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Research Article
Hemiaustroboletus, a new genus in the subfamily Austroboletoideae (Boletaceae, Boletales)
expand article infoOlivia Ayala-Vásquez‡, Jesús García-Jiménez‡, Elvira Aguirre-Acosta§, Rigoberto Castro-Rivera|, Rodolfo Enrique Ángeles-Argáiz§, Ángel Emmanuel Saldivar, Roberto Garibay-Orijel§
‡ Tecnológico Nacional de México, Instituto Tecnológico de Ciudad Victoria, Ciudad Victoria, Mexico
§ Universidad Nacional Autónoma de México, Ciudad de México, Mexico
| CIBA, Instituto Politécnico Nacional, Tlaxcala, Mexico
¶ Universidad de Guadalajara, Jalisco, Mexico

Corresponding author: Roberto Garibay-Orijel ( rgaribay@ib.unam.mx )

Academic editor: Olivier Raspé
© 2022 Olivia Ayala-Vásquez, Jesús García-Jiménez, Elvira Aguirre-Acosta, Rigoberto Castro-Rivera, Rodolfo Enrique Ángeles-Argáiz, Ángel Emmanuel Saldivar, Roberto Garibay-Orijel.
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: Ayala-Vásquez O, García-Jiménez J, Aguirre-Acosta E, Castro-Rivera R, Ángeles-Argáiz RE, Saldivar ÁE, Garibay-Orijel R (2022) Hemiaustroboletus, a new genus in the subfamily Austroboletoideae (Boletaceae, Boletales). MycoKeys 88: 55-78. https://doi.org/10.3897/mycokeys.88.73951
Open Access

Abstract

The present study describes Hemiaustroboletus gen. nov. in the subfamily Austroboletoideae (Boletaceae). Hemiaustroboletus is supported by morphological and molecular data using LSU and RPB2 regions. Additionally, its geographic distribution and intraspecific variation were inferred using ITS sequences. The genus is characterised by pileate-stipitate basidiomata; purple, brown, reddish-brown, orange-brown to dark brown vinaceous pileus; whitish or lilac to vinaceous context and a subclavate stipe. Microscopically, it is characterised by ornamented, slightly verrucose, cracked to perforated brown basidiospores. Two species are described within the genus, Hemiaustroboletus vinaceobrunneus sp. nov. and H. vinaceus sp. nov. Hemiaustroboletus vinaceus sp. nov. is morphologically similar to Austroboletus gracilis, which suggests they may have been confused in the past. This study presents the phylogenetic placement, microscopic structures, detailed morphological descriptions and illustrations of both new species.

Keywords

Mexico, mycodiversity, neotropics, new taxa

Introduction

Boletaceae is the most diverse family within the Boletales; it has a wide distribution in both temperate and tropical regions (Binder and Hibbett 2006; Wu et al. 2014). Most species of this family are ectomycorrhizal with members of Betulaceae, Casuarinaceae, Dipterocarpaceae, Ericaceae, Fabaceae, Fagaceae, Mimosaceae, Myrtaceae, Pinaceae, Polygonaceae, and Salicaceae (Tedersoo et al. 2010; Smith et al. 2013; Wu et al. 2016). Currently, 98 genera are recognised in this family (He et al. 2019; Vadthanarat et al. 2019; Hosen and Yang 2021). Its members are characterised by fleshy, epigeous pileate-stipitate basidiomata or hypogeous to subhypogeous gastroid basidiomata, with tubular or lamellar hymenophore; elliptical, cylindrical, fusoid, subfusoid, ovoid, subglobose to globose, smooth or ornamented basidiospores; spore ornamentation ranging from striated, reticulate, echinulate, filiform and perforated to verrucose (Singer et al. 1991; Wu et al. 2014; Halling et al. 2015; Ayala-Vásquez et al. 2018).

Wu et al. (2014) proposed six subfamilies for Boletaceae, of which Austroboletoideae includes Austroboletus (Corner) Wolfe, Fistulinella Henn., Mucilopilus Wolfe and Veloporphyrellus L.D. Gómez & Singer, with Austroboletus as the type genus. This subfamily is distinguished by pileate-stipitate basidiomes; smooth, furfuraceous, tomentose, dry or viscous pileus, with or without a marginal veil and whitish context that does not change colour when cut. The hymenophore is tubular, whitish or pink with purple tinge, immutable or rarely brown when cut. The stipe is smooth, reticulate or squamose with a whitish basal mycelium. The basidiospores are smooth or ornamented, perforated, verrucose to smooth, grey-violet, yellowish, yellow brown, ochraceous in potassium hydroxide (KOH) and yellow-brown, yellow-cinnamon to ochraceous in Melzer’s reagent. The pileipellis is formed by a trichoderm or ixotrichoderm. The hymenophoral trama is boletoid. Austroboletoideae species are mainly associated with Fagaceae and Pinaceae hosts in temperate, subtropical to tropical regions.

In recent years, various authors (Wu et al. 2014; Wu et al. 2016; Gelardi et al. 2020; Kuo and Ortiz-Santana 2020) have recognised the polyphyly of Austroboletus, which is divided into the Austroboletus s.s., Austroboletus s.l. and the A. gracilis s.l. independent clades. This study focuses on the phylogenetic placement and taxonomy of the A. gracilis s.l. clade, placing it in the new genus Hemiaustroboletus with two new species, Hemiaustroboletus vinaceobrunneus and H. vinaceus.

