW598537 / / Xu et al. 2022
Melanogaster spinisporus BJTC FAN938 China MW598546 / / Xu et al. 2022
Melanogaster spinisporus BJTC FAN941-A China MW598548 / / Xu et al. 2022
Melanogaster spinisporus BJTC FAN941-B China MW598549 / / Xu et al. 2022
Melanogaster subglobisporus HMAS83329 China MW598534 / / Xu et al. 2022
Melanogaster truncatisporus TJ83 China ON427478 ON427491 ON533871 This study
Melanogaster truncatisporus TJ87 (TYPE) China ON427479 ON427492 ON533872 This study
Melanogaster truncatisporus TJ109 China ON427480 ON427493 ON533873 This study
Melanogaster truncatisporus L5346 China ON427481 ON427494 / This study
Melanogaster tuberiformis B-1295 Romania AJ555527 / / Unpublished
Melanogaster tuberiformis JC110130BT Spain MN594289 MN594302 MN594773 Unpublished
Melanogaster variegatus 23640 Hungary AJ555522 / / Unpublished
Melanogaster variegatus B-1438 Hungary AJ555523 / / Unpublished
Melanogaster variegatus B-1688 Hungary AJ555524 / / Unpublished
Melanogaster variegatus B-1225 Hungary AJ555533 / / Unpublished
Melanogaster variegatus B-1348 Hungary AJ555534 / / Unpublished
Melanogaster variegatus B-2312 Hungary AJ555535 / / Unpublished
Melanogaster variegatus JC180617BT Spain MN594290 MN594303 MN594774 Unpublished
Uncultured Melanogaster MFT57 Germany FJ403505 / / Pena et al. 2010
Paragyrodon sphaerosporus MB06-066 USA GU187540 GU187593 GU187803 Binder et al. 2010
Paxillus involutus Bel10.4 France KF261366 / JQ436854 Jargeat et al. 2014

Note: The symbol “/” means that the sequence was not available, and sequences newly generated for this study are in bold.

Subdivision of the genus Melanogaster based on morphological characteristics

In order to explore the correspondence between morphological features on the subdivision of Melanogaster from China (including the three new species described herein) and other parts of the world, the main morphological features (including the number of peridium layers, average thickness of peridium, average length, and width of basidiospores) were selected for statistical analysis (Table 2). Using the three sections (Melanogaster, Rivulares, and Variegati) as the dependent variable (Y), and the morphological features of the species as independent variables (X), we conducted a visual analysis of subdivision of different species using Orthogonal partial least squares discriminant analysis (OPLS-DA) using SIMCA 14.0.

Table 2.
Download as
CSV
XLSX

The main morphological characterization of Melanogaster species.

Species Peridium Basidiospores Country
layers Min. (μm) Max. (μm) Lm (μm) Wm (μm)
M. quercicola 2 450 600 13.1 6.5 China
M. utriculatus 2 300 400 13 9 Japan
M. fusisporus 2 250 420 12.3 5.2 China
M. shanxiensis 2 180 420 12.2 6.1 China
M. obovatus 2 350 600 12.2 6 China
M. macrosporus 1 85 315 11.5 5.7 Hungary
M. panzhihuaensis 1 150 220 10.4 6.1 China
M. natsii 2 200 250 10 6 China
M. coccolobae 2 170 230 9.9 7.5 Mexico
M. subglobisporus 2 130 300 9.8 7 China
M. spinisporus 1 360 750 9.6 7.3 China
M. cyaneus 2 100 400 9.5 7 China
M. tomentellus 1 250 350 9.4 4.5 China
M. ovoidisporus 2 250 450 7.1 4.3 China
M. truncatisporus 2 200 450 7 4.5 China
M. minobovatus 2 350 500 6.1 4.7 China
M. broomeanus 1 150 400 6 3.9 China
M. luteus 2 100 250 6 2.75 France, Montenegro
M. minysporus 1 160 240 5.5 4 Mexico
M. rivularis 2 100 135 5.5 2.9 France
M. diqingensis 2 100 320 3.8 3.2 China

Notes: The new species introduced in this study are presented in bold.

Results

Molecular data analyses

The multi-gene dataset (ITS, 5.8S, nrLSU and RPB2) contained 27 specimens (7 novel specimens from our collections) and had an aligned length of 2,295 characters. ML and BI yielded identical tree topologies and only the tree inferred from the ML analysis is shown (Fig. 1). The ITS dataset contained 79 specimens, of which 7 were newly sequenced, and had an aligned length of 683 characters. ML and BI analyses produced identical tree topologies and only the tree derived from the ML analysis is shown (Fig. 2). The four-gene dataset and ITS (ITS1-2 and 5.8S) resulted in ML trees with a log-likelihood of -9919.6 and -5052.3, respectively. The general topology of the trees (Figs 1, 2) is congruent with those already published by Moreau et al. (2011, 2013). The phylogenetic tree derived from the four-gene dataset (Fig. 1), validates the classification of our specimens in Melanogaster. Our 7 specimens were resolved as 3 strongly supported species-level clades or branches, different from all known species included in the analysis (Fig. 2). Furthermore, two sequences (Melanogaster sp. MVC 753 FLASF 65878 and Melanogaster sp. MES 1003) from Chile, a sequence (Melanogaster sp. JC110118BT) from Spain, two sequences (Melanogaster sp. OSC AHF420 from France, Melanogaster sp. OSC LG1042 from the USA), and two sequences (Melanogaster sp. OK 2022a oka331 and Melanogaster sp. OK 2022a oka332) from Turkey formed four distinct, species-level clades/lineages labelled M. sp1 to M. sp4 (Fig. 2). These clades exhibited less than 98.4% similarity (M. sp1 = 98.3%, M. sp2 = 93.47%, M. sp3 = 94.06%, and M. sp4 = 96.63%) in their ITS sequences compared to other species of Melanogaster. This observation suggests the likely presence of four additional undescribed species.

Figure 1. 

The phylogram of Melanogaster and closely related genera obtained with RAxML (including ITS1, ITS2, 5.8S, nrLSU and RPB2). Paragyrodon sphaerosporus MB06 066 and Paxillus involutus Bel10 10 were selected as the outgroup. Nodes were annotated with ML BS > 70%, Bayesian PP > 0.90, or “-” in case of non-significant support value. New species are in bold font.

Figure 2. 

The ITS phylogram of Melanogaster obtained with RAxML (including ITS1, ITS2, and 5.8S). The six Alpova cessions were chosen as the outgroup. Nodes were annotated with ML BS > 70%, Bayesian PP > 0.90, or “-” in case of non-significant support value. New species are in bold font. The sections are as defined by Moreau et al. 2011 on the basis of basidiospore length.

Taxonomy

Melanogaster cyaneus T. J. Yuan, Shu H. Li, & Raspé, sp. nov.

MycoBank No: 901868
Fig. 3a–d

Diagnosis

Melanogaster cyaneus is diagnosed by its blue or bluish gleba, rhizomorphs on the base, thinner peridium (100–400 μm), and longer and wider basidiospores (6.2–15 × 4.6–9.0 μm).

Etymology

The epithet cyaneus refers to the blue or bluish gleba.

Holotype

China. Sichuan Province: Panzhihua City, Yanbian County, Shuanglong village, 26°49'12"N, 101°33'7.1028"E, elevation 1,970 m, in mainly reddish brown soils under Castanea mollissima Bl., 16 Aug. 2020, collected by M. Yang (KUN- HKAS129200, holotype; YAAS-TJ75-1, isotype).

Description

Basidiomata 2.5–4.0 × 1.5–2.5 cm, hypogeous or semi-hypogeous, subglobose to ellipsoidal, occasionally elongate; light brown to yellowish brown, with mycelial strands attached in the base (Fig. 3a). Peridium two-layered, outer layer 20–75 μm thick, composed of interwoven hyphae, orange-yellow to reddish, 2–4 μm broad, thick-walled, clamp connections present; inner layer 145–335 μm thick, composed of interwoven, strongly gelatinized hyphae, 2–5 μm broad, pale-yellow, intermixed with massive inflated cells, 6–12 μm broad, with clamp connections present. Gleba solid, gelatinous, milk-white when immature, blue or bluish at mature; trama plates pale-yellow, of gelatinized hyphae (Fig. 3b); locules small, filled with black spores (Fig. 3c). Basidia exhibit limited revival, appearing clavate, hyaline, 4-spores, randomly distributed, gelatinized at maturity. Basidiospores subglobose to globose, 6.2–15 × 4.6–9.0 μm (Lm × Wm= 9.5 ± 3.0 × 7.0 ± 2.0, Q= 1.0–2.0, Qm =1.4 ± 0.6, n = 75), smooth, hyaline when immature, light yellow to reddish at maturity, surfaces display distinctive spots, and optical microscopy reveals a notable hilar appendage, 0.5–1.0 μm in diam (Fig. 3d).

Other material examined

China, Sichuan province, Panzhihua City, Yanbian County, Shuanglong village, 26°49'12"N, 101°33'7.1029"E, elevation 2,000 m, under Castanea mollissima Bl. in evergreen hill forest, in mainly reddish-brown soils, 16 Aug. 2020, collected by M. Yang (YAAS TJ75-2).

Notes

M. broomeanus Berk (Türkoğlu and Castellano 2013; Uzun et al. 2014), M. shanxiensis and M. obovatisporus (Liu et al. 1989), are similar to M. cyaneus in morphology. M. cyaneus (basidiomata light brown to yellowish brown), M. broomeanus (basidiomata yellow-brown to deep brown), M. shanxiensis (basidiomata brown to rust-brown) and M. obovatisporus (basidiomata brown to brownish-black), but the specimens of M. cyaneus clustered in an independent clade with strong support (BS = 100%, PP = 1.0; Fig. 2), supporting it as a distinct species. Additionally, DNA analysis revealed M. cyaneus shared less than 92% ITS similarity with other Melanogaster species.