Materials and methods

To resolve the systematics and taxonomy of the new genus Hemiaustroboletus, we conducted an exhaustive sampling of an area with high bolete diversity according to García-Jiménez et al. (2013). The sampling was carried out over the last 10 years including the different biogeographic areas of Mexico: Nearctic, Neovolcanic Axis and Neotropic. The collection trips were conducted in the States of Chiapas, Chihuahua, Estado de Mexico, Jalisco, Michoacan and Oaxaca, in six vegetation types in temperate and subtropical forests during the rainy season from June to October from 2010 to 2019. The samples were characterised at macro- and micromorphological level and three genetic markers were sequenced and analysed.

Morphological study

Morphological characters were described according to Largent (1986) and Lodge et al. (2004). Chemical reactions with KOH and ammonium hydroxide (NH₄OH) were characterised. Photographs of basidiomata were taken in situ, as well as data on the botanical composition of the sites. The colours for taxonomic descriptions were based on Kornerup and Wanscher (1978). Microscopic characters of 30 basidiospores, basidia, pleurocystidia, cheilocystidia, pileipellis cells and stipitipellis were measured by optical microscopy (Carl Zeiss GmbH 37081, Germany). The Q index (length/width) was estimated for the basidiospores. Ornamentation of basidiospores was observed by scanning electron microscopy (SEM) (Hitachi Su 1510, Hitachi, Japan). The specimens were deposited at the “Herbario Nacional de México” of the “Instituto de Biología, Universidad Nacional Autónoma de México” (MEXU), at the “Herbario José Castillo Tovar del Tecnológico de Ciudad Victoria” (ITCV) and at the “Herbario del Instituto de Botánica, Universidad de Guadalajara” (IBUG).

DNA Extraction, PCR and Sequencing

Samples of dehydrated basidiomata were used for DNA extraction. The DNA was extracted using the DNeasy Power-Soil kit (QIAGEN). Cell lysis was performed by grinding samples in mortar with liquid nitrogen. Three nuclear loci (ITS, LSU and RPB2) were amplified with Platinum Taq DNA Polymerase (Invitrogen-Thermo Fisher Scientific) and Taq & Load PCR Mastermix (MP Biomedicals) in a thermocycler (BIO-RAD). The PCR parameters were as follows: 95 °C initial denaturation for 4 min; 35 cycles of denaturation at 94 °C for 60 s, alignment at 54 °C for 60 s, extension at 72 °C for 60 s and a final extension at 72 °C for 10 min. The primers ITS1/ITS4 (White et al. 1990) were used for the ITS region; LROR/LR5 (Vilgalys and Hester 1990) for LSU; and RPB2-B-F2/RPB2-B-R (Wu et al. 2014) for the partial RPB2 gene. The amplification was examined by 1% agarose gel electrophoresis; gels were stained with GelRed (Biotium) and observed under an UVP Multidoc-It transilluminator (Analytikjena). Only PCR products generated with Taq-Platinum required LB loading buffer. PCR products with successful amplification were cleaned with ExoSAP-IT (Thermo Fisher Scientific) diluted 1:1 with ddH2O and incubated at 37 °C for 45 min and 80 °C for 15 min. Sanger sequencing was performed at the “Laboratorio de secuenciación genómica de la biodiversidad y la salud, Instituto de Biología, Universidad Nacional Autónoma de México”. Samples were sequenced in both directions with PCR primers using BigDye Terminator v.3.1 (Thermo Fisher Scientific).

Phylogenetic analyses

Hemiaustroboletus species produce scarce fruit bodies; from 606 Boletales specimens collected, just eight (1.32%) belonged to this genus. Three materials corresponded to H. vinaceus, four to H. vinaceobrunneus and two were determined as Hemiaustroboletus sp. The three loci of the holotype of H. vinaceus (IBUG-AES334) and one more collection (ITCV-AV524, MEXU-30103) were sequenced; we only recovered ITS and RPB2 loci from a third specimen (IBUG-AES364) (Table 1). The three loci of the holotype of H. vinaceobrunneus (ITCV-AV868, MEXU-30051) and one additional material (ITCV-AV845, MEXU-30052) were sequenced; only the ITS and RPB2 loci were sequenced for a third collection (ITCV-AV1168, MEXU-30053). ITS locus was also sequenced for one Hemiaustroboletus sp. collection (ITCV-AK_3508) (Table 1).

Table 1.
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List of species, geographic origin and GenBank accession numbers of ITS, LSU and RPB2 sequences used in the phylogenetic analyses.

Taxa Voucher Country ITS LSU RPB2 Reference
Aureoboletus betula USA MK601736 MK766298 Kuo and Ortiz-Santana (2020)
A. garciae MEXU:29006 Mexico MH337251 MT228983 Haelewaters et al. (2020)
Austroboletus amazonicus 1839_ AMV Colombia KF937307 KF714508 Vasco-Palacios et al. (2014)
A. amazonicus 1914_ AMV Colombia KF937308 KF714509 Vasco-Palacios et al. (2014)
A. austrovirens BRI:AQ0795791 Australia KP242211 KP242225 KP242133 Fechner et al. (2017)
A. austrovirens BRI:AQ0794622 Australia KP242210 Fechner et al. (2017)
A. austrovirens MEL:2382920a Australia KP242284 KP242113 Fechner et al. (2017)
A. austrovirens BRI:AQ0794609 Australia KP242226 KP242131 Fechner et al. (2017)
A. austrovirens BRI:AQ0794171 Australia KP242227 KP242133 Fechner et al. (2017)
A. eburneus REH9487 Australia JX889668 Vasco-Palacios et al. (2014)
A. dictyotus HKAS59804 China JX901138 Hosen et al. (2013)
A. fusisporus HKAS75207 China JX889719 JX889720 Hosen et al. (2013)
A. fusisporus JXSB0351 China MK765810 GenBank
A. gracilis 112-96 USA DQ534624 Binder and Hibbett (2006)
A. gracilis TM03_434 Canada EU522815 Porter et al. (2008)
A. gracilis var. gracilis CFMR BOS-547 USA MK601715 MK766277 Kuo and Ortiz-Santana (2020)
A. gracilis var. flavipes CFMR BOS-562 USA MK601714 Kuo and Ortiz-Santana (2020)
A. gracilis ACAD11344F Canada MH465078 Young et al. (2019)
A. gracilis SFC20140823-02 South Korea MN794901 GenBank
A. gracilis NAMA 2017-106 USA MH979242 GenBank
A. gracilis 310751 México MH167935 GenBank
A. gracilis CNV35 USA MT345212 Victoroff (2020)
A. cf. gracilis JLF6600 USA MN174796 GenBank
A. lacunosus REH9146 Australia JX889669 Vasco-Palacios et al. (2014)
A. lacunosus MEL2233764 Australia KC552056 GenBank
A. mucosus TH6300 Guyana AY612798 Drehmel et al. (2008)
A. mutabilis BRI:AQ0795793 Australia KP242169 KP242263 KP242098 Fechner et al. (2017)
A. mutabilis BRI:AQ0669270 Australia KP242266 KP242097 Fechner et al. (2017)
A. mutabilis BRI:AQ0796266 Australia KP242262 KP242099 Fechner et al. (2017)
A. niveus 312 New Zealand DQ534622 Binder and Hibbett (2006)
A. niveus MEL2053830 Australia KC552016 KC552058 Orihara et al. (2016)
A. novae-zelandiae PDD:72542 New Zealand HM060327 GenBank
A. rarus BRI:AQ0794045 Australia KP242197 KP242236 KP242086 Fechner et al. (2017)
A. rostrupii TH8189 Guyana JN168683 Smith et al. (2011)
Austroboletus sp. BRI:AQ0794156 Australia KP242235 KP242115 GenBank
Austroboletus sp. BRI:AQ0794222 Australia KP242234 KP242106 GenBank
Austroboletus sp. BRI:AQ0794271 Australia KP242259 KP242102 GenBank
Austroboletus sp. HKAS 57756 China KF112383 KF112764 Wu et al. (2014)
Austroboletus sp. HKAS 59624 China KF112485 KF112765 Wu et al. (2014)
Austroboletus sp. HKAS 74743 China KT990527 KT990367 Wu et al. (2014)
Austroboletus sp. PERTH6658407 Australia KP242277 KP242126 GenBank
Austroboletus sp. BRI:AQ0794242 Australia KP242087 GenBank
Austroboletus sp. OR0891 Thailand MH614753 Vadthanarat et al. (2019)
Austroboletus sp. OTAFUNNZ2013434 New Zealand KP191670 GenBank
A. subflavidus JBSD130771 Dominican Republic MT580902 MT590754 Gelardi et al. (2020)
A. subflavidus JBSD130772 Dominican Republic MT580903 MT590755 Gelardi et al. (2020)
A. subflavidus CFMR BZ-3178 Belize MK601716 MK766278 Kuo and Ortiz-Santana (2020)
A. subvirens KPM-NC-0017836 Japan JN378518 Orihara et al. (2012)
A. viscidoviridis Perth 7588682 Australia KP242282 KP242128 Fechner et al. (2017)
Boletellus indistinctus HKAS77623 China KT990531 KT990371 Wu et al. (2016)
Boletellus sp. HKAS80554 KT990535 KT990374 Wu et al. (2016)
Boletus harrisonii MICH: KUO-09071204 USA MK601718 MK766280 Kuo and Ortiz-Santana (2020)
Boletus sp. dd08055 China FJ810161 GenBank