Figure 3. 

Photographs of Melanogaster species. Melanogaster cyaneus (YAAS-TJ75-1, holotype) a basidiomata b LM of Peridium c LM of basidiospores d SEM of basidiospore. Melanogaster diqingensis (YAAS-WXH_9068, holotype) e basidiomata f LM of Peridium g LM of basidiospores h SEM of basidiospore. Melanogaster truncatisporus (YAAS-TJ87, holotype) i basidiomata j LM of peridium k LM of basidiospores l SEM of ascospore. Scale bars: 2 cm (a, e, i); 20 μm (b, f, g); 10 μm (c, g, k); 1 μm (d, h, l).

Melanogaster diqingensis T. J. Yuan, Shu H. Li, & Raspé, sp. nov.

MycoBank No: 901869
Fig. 3e–h

Diagnosis

Melanogaster diqingensis is diagnosed by the combination of medium-sized, pale-yellow to orange or brown-yellow basidiomata with scaled, lobed or concave surface, pale yellow gleba, and obovate to subglobose, smooth basidiospores (3.0–5.1 × 2.0–4.0 μm, Q = 1.0–1.8).

Etymology

The epithet diqingensis refers to the prefecture of the type locality.

Holotype

China. Yunnan Province: Diqing Autonomous Prefecture, Shangri-La County, Baishuitai village, 27°30'14.2236"N, 100°2'50.5716"E, elevation 2,380 m, in brown soil under Quercus aquifolioides Rehd. et Wils., 25 Sep. 2020, collected by X. H. Wang (KUN- HKAS121212, holotype; YAAS-WXH_9068, isotype).

Description

Basidiomata 0.5–2.5 × 0.2–2.2 cm, hypogeous, globose, subglobose or ellipsoidal, pale-yellow to orange or brown-yellow, scaled, lobed or concave in the surface, without visible rhizomorphs (Fig. 3e). Odor faint. Peridium two-layered, outer layer 40–80 μm thick, composed of interwoven hyphae, 3.5–7.5 μm broad, with fusoid to cylindrical terminal cells, deeply orange-yellow walls toward surface, with clamp connections; inner layer 150–260 μm thick, composed of interwoven hyphae, 5–10 μm broad, with inflated cells, 7.5–15 μm broad, bright pale yellow, with abundant clamp connections. Gleba solid, milk-white when immature, pale yellow at maturity, hard when dried; trama plates of hyaline or yellowish, gelatinized hyphae (Fig. 3f); locules 2–3 mm in diam (Fig. 3g). Basidia exhibit limited reviving, appearing clavate, hyaline, 4–spored. Basidiospores obovate to subglobose, smooth, 3.0–5.1 × 2.0–4.0 μm (Lm × Wm= 3.8 ± 1.0 × 3.2 ± 0.8, Q= 1.0–1.8, Qm =1.2 ± 0.6, n = 75), hyaline (immature) to light yellow, dark brown (mature) in KOH 5%, with truncate-cupped base and very short hilar appendage in optical microscopy, 0.5–1.5 μm in diam (Fig. 3h).

Notes

Six Melanogaster species, namely M. subglobisporus, M. natsii, M. spinisporus (Wang et al. 1995), M. rivularis, M. luteus=M. microsporus (Moreau et al. 2011), are similar to M. diqingensis in morphology and related by phylogeny. The colors of their basidiomata are similar to M. diqingensis, i.e. rust brown to deep brown in M. subglobisporus, yellow-brown in M. natsii, grayish brown to light brown in M. spinisporus, and bright golden yellow in M. minysporus, but the basidiomata of all of latter species are without scales, lobes or concave area, by which they were easily differentiated from M. diqingensis. M. rivularis (anthracite-black gleba), and M. luteus (club-shaped or cylindro-elliptical to cylindrical basidiospores) also are easily differentiated from M. diqingensis. The thickness of the peridium (≤ 300 μm) is similar among M. subglobisporus, M. ovoidisporus, and M. coccolobae, However, the size of basidiospores provide a clear distinction between these species (M. subglobisporus 8–11 × 7–9 μm, M. ovoidisporus 5–7 × 3.5–5.8 μm and M. coccolobae 6.2–12 × 5.2–10 μm). Phylogenetically, M. diqingensis and M. subglobisporus formed an independent and strongly supported clade (BS = 78%, PP = 1.0; Fig. 2), but M. diqingensis shared less than 93.2% ITS similarity with M. subglobisporus, supporting M. diqingensis as a distinct species.

Melanogaster truncatisporus T. J. Yuan, Shu H. Li, & Raspé, sp. nov.

MycoBank No: 901870
Fig. 3i–l

Diagnosis

Melanogaster truncatisporus is diagnosed by the combination of medium-sized basidiomata with orange-yellow peridium that becomes reddish brown to dark brown in age, and truncate basidiospores.

Etymology

The epithet truncatisporus refers to the truncate basidiospores.

Holotype

China. Yunnan Province: Nujiang Autonomous Prefecture, Lanping County, Zhongpai township, Xinchangping village, 26°54'15"N, 99°10'32"E, elevation 1990 m, in mainly brown soils under Castanea mollissima and Pinus yunnanensis Franch., 26 Oct. 2020, collected by T. J. Yuan (KUN-HKAS129199, holotype; YAAS-TJ87, isotype).

Description

Basidiomata 1.5–3.0 × 0.4–2.3 cm, hypogeous or semi-hypogeous, subglobose to oval, occasionally irregular-elongated, yellowish when young, reddish brown to dark brown at maturity, smooth or slightly velvety surface, lobed or indented at the base, attached mycelial strands, occasionally extending to the surface, dark brown, rhizomorphs not distinct (Fig. 3i). Peridium two-layered, outer layer 50–100 μm thick, composed of interwoven hyphae, orange-yellow, with clamp connections, and fusoid to cylindrical terminal cells, 4–5 μm broad; inner layer 150–350 μm thick, composed of interwoven hyphae, 3–5 μm broad, pale yellow, intermixed with massive inflated cells, ellipsoidal or irregular, 3–20 μm broad. Gleba solid, pale brown when young, blackish brown to black at maturity, separated by white or pale yellow trama when young, which becomes deep brown at maturity, hard when dried; trama plates of hyaline or yellowish gelatinized hyphae (Fig. 3j); locules 2–4 mm in diameter, polygonal to irregular (Fig. 3k). Basidia poorly recovered, hyaline, 4–spored, occurring randomly in the locules (Fig. 3k). Basidiospores subglobose to globose or irregularly elongate-pyriform, 3.5–9.5 × 3.0–7.0 μm (Lm × Wm = 7.0 ± 2.5 × 4.5 ± 2.0, Q = 1.0–2.5, Qm = 1.5 ± 1.0, n = 65), hyaline when immature, becoming dark brown at maturity, smooth, with truncate-cupped base and short hilar appendage,1–2 μm in diam in optical microscopy (Fig. 3l).

Habitat, phenology, and distribution

hypogeous to semi-hypogeous under Castanea mollissima and Pinus yunnanensis, in mixed forest, in late autumn. So far found in Lanping and Gongshan counties, Yunnan Province, China.

Other material examined

China. Yunnan Province: Nujiang autonomous Prefecture, Lanping County, 26°54'17"N, 99°10'31"E, elevation 2,030 m, in mainly brown soils under Castanea mollissima and Pinus yunnanensis, 26 Oct. 2020, collected by T. J. Yuan (YAAS TJ83 and YAAS TJ109); China. Yunnan Province: Nujiang Autonomous Prefecture, Gongshan County, 28°1'19"N, 98°37'2"E, elevation 1,800 m, in mainly brown soils under Castanea mollissima and Pinus yunnanensis, 25 Sep. 2020, collected by Li, S. H. (YAAS L5346).

Notes

Four Melanogaster species, namely M. minysporus (Cázares et al. 2008), M. broomeanus Berk (Türkoğlu and Castellano 2013, Uzun et al. 2014), M. obovatisporus (Liu et al. 1989), and M. variegatus (Krisztián 2008), are similar in morphology and related to M. truncatisporus by phylogeny. M. truncatisporus can be easily differentiated by its peridium thickness (M. truncatisporus, 200–450 μm vs M. minysporus, 160–240 μm) and the size of basidiospores (M. truncatisporus, 3.5–9.5 × 3.0–7.0 μm vs M. minysporus, 5–6.5 × 3–5 μm). M. truncatisporus has a two-layered peridium and M. broomeanus has a single-layered peridium. The difference in basidiospore size is evident, with 3.5–9.5 × 3.0–7.0 μm for M. truncatisporus and 7–9 × 4 μm for M. broomeanus. Also, M. truncatisporus basidia typically contain 4 spores, whereas those of M. obovatisporus consistently contain 8 spores. Basidiospore size (especially minimum size) is also a diagnostic character to separate M. truncatisporus from M. variegatus (3.5–9.5 × 3.0–7.0 μm for the former, and 7.5–10 × 5.5–8 μm for the latter). Phylogenetically, the specimens of M. truncatisporus clustered in an independent clade with strong support (BS = 100%, PP = 1.0; Fig. 2), supporting it as a new species. Additionally, M. truncatisporus (holotype ITS sequence ON427479) shared less than 93.2% similarity with ITS sequences of other Melanogaster species.

Subdivision of the genus Melanogaster in sections based on morphology

The main characters to identify Melanogaster species are the number of layers of the peridium, thickness of the peridium, and average length and width of basidiospores (Table 2). A discriminant analysis based on five variables, namely the number of layers of the peridium, the minimum and maximum thickness of the peridium, and the length and width of basidiospores, was performed by OPLS-DA (Fig. 4). In this analysis, the three sections, Melanogaster, Variegati, and Rivulares, were separated in the 3D principal component space by the average length and average width of basidiospores, and the minimum thickness of the peridium. The cumulative contribution of the first three principal components exceeded 80.5%. The primary factors determining the distribution of species in the 3-dimensional space are the average length and width of spores. The three new species (M. cyaneus, M. diqingensis, and M. truncatisporus) were placed into the section Melanogaster, Rivulares, and Variegati by OPLS-DA, respectively.