Boletus sp. MHM165 Mexico EU569243 Morris et al. (2008)
Boletales sp. B0229 Canada KY825985 GenBank
Fistulinella campinaranae var. scrobiculata AMV1980 Colombia KF714520 Vasco-Palacios et al. (2014)
F. gloeocarpa JBSD130769 Dominican Republic MT580906 MT590756 Gelardi et al. (2020)
F. gloeocarpa CFMR:B4 Bahamas MT580904 Gelardi et al. (2020)
F. gloeocarpa CFMR:B10 Bahamas MT580905 Gelardi et al. (2020)
F. prunicolor REH9502 Australia JX889648 MG212630 Halling et al. (2012)
F. olivaceoalba HKAS 53432 Vietnam MH745969 GenBank
F. olivaceoalba LE312004 Vietnam MH718396 GenBank
F. ruschii CORT:TJB-8329 USA MT580907 Gelardi et al. (2020)
F. viscida 238 25S New Zealand AF456826 Vasco-Palacios et al. (2014)
F. cinereoalba TH8471 Guyana GQ477439 KT339237 GenBank
Hemiaustroboletus vinaceobrunneus MEXU_30051 Holotype Mexico MN178797 MN200222 MT887617 This study
H. vinaceobrunneus MEXU_30052 Isotype Mexico MN178798 MN200223 MT887618 This study
H. vinaceobrunneus MEXU_30053 Isotype Mexico MN178799 MT887619 This study
H. vinaceus AV524 Paratype Mexico MN178802 MN200225 MT887622 This study
H. vinaceus AES334 Holotype Mexico MN178800 MN200224 MT887620 This study
H. vinaceus AES364 Isotype Mexico MN178801 MT887621 This study
Hemiaustroboletus sp. AK_3508 Mexico MN178803 This study
Hemileccinum subglabripes MICH: KUO-08301402 USA MK601739 MK766301 Kuo and Ortiz-Santana (2020)
Hortiboletus rubellus MICH: KUO-06081002 USA MK601741 MK766303 Kuo and Ortiz-Santana (2020)
H. amygdalinus HKAS54166 China KT990581 KT990416 Wu et al. (2016)
Hourangia cheoi Tang572 China KP136953 KP136985 Zhu et al. (2015)
Imleria badia MICH: KUO-09110404 USA MK601743 MK766305 Kuo and Ortiz-Santana (2020)
Mucilopilus castaneiceps HKAS 75045 China KF112382 KF112735 Wu et al. (2016)
M. castaneiceps HKAS50338 China KT990555 KT990391 Wu et al. (2016)
M. castaneiceps HKAS71039 China KT990547 KT990385 Wu et al. (2016)
Parvixerocomus pseudoaokii HKAS 80480 China KP658468 KP658470 Wu et al. (2016)
Porphyrellus castaneus HKAS52554 China KT990697 KT990502 Wu et al. (2016)
P. porphyrosporus MB97-023 Germany DQ534643 GU187800 Binder and Hibbett (2006)
P. orientifumosipes HKAS53372 China KT990629 KT990461 Wu et al. (2016)
Tengioboletus sp. HKAS 77869 China KT990658 KT990483 Wu et al. (2016)
Strobilomyces confusus CFMR:DR-3024 Dominican Republic MK601809 MK766365 Kuo and Ortiz-Santana (2020)
Tylopilus felleus CFMR: BOS-780 USA MK601814 MK766370 Kuo and Ortiz-Santana (2020)
T. sordidus MICH: KUO-06240801 MK601815 MK766371 Kuo and Ortiz-Santana (2020)
Tylopilus sp. HKAS 50229 China KF112423 KF112734 Wu et al. (2014)
Uncultured mycorrhizal BOLETE1 USA AY656925 Walker et al. (2005)
Uncultured mycorrhizal clon N_1 South Korea AB571507 Obase et al. (2012)
Uncultured Boletus isolate: YM490 Japan LC175482 Miyamoto et al. (2018)
Uncultured Boletus Clon ZE2 China GU391428 Ma et al. (2010)
Veloporphyrellus alpinus KUN:HKAS68301 China JX984537 Li et al. (2014)
V. pseudovelatus KUN: HKAS59444 China JX984542 Li et al. (2014)
V. pseudovelatus KUN:HKAS52244 China JX984531 Li et al. (2014)
V. conicus CFMR:BZ1670 Belize JX984543 Li et al. (2014)
V. conicus CFMR:BZ1705 Belize JX984544 Li et al. (2014)
V. pantoleucus F:Gomez21232 Costa Rica JX984548 Li et al. (2014)
V. velatus KUN: HKAS63668 China JX984546 Li et al. (2014)
V. aff. velatus HKAS 57490 China KF112380 KF112733 Wu et al. (2014)
V. vulpinus LE315544 Vietnam MN511177 MN511170 GenBank
V. vulpinus LE315549 Vietnam MN511180 GenBank
V. vulpinus LE315546 Vietnam MN511179 GenBank
V. vulpinus Vietnam MN511178 GenBank
Xerocomellus chrysenteron HKAS:56494 China KF112357 KF112685 Wu et al. (2014)