Figure 4. 

OPLS-DA discriminant analysis of three sections including Chinese species, based on their main morphological characteristics. Lm = mean spore length; Wm = mean spore width; Peridium Tmin = minimum thickness of peridium.

Discussion

In this paper, we introduce three novel species of hypogeous fungi belonging to Melanogaster (Paxillaceae, Boletales): M. cyaneus, M. diqingensis, and M. truncatisporus. The first two species were discovered in Yunnan Province, while the latter was collected in Sichuan Province, in southwest China. Morphologically, M. cyaneus stands out with its distinctive blue or bluish gleba and light brown to yellowish brown basidiomata. M. diqingensis can be identified by its pale yellow to brown-yellow basidiomata, featuring a scaled, lobed, or concave surface. M. truncatisporus is characterized by its distinct pale yellow to deeply orange-yellow peridium and subglobose to globose or pyriform basidiospores. These characteristics differentiate them from other Melanogaster species found in China. Phylogenetically, our analysis based on an ITS rDNA dataset, including ITS1-2 and 5.8S, revealed that specimens of M. cyaneus and M. truncatisporus clustered into two independent clades with strong support (BS = 100%, PP = 1.0). M. diqingensis and M. subglobisporus formed a separate and well-supported clade (BS = 78%, PP = 1.0; Fig. 2). Moreover, these three species shared less than 93.2% ITS similarity with other Melanogaster species. Both the phylogenetic tree and morphological features concur to support these three species as new additions to Melanogaster.

Knapp (1954) and Moreau et al. (2011) proposed a division of European Melanogaster species into three sections based on the average length of basidiospores. These sections were defined as follows: section Melanogaster, with basidiospores longer than 10 μm; section Variegati, with basidiospores measuring 6–10 μm; and section Rivulares, with basidiospores ranging from 5 to 6 μm. Our multivariate statistical analysis based on wider taxon sampling and three morphological characters instead of only spore length allowed to separate species in line with Moreau et al. (2011) sections (Fig. 4). However, in the phylogenetic tree (Fig. 2), the three morphologically delineated sections were not monophyletic. For example, M. panzhihuaensis described from China was placed within the Melanogaster section based on morphology (Fig. 4), but shared a sister relationship with M. minobovatus, also from China, which was assigned to section Rivulares by the multivariate morphological analysis (Fig. 4). This placement is distinct from other species classified within the Melanogaster section, such as M. ambiguus (Spain and China), M. intermedius (UK), M. euryspermus (USA), M. tuberiformis (Spain), M. natsii (China), M. subglobisporus (China), and M. macrosporus (Hungary) (Wang et al. 1995; Frank et al. 2006; Brock et al. 2009; Alvarado et al. 2021; Xu et al. 2022).

A set of 19 Melanogaster sequences from Germany, France, and the USA (as detailed in Table 1) were included in our phylogenetic analyses. The phylogenetic position of certain specimens based on these sequences was problematic, particularly concerning M. intermedius B-1770, and M. variegatus 23640 and B-1688 from Hungary, as well as M. natsii OSC82168 JMT7491 and OSC158336 MES297 from the USA. All those accessions belonged in species-level clades including other accessions identified as different species. The ITS rDNA sequences of Melanogaster sp. (Melanog002FRA, Melanog006FRA, Melanog007FRA, Melanog008FRA, Melanog011FRA, and Melanog012FRA), along with Melanogaster sp. (Melanog018FRA and Melanog019FRA) from France, clustered with M. rivularis and M. luteus clades, respectively. This observation is substantiated by robust support from high bootstrap values (BS = 97 and BS = 93) and posterior probability from Bayesian inference (PP = 1.0) in Fig. 2. Consequently, we recommend classifying the first six unidentified specimens as M. rivularis species and the last two specimens as M. luteus.

Melanogaster sp. MVC 753 FLASF 65878 (Chile) and Melanogaster sp. MES 1003 (Chile) exhibited less than 98.3% similarity in the ITS region when compared to M. macrosporus. Furthermore, an ITS sequence (Melanogaster sp. JC110118BT) from Spain, two ITS sequences (Melanogaster sp. OSC AHF420 from France and Melanogaster sp. OSC LG1042 from the USA), and two ITS sequences (Melanogaster sp. OK 2022a oka331 and Melanogaster sp. OK 2022a oka332) from Turkey exhibited less than 96.7% similarity to ITS sequences of other Melanogaster species. Consequently, those accessions represent four distinct species-level lineages, designated as M. sp1 to M. sp4, respectively (Fig. 2). This observation suggests the presence of four undescribed species. More research is therefore needed to validate and properly describe those putative novel species.

Key to the Melanogaster species from China

1a Basidiospores verrucose 2
1b Basidiospores smooth 3
2a Basidiomata brown to deep brown, peridium 250–350 μm thick, surface minutely green tomentose; basidiospores elongate fusiform M. tomentellus
2b Basidiomata light brown with some red-brown or brick-red when fresh, peridium 360–750 μm thick, basidiospores ellipsoid to obovoid, subglobose M. spinisporus
3a Basidiospores light yellow or yellow 4
3b Basidiospores brown to dark brown or reddish brown 5
4a Basidiomata brick-red with dark green to black-green gleba at maturity M. obovatus
4b Basidiomata red-brown to brown with yellow-green gleba at maturity M. quercicola
5a Basidiospores mostly longer than 10 μm 6
5b Basidiospores mostly shorter than 10 μm 7
6a Basidiomata yellow-brown to brown, gleba brown, basidiospores fusiform M. fusisporus
6b Basidiomata brown to rust-brown, gleba brownish, basidiospores long obovoid M. shanxiensis
6c Basidiomata light brown to yellowish brown, gleba bluish, basidiospores subglobose to globose M. cyaneus
6d Basidiomata yellow-brown, gleba brown to dark brown, basidiospores limoniform or ellipsoid M. natsii
7a Basidiospores oblong to cylindrical M. broomeanus
7b Basidiospores obovate or obovate to subglobose 8
7c Basidiospores subglobose to globose 9
8a Basidiospores red-brown, 8.7–12.4 × 5.2–7.6 µm M. panzhihuaensis
8b Basidiospores dark brown to brownish, 6.5–8 × 3.8–4.7 µm M. obovatisporus
8c Basidiospores yellowish brown to brown, 8.7–10.6 × 6.6–8.0 µm M. subglobisporus
8d Basidiospores dark brown, 5.1–6.9 × 4.2–5.1 μm M. minobovatus
9a Basidiomata reddish brown to dark brown, gleba brownish-black or black, basidiospore 3.5–9.5 × 3.0–7.0 μm M. truncatisporus
9b Basidiomata pale yellow to orange, gleba pale yellow, basidiospore 3.0–5.1 × 2.0–4.0 μm M. diqingensis

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This study was financially supported by grants from the Central guidance for local scientific and technological development funds (Project ID: 202307AB110001); the National Natural Science Foundation of China to Shu-Hong Li (No. 31560009); the Biodiversity Survey and Assessment Project of the Ministry of Ecology and Environment, China to Xiang-Hua Wang (2019HJ2096001006); the Science and Technology Talents and Platform Plan from the Yunnan Provincial Science and Technology Department (No. 2017HB084); Anhui Province University Natural Science Research Foundation (No. gxyq2018097 & 1908085MC61); the Exploring and Studying of Wild Edible and Valuable Hypogeous Fungi in Nujiang and Diqing Prefectures (YN2020019); the Project of Industrial Technology System (CARS-20); and the Thesis Writing Grant of Mae Fah Luang University, Thailand, to Tianjun Yuan.

Author contributions

Tian-Jun Yuan performed the research and wrote the manuscript, Olivier Raspé supervised the work and revised the manuscript, Hong-Mei Luo and Kai-Mei Su collected specimens, Shu-Hong Li provided the fundings.

Author ORCIDs

Olivier Raspé https://orcid.org/0000-0002-8426-2133

Data availability

All of the data that support the findings of this study are available in the main text, or in publicly accessible repositories as indicated in the text.