We conducted two sets of phylogenetic analyses, the first one to reconstruct the phylogenetic relationships of Hemiaustroboletus gen. nov. and the second one to complement its taxonomic concept with biogeographic and ecological information. The first analysis used the LSU and RPB2 markers in a concatenated matrix, while the second used ITS in order to leverage GenBank data.

Individual LSU and RPB2 alignments were concatenated into a single matrix (83 taxa, 1335 characters) with GENEIOUS PRIME V.2019.0.4 (Biomatters Ltd). Alignments and concatenation were performed with the MAFFT algorithm (Katoh et al. 2002) using GENEIOUS PRIME V.2019.0.4. Sequences representing the subfamilies Austroboletoideae, Boletoideae and Xerocomoideae came from: 83 LSU sequences, 56 rpb2 sequences, 30 ITS sequences from published works and unpublished sequences available in GenBank (Table 1).

The best-fit evolutionary model was estimated with JMODELTEST 2 (Darriba et al. 2012) using CIPRES SCIENCE GATEWAY V. 3.3 (Miller et al. 2010) for each marker separately. For all three markers, the best model was GTR+G+I. We used the LSU-RPB2 dataset to make evolutionary inferences within Austroboletoideae and the ITS dataset to make biogeographic/ecological inferences for Hemiaustroboletus.

The phylogenetic hypotheses (LSU-RPB2) were constructed with Bayesian Inference (BI) and Maximum Likelihood (ML) on a partitioned alignment with same evolutionary model for both markers. Bayesian posterior probability phylogeny was performed using MrBayes algorithm (Ronquist et al. 2012) using two separate Monte Carlo four chains starting from random trees for 10 million generations each (final standard deviation ± 0.224), trees were sampled every 100 generations. The first 25% of samples were discarded as burn-in. ML analyses were performed using the RAxML algorithm (Stamatakis 2014) with 1000 bootstrap replicates. For both analyses, members of subfamilies Boletoideae and Xerocomoideae were used as outgroup. The second analysis (ITS) was performed with the same parameters including Veloporphyrellus and Austroboletus without outgroup. The resulting phylogenetic trees were edited with FIGTREE V.1.4.3 (Rambaut 2009).

Average intrageneric and intergeneric nucleotide similarities between the genera within Austroboletoidеae were obtained separately for RPB2, LSU and ITS alignments as follows. For each alignment a nucleotide similarity matrix was computed in GENEIOUS 10.2.6 (Biomatters Ltd). Sequences belonging to genera outside Austroboletoidеae were removed and then the mean nucleotide similarity was calculated amongst all pairwise comparisons between sequences of each pair of genera.

Results

Phylogenetic analyses of LSU-RPB2 concatenated alignment showed that Hemiaustroboletus is a supported monophyletic group, belonging to the Austroboletoideae (BPP = 0.98, MLB = 47%). Additionally, H. vinaceobrunneus (BPP = 1, MLB = 100%) and H. vinaceus (BPP = 1, MLB = 96%) were supported monophyletic species (Fig. 1). The ITS analyses showed that Hemiaustroboletus forms ectomycorrhizae with Fagaceae, particularly Quercus and also with Pinus in temperate, subtropical and tropical forests. It distributes in North America (Mexico, USA and Canada) and Asia (China, Japan and Korea) (Fig. 2). These analyses also showed that Austroboletus gracilis s.l. is a widely-used name mainly applied to designate Hemiaustroboletus species.

Figure 1. 

Phylogenetic placement of Hemiaustroboletus gen. nov. in the Austroboletoideae subfamily (Boletaceae) using LSU and RPB2 markers in a concatenated and partitioned matrix. The tree shows the topology of Bayesian analysis, with both MLB (≥ 70%) and BPP (≥ 0.7) clade support given. New genera and new species are indicated in the rectangles; taxa and/or branches in purple correspond to Hemiaustroboletus gen. nov.; remaining Austroboletoideae (blue); Boletoideae (green); Xerocomoideae (mustard). Background colours correspond to subfamilies; grey bars correspond to families.

Figure 2. 

Phylogenetic tree of Hemiaustroboletus displaying geographic distribution using voucher and environmental ITS nrDNA sequences. The tree shows the topology of Bayesian analysis, with both MLB (≥ 70%) and BPP (≥ 0.7) clade support given. Taxa and branches in purple correspond to Hemiaustroboletus gen. nov. and those in blue to Veloporphyrellus and Austroboletus.

Taxonomy

Hemiaustroboletus Ayala-Vásquez, García-Jiménez & Garibay-Orijel, gen. nov.

MycoBank No: 838460

Diagnosis

Hemiaustroboletus is characterised by small and medium basidiomata with slightly ornamented pileus surface, stipe fibrillose to striated without veil, slightly verrucose or cracked to pitted basidiospores and pileipellis formed by an ixotrichoderm or trichoderm.

Etymology

From the Latin hemi “almost or half”, Austroboletus the generic epithet refers to the morphological affinities with this genus.

Generic type

Hemiaustroboletus vinaceobrunneus Ayala-Vásquez, García-Jiménez & Garibay-Orijel sp. nov.

Generic Description

Epigeous, stipitate-pileate basidiomata. Pileus reddish-brown, violet-brown, dark violet, reddish-brown, orange-brown, yellow-brown, cinnamon, dry surface, finely velvety, velutinous, rivulose, granular-tomentose, subtomentose, minutely areolate. Hymenophore tubular, circular to angular pores, whitish, pink-purple, lilac, magenta-grey, brown-violet to pinkish-brown, with or without change brown when cut. Context whitish to pale red. Stipe subclavate, tomentose, pruinose, granular furfuraceous, striate surface, longitudinally fibrous, very finely reticulated in tapering towards apex. Whitish basal mycelium. Basidiospores ornamented, slightly verrucose, cracked to pits, fusoid, oval-elliptical, cylindrical to subfusoid, oblong, ovoid-oblong. Cystidia clavate, sphaeropedunculate, subfusoid. Pileipellis an ixotrichoderm or trichoderm; terminal cells cylindrical, fusoid, ventricose-rostrate with or without encrustations in the wall. Caulocystidia fusoid, cylindrical to subclavate and tetrasporic caulobasidia.

Distribution

Canada, China, Japan, Mexico, South Korea and United States.