References

  • Alfaro ME, Zoller S, Lutzoni F (2003) Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. Molecular Biology and Evolution 20(2): 255–266. https://doi.org/10.1093/molbev/msg028
  • Alvarado P, Cabero J, Moreno-Mateos D, Vizzini A, Alonso J, Lebeuf R, Siquier JL, Vidal JM (2021) Phylogenetic relationships among false truffle genera of Paxillaceae – Alpova, Melanogaster, Neoalpova, and Paralpova, gen. nov. Mycologia 113(4): 828–841. https://doi.org/10.1080/00275514.2021.1911552
  • Arraiano-Castilho R, Bidartondo MI, Niskanen T, Clarkson JJ, Brunner I, Zimmermann S, Senn-Irlet B, Frey B, Peintner U, Mrak T, Suz LM (2021) Habitat specialisation controls ectomycorrhizal fungi above the treeline in the European Alps. The New Phytologist 229(5): 2901–2916. https://doi.org/10.1111/nph.17033
  • Binder M, Larsson KH, Matheny PB, Hibbett DS (2010) Amylocorticiales ord. nov. and Jaapiales ord. nov.: Early diverging clades of Agaricomycetidae dominated by corticioid forms. Mycologia 102(4): 865–880. https://doi.org/10.3852/09-288
  • Castellano MA, Trappe JM, Maser Z, Maser S (1986) Key to spores of the genera of hypogeous fungi of North America, with reference to animal mycophagy. Mad River Press, Eureka, USA, 185 pp.
  • Cázares E, Guevara G, García J, Trappe JM (2008) Melanogaster minysporus sp. nov., a new sequestrate member of the Boletales from Mexico. Revista Mexicana de mcologia 28: 67–69.
  • Cubeta MA, Echandi E, Abernethy T, Vilgalys R (1991) Characterization of anastomosis groups of binucleate Rhizoctonia species using restriction analysis of an amplified ribosomal RNA gene. Phytopathology 81(11): 1395–1400. https://doi.org/10.1094/Phyto-81-1395
  • de la Fuente JI, Martínez-González CR, Oros-Ortega I, Guevara G, Bandala VM, Córdova-Lara I, Vela-Hernández RY, López CY, García Jiménez J (2021) Melanogaster coccolobae sp. nov. (Paxillaceae, Boletales), a tropical hypogeous fungus from the urban areas of Quintana Roo, Mexico. Acta Botánica Mexicana 128: e1896. https://doi.org/10.21829/abm128.2021.1896
  • Frank JL, Barry S, Southworth D (2006) Mammal mycophagy and dispersal of mycorrhizal inoculum in Oregon white oak woodlands. Northwest Science 80(4): 264–273.
  • Grubisha LC, Trappe JM, Molina R, Spatafora JW (2001) Biology of the ectomycorrhizal genus Rhizopogon. V. Phylogenetic relationships in the Boletales inferred from LSU rDNA sequences. Mycologia 93(1): 82–89. https://doi.org/10.1080/00275514.2001.12061281
  • Krisztián H (2008) Különböző stressztűrőképességű nagygombanemzetségek Kárpát-medencei leletanyagának molekuláris azonosítása és rendszerezése. Doktori értekezés, Biológia Doktori Iskola.
  • Hayward J, Tourtellot SG, Horton TR (2014) A revision of the Alpova diplophloeus complex in North America. Mycologia 106(4): 846–855. https://doi.org/10.3852/13-360
  • He MQ, Zhao RL, Hyde KD, Begerow D, Kemler M, Yurkov A, McKenzie EHC, Raspé O, Kakishima M, Sánchez-Ramírez S, Vellinga EC, Halling R, Papp V, Zmitrovich IV, Buyck B, Ertz D, Wijayawardene NN, Cui BK, Schoutteten N, Liu XZ, Li TH, Yao YJ, Zhu XY, Liu AQ, Li GJ, Zhang MZ, Ling ZL, Cao B, Antonín V, Boekhout T, da-Silva BDB, Crop ED, Decock C, Dima B, Dutta AK, Fell JW, Geml J, Ghobad-Nejhad M, Giachini AJ, Gibertoni TB, Gorjón SP, Haelewaters D, He SH, Hodkinson BP, Horak E, Hoshino T, Justo A, Lim YW, Menolli Jr N, Mešić A, Moncalvo JM, Mueller GM, Nagy LG, Nilsson RH, Noordeloos M, Nuytinck J, Orihara T, Ratchadawan C, Rajchenberg M, Silva-Filho AGS, Sulzbacher MA, Tjalčec Z, Valenzuela R, Verbeken A, Vizzini A, Wartcgow F, Wei TZ, Wei M, Zhao CL, Kirk PM (2019) Notes, outline and divergence times of Basidiomycota. Fungal Diversity 99(1): 105–367. https://doi.org/10.1007/s13225-019-00435-4
  • Hofstetter V, Clémençon H, Vilgalys R, Moncalvo JM (2002) Phylogenetic analyses of the Lyophylleae (Agaricales, Basidiomycota) based on nuclear and mitochondrial rDNA sequences. Mycological Research 106(9): 1043–1059. https://doi.org/10.1017/S095375620200641X
  • Jargeat P, Chaumeton JP, Navaud O, Vizzini A, Gryta H (2014) The Paxillus involutus (Boletales, Paxillaceae) complex in Europe: Genetic diversity and morphological description of the new species Paxillus cuprinus, typification of P. involutus ss, and synthesis of species boundaries. Fungal Biology 118(1): 12–31. https://doi.org/10.1016/j.funbio.2013.10.008
  • Katoh K, Standley DM (2013) MaFFT Multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30(4): 772–780. https://doi.org/10.1093/molbev/mst010