Ecology

Temperate and subtropical forests, with conifers and broadleaf trees (Abies spp., Quercus spp., Pinus spp.) from 2000 to 3000 m alt.

Hemiaustroboletus vinaceobrunneus Ayala-Vásquez, García-Jiménez & Garibay-Orijel, sp. nov.

MycoBank No: 838461
Figs 3, 4, 5B, D

Diagnosis

Pileus vinaceous to brown, pores whitish to pinkish at maturity, vinaceous context; longitudinally fribrillose stipe; basidiospores (10) 11–17 (–21) × 4–5 (–7) µm, slightly verrucose to cracked, fusoid to cylindrical; pleurocystidia ventricose-rostrate to fusoid, cheilocystidia sphaeropedunculate.

Figure 3. 

Hemiaustroboletus vinaceobrunneus A, C basidiomata (MEXU-30052 Holotype) B, D pileus (MEXU-30053, MEXU-30051, Isotype) E hymenophore (MEXU-30052 Holotype) F, G context (MEXU-30052 Holotype). Scale bar: 10 mm (A–G).

Holotype

Mexico. Oaxaca State, Santa Catarina Ixtepeji Municipality, La Cumbre Town, Peña Prieta site, 17°11'11.34"N, 96°38'00"W (DMS), 2800 m alt., 19 July 2017, Ayala-Vásquez (MEXU-30051; isotype ITCV-AV868).

Figure 4. 

Hemiaustroboletus vinaceobrunneus (AV845-ITCV, MEXU-30052 Holotype) A basidiospores B basidia C pleurocystidia D cheilocystidia E pileipellis F caulocystidia. Scale bars: 10 µm (A–F).

Etymology

The name refers to the colour of the pileus, from the Latin “vinosus” vinaceous when young and “brunneus” brown when mature.

Figure 5. 

Basidiospore ornamentation of Hemiaustroboletus revealed by SEM A, C Hemiaustroboletus vinaceus (AV868-ITCV, MEXU-30051, Holotype) B, D Hemiaustroboletus vinaceobrunneus (AV1168-ITCV, MEXU-30053 Isotype).

Description

Basidiomata stipitate-pileate. Pileus 36–40 mm diameter, convex when young becoming plano-convex, reddish-vinaceous (13B6) when young, orange brown (7C8), reddish-brown (8D8-8E8) to dark brown (7F8) with some ruby tones (12E8) at maturity, dry surface, subtomentose, rivulose to areolate, whitish context, decurved margin. Hymenophore slightly depressed around the stipe to subadnate, pores 1–1.2 mm diameter, circular to subangular, whitish when young, pink to red-whitish (11A3-11A2) at maturity, tubes 6 mm length, of pores concolorous, unchanging when cut or touched, tubes detachable from the context. Context 4–8 mm thick, whitish, with some shades of pale red, vinaceous at the edge of the pileus and at the apex of the stipe at maturity. Stipe 45–65 × 8–10 mm, subclavate, reddish-vinaceous (13B6), orange-brown (7C8) to brown (7D8 -7E8) at the apex and part of the base, orange in the middle area (6B8) to orange-brown (6C8), rest of the base whitish; surface furfuraceous, longitudinally fibrillose. Whitish mycelium. Chemical reactions pileus negative in KOH, the context and the hymenophore slightly become pale violet (16A2) and the stipe becomes pale brown (6D4). When ammonium hydroxide (NH4OH) is applied, the pileus becomes brown-violet (11F8-11F7), the hymenophore and context pale orange (5A2) and the stipe pale violet (16A2).

Basidiospores 10–15 (–20) × 4–5 (–7) µm, X = 14.04 × 4.96 µm, std = 3.46 × 0.99 µm, (n = 30, Q = (2.2) 2.4–2.5 (2.8), (holotype); (10–) 11–15 (–21) × 4.5–7 (–8) µm, X = 13.78 × 6.07 µm, std = 3.74 × 1.3 µm, Q = (2.2) 2.4–2.6 (2.8) (paratype MEXU-30052); (10–) 11–15 (–17) × (4–) 4.5–5.5 (–6) µm, X = 13.15 × 4 µm, std = 2.62 × 0.64 µm, Q = (2.2) 2.6–2.9 (3) µm, (paratype ITCV-AV1121), cylindrical to subfusoid, slightly verrucose to cracked, brown-orange in KOH, inamyloid in Melzer’s reagent. Basidia 30–33 (–49) × 9–11 (–12) µm, clavate, hyaline in KOH, pale yellow in Melzer’s reagent, with granular content, tetrasporic. Pleurocystidia 31–45 × 8–11 µm, ventricose to fusoid, some mammillate, hyaline in KOH, yellowish in Melzer’s reagent, thick walled (1–1.5 µm). Cheilocystidia 42–70 (–86) × 9–15 (–17) µm, clavate with septa (1–2 µm thick), sphaeropedunculate, some mammillate, hyaline in KOH, yellowish in Melzer’s reagent, thick-walled (1–1.5 µm). Hymenophoral trama boletoid; hyphae cylindrical 3–15 µm diameter, with gelatinous wall some with smooth walls, hyaline to yellowish in KOH and Melzer’s reagent. Pileipellis a trichoderm with terminal cells (22–) 35–75 (–105) × 8–14 (–21) µm, cylindrical to subclavate, hyaline in KOH, yellowish in Melzer’s reagent, embedded in a gelatinous substance and with visible contents in Melzer’s reagent, thick-walled (1–1.5 µm). Caulocystidia 20–64 (–140) × 6–14 (–16) µm, fusoid, cylindrical to sphaeropedunculate with one to two septa, hyaline to yellowish KOH with visible contents visible in Melzer’s reagent. Caulobasidia 25–30 × 7–8 µm tetrasporic, concolorous with the caulocystidia. Clamp connections absent.

Habit and habitat

Solitary, in Abies guatemalensis, Pinus pseudostrobus and Quercus laurina mixed forest, putatively associated with Quercus laurina, from 2800 to 3000 m alt.

Known distribution

Currently only known from Oaxaca State, southeast Mexico.

Additional materials examined

Mexico, Oaxaca State, Santa Catarina Ixtepeji Municipality, La Cumbre Town, East of cottage site, 17°11'30"N, 96°38'18"W (DMS), 2903 m alt., 18 July 2017, Ayala-Vásquez (MEXU-30052; ITCV-AV845); Cabeza de Vaca site, 17°11'10"N, 96°38'28"W (DMS), 3038 m alt., 18 July 2017, Ayala-Vásquez (ITCV-AV1121), Cabeza de Vaca site, 15 August 2018, Ayala-Vásquez (MEXU-30053; ITCV-AV1168).

Remarks

Hemiaustroboletus vinaceobrunneus differs from H. vinaceus by its context with vinaceous tones especially at maturity and a whitish-pink to pale red hymenophore; the stipe is orange-brown; basidiospores are 10–15 (–20) × 4–5 (–7) µm, finely verrucose to cracked, lodged to sphaeropedunculate cheilocystidia, caulocystidia fusoid, cylindrical to sphaeropedunculate with a septum. In contrast, H. vinaceus has a whitish context with slight yellowish-brown tones near the epicutis, has shorter basidiospores (9–) 10–14.4 (–16) × 4–5(–8) µm, cylindrical to clavate queilocystidia and caulocystidia fusoid or clavate. In the field, the former can be mistaken for Gyroporus purpurinus because of the colours and size of the basidiomata, but G. purpurinus has a hollow stipe (Davoodian and Halling 2013), while H. vinaceobrunneus has a compact context.

Hemiaustroboletus vinaceus Ayala-Vásquez, García-Jiménez & Saldivar, sp. nov.

MycoBank No: 838462
Figs 5A, C, 6, 7

Diagnosis

Pileus dark violet to dark brown, whitish context; hymenophore pink-purple to violet-brown; stipe surface tomentose to longitudinally fribrillose; basidiospores 9–13 × 4–5 µm, surface with cylindrical pits; pleurocystidia and cheilocystidia fusiform-ventricose to lanceolate.

Figure 6. 

Hemiaustroboletus vinaceus (AES334-IBUG, Holotype) A, B basidiomata C hymenophore D context E pileus surface. Scale bar: 10 mm (A–E).

Holotype

Mexico, Jalisco State, Tequila Municipality, Tequila Volcano site, between 11 and 12 km on the road uphill to the antenna station, 20°48'35"N, 103°51'46"W (DMS), 2144 m alt., 18 August 2019, Á.E. Saldivar (IBUG-AES334).

Figure 7. 

Hemiaustroboletus vinaceus (AES334-IBUG, Holotype) A basidiospores B basidia C pleurocystidia D cheilocystidia E pileipellis F caulocystidia. Scale bars: 10 µm (A–F).

Etymology

The name refers to the colour of the pileus from the Latin “vinosus” vinaceous.

Description

Pileus 35–70 mm in diameter, convex when young, becoming plano-convex with age, dark violet (16F6-16F4), violet-brown (11F5-11F8), orange-brown (5E7), with lighter shades of dark brown (6F5-6F8) lighter towards margin, whole edge, straight, dry surface, finely scamose, slightly areolate at the centre. Hymenophore adnate, slightly depressed, pores 0.5–2 mm in diameter, subangular to angular, pink-purple (14A4), lilac (14B4–14C4), magenta-grey (14C4–14D4), ruby-grey (12C4–12D4), colour unchanging when injured, tubes 7–10 mm, concolorous with the pores. Context 7–12 mm thick, solid, whitish, with slight yellowish-brown tones near the epicutis. Stipe 62–77 × 8–9 mm, central, cylindrical, with wider base, surface with longitudinal striations, whitish at the apex, yellowish-brown (5D5-5E5), orange-brown (5C5) shades in the middle, base with yellowish (5B6) to whitish shades; whitish context, unchanged when cut. Whitish basal mycelium. Odour pleasant. Taste slightly acidic. Chemical reactions: KOH reddish-brown in pileus, brown in hymenophore, slightly pinkish in context, yellowish-brown in stipe. NH4OH orange with violet tones on pileus, yellow in hymenophore, pale yellow in context, red-orange in stipe.