  • Knapp A (1954) Die Europäischen Hypogaeen-Gattungen und ihre Gattungstypen. II. Teil (Fortsetzung). Die Gattungstypen der Melanogastraceae und weitere spezies. Schweizerisches Zeitschrift für Pilzkunde 32(8): 117–130.
  • Lacheva M (2015) New records of Melanogaster species (Fungi, Boletales) in Bulgaria. Journal of Biodiversity and Environmental Sciences 6(1): 494–500.
  • Liu B, Tao K, Chang MC (1989) Two new species of Melanogaster from China. Acta Mycologica Sinica 8: 210–213.
  • Matheny PB (2005) Improving phylogenetic inference of mushrooms with RPB1 and RPB2 nucleotide sequences (Inocybe, Agaricales). Molecular Phylogenetics and Evolution 35(1): 1–20. https://doi.org/10.1016/j.ympev.2004.11.014
  • Matheny PB, Wang Z, Binder M, Curtis JM, Lim YW, Nilsson RH, Hughes KW, Hoftsetter V, Ammirati JF, Schoch CL, Langer E, Langer G, McLaughlin DJ, Wilson AW, Frøslev T, Ge ZW, Kerrigan RW, Slot JC, Yang ZL, Baroni TJ, Fischer M, Hosaka K, Matsuura K, Seidl MT, Vauras J, Hibbett DS (2007) Contributions of rpb2 and tef1 to the phylogeny of mushrooms and allies (Basidiomycota, Fungi). Molecular Phylogenetics and Evolution 43(2): 430–451. https://doi.org/10.1016/j.ympev.2006.08.024
  • Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA, 1–8. https://doi.org/10.1109/GCE.2010.5676129
  • Montecchi A, Sarasini M (2000) Funghi Ipogei d’Europa. Associazione Micologica Bresadola, Fondazione Centro Studi Micologici. Vicenza, Italy, 714 pp.
  • Moreau PA, Rochet J, Richard F, Chassagne F, Manzi S, Gardes M (2011) Taxonomy of Alnus-associated hypogeous species of Alpova and Melanogaster (Basidiomycota, Paxillaceae) in Europe. Cryptogamie. Mycologie 32(1): 33–62. https://doi.org/10.7872/crym.v32.iss1.2012.033
  • Moreau PA, Stéphane W, Branislav P, Jargeat P, Manzi S, Vizzini A (2013) Alpova komoviana (Boletales, Paxillaceae), a new sequestrate fungus from montenegro, with a revised phylogeny of the genus in europe. Mycological Progress 12(1): 109–119. https://doi.org/10.1007/s11557-012-0818-x
  • Page RD (2001) TreeView. Glasgow University, Glasgow.
  • Pena R, Offermann C, Simon J, Naumann PS, Geßler A, Holst J, Dannenmann M, Mayer H, Kögel-Knabner I, Rennenberg H, Polle A (2010) Girdling affects ectomycorrhizal fungal (EMF) diversity and reveals functional differences in EMF community composition in a beech forest. Applied and Environmental Microbiology 76(6): 1831–1841. https://doi.org/10.1128/AEM.01703-09
  • Perič B, Moreau PA (2010) Melanogaster luteus, un hypogé rare retrouvé au Monténégro. Mycologia Montenegrina 12: 77–83.
  • Stamatakis A, Ludwig T, Meier H (2005) RAxML-III: A fast program for maximum likelihood-based inference of large phylogenetic trees. Bioinformatics (Oxford, England) 21(4): 456–463. https://doi.org/10.1093/bioinformatics/bti191
  • Suz LM, Kallow S, Reed K, Bidartondo MI, Barsoum N (2017) Pine mycorrhizal communities in pure and mixed pine-oak forests: Abiotic environment trumps neighboring oak host effects. Forest Ecology and Management 406: 370–380. https://doi.org/10.1016/j.foreco.2017.09.030
  • Svrček M (1958) VIII. řad Melanogastrales-Černouškotvaré. Flora ČSR B(1). Gasteromycetes. Praha, Czechoslovakian Academy, 527–556, 795–802.
  • Trappe JM (1975) A revision of the genus Alpova with notes on Rhizopogon and the Melanogastraceae. Beihefte zur Nova Hedwigia 51: 279–309.
  • Trappe JM, Molina R, Luoma DL, Cázares E, Pilz D, Smith JE, Castellano MA, Miller SL, Trappe MJ (2009) Diversity, ecology, and conservation of truffle fungi in forests of the Pacific northwest. USDA general technical report, Forest Service, Pacific northwest Research Station. Portland, Oregon, 196 pp. https://doi.org/10.2737/PNW-GTR-772
  • Uzun Y, Acar I, Akata I (2014) Notes on Turkish Melanogaster. Ot Sistematik Botanik Dergisi 22: 113–118.
  • Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 72(8): 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990
  • Vizzini A, Zotti M, Ryman S, Ghignone S (2010) Typification of Octaviania rubescens (Paxillineae, Boletales) and phylogenetic hypotheses for genus Alpova. Mycologia 102(4): 967–975. https://doi.org/10.3852/09-256
  • Wang Y, Chang MC, Tao K, Liu B (1995) New species and new varieties of the genus Melanogaster from China. Journal of Shangxi University. Natural Sciences Education 18(4): 449–453.
  • Wang Y, Castellano MA, Trappe JM (2005) Melanogaster utriculatus sp. nov. from Japan. Mycotaxon 93: 315–317.
  • Xu YY, Yan XY, Li T, Zhao TY, Lv JC, Fan L (2022) The taxonomic revision of Melanogaster (Paxillaceae, Boletales) in China based on molecular and morphological evidence. Mycological Progress 21(12): e98. https://doi.org/10.1007/s11557-022-01842-9
login to comment