Basidiospores 9–13 (–14.5) × 4–5 (–8) µm, X = 12.14 × 5.2 µm, std = 2.08 × 1.36 µm, (n = 35), Q = (1.8) 2.1–2.2 (2.5) (holotype); (10–) 12–14 × 4–5 (–7) µm, X = 11.94 × 5.14 µm, std = 1.60 × 1.13 µm, (n = 35), Q = (2.2) 2.3–2.4 (2.5), (paratype MEXU-30103); (10–) 14–15 (–16) × (4–) 5–6 (–7) µm, X = 14.29 × 5.8 µm, std = 1.69 × 0.76 µm, (n = 40), Q = (2.2) 2.3–2.5 (2.6), (paratype colpos-CP5); subfusiform to cylindrical, slightly rough or dotted, apex rounded to subacute, with suprahilar depression, yellowish. Basidia 27–34 × 7–15.2 µm, claviform, bisporic, tetrasporic, with sterigma 2–4 × 0.5–1 µm, thin-walled, hyaline in KOH, yellow in Melzer’s reagent. Pleurocystidia 28–50 × 6.4–11 µm, fusoid-ventricose, slightly lanceolate, with content hyaline in KOH, yellow in Melzer’s reagent, with walls 0.5 μm thick. Cheilocystidia 25–61 × 6.4–11 µm, subclavate, hyaline in KOH, yellow in Melzer’s reagent, thin-walled. Hymenophoral trama divergent, with central and lateral hyphae tubular, 2–6 µm wide, hyaline in KOH, yellow in Melzer’s reagent, thin-walled; septa without clamp connections. Pileipellis a trichoderm with terminal cells 32–92 × 5–11 µm, cylindrical to subclaviform, hyaline in KOH, yellow in Melzer’s reagent, thin-walled. Caulocystidia 29–95 × 14–17 (–19) µm, subclaviform to claviform, thin-walled, with yellow visible contents in Melzer’s reagent, hyaline in KOH.

Habit and habitat

Pinus-Quercus forests and Quercus forests, associated with Q. liebmanii and other Quercus spp.

Known distribution

Currently only known from Neovolcanic Axis and Sierra Madre del Sur, Mexico.

Additional material examined

Mexico, Jalisco State, Tequila Municipality, Tequila Volcano site, km 11–12 on the road uphill to the antenna station, 20°48'14"N, 103°51'37"W (DMS), 2144 m alt., 18 September 2019, A.E. Saldivar (IBUG-AE364); Oaxaca State, San Antonio de la Cal Municipality, Las Peñas site, 17°01'11"N, 96°40'33"W (DMS), 2160 m alt., 4 October 2014, Ayala-Vásquez (MEXU-30103; ITCV-AV524, duplicated ENCB); Michoacan State, Road Morelia, Ciudad Hidalgo Town, km 40, 21 July 1983, García-Jiménez (ITCV-3662), Mil Cumbres Town, 9 August 1969, R. Singer M8993 (F). Estado de México State, Ocuilan, San Juan Atzingo Town, mixed forest, 15 July 2021, mycoredes (Colpos- CP5).

Remarks

Hemiaustroboletus vinaceus differs from H. vinaceobrunneus due to its dark violet pileus, lilac to violet hymenophore, yellow stipe in the basal area and whitish apex. It has short, perforated basidiospores 9–13 (–14.4) × 4–5 (–8) µm, caulocystidia clavate to fusoid and pileipellis formed by a trichoderm with terminal cell cylindrical or subclavate, thin-walled. In contrast, H. vinaceobrunneus has a pileipellis formed by a trichoderm with encrustations. Hemiaustroboletus vinaceus is easily confused with Austroboletus gracilis sensu Wolfe (1979), because of its macroscopic characteristics and basidiospore ornamentation, but A. gracilis differs by pileus red-brown, brown-orange, having a total or partial reticulum on the stipe surface; longer basidiospores 10–19.5 × 4.5–9 µm, rugulose- punctate, elliptical to ovoid-elliptical. Austroboletus var. gracilis (Peck) Wolfe differs from H. vinaceus by pileus surface dry, finely velvety, when young, sometime rimose, reddish-brown, cinnamon or yellow-brown; stipe surface anastomosing lines, narrow reticulation overall or at least on the upper half; basidiospores 10–17 × 5–8 µm, narrowly ovoid to subelliptical. Austroboletus gracilis var. laevipes is distinguished by the smooth stipe, pileus yellow-ochraceous to yellow-brown, stipe subclavate, striate, finely pruinose, neither ribs nor reticulated surface, pale yellow or yellow-brown, basidiospores 11.2–14 × 5–8µm, oval-elliptical in face view, inequilateral in profile (Bessette et al. 2000). Austroboletus gracilis var. pulcherripes Both & Bessette differs from H. vinaceus by a white hymenium when young, becoming pinkish to pale cocoa at maturity; stipe clavate, surface dry, coarsely reticulated on the upper two- thirds, reticulated, finely tomentose; basidiospores 13–19 × 5–8 µm, smooth to rugose-punctate, ovoid-elliptical, narrowly ovoid, inequilateral profile.

Discussion

According the phylogenetic analysis, our collections are nested within the Austroboletoideae close to Veloporphyrellus. Recognising the Hemiaustroboletus genus contributes to solving the systematics within Austroboletoideae since previous works have shown that Austroboletus and Veloporphyrellus, as currently morphologically circumscribed, are polyphyletic (Wu et al. 2016; Gelardi et al. 2020; Kuo and Ortiz-Santana 2020). For example, Wu et al. (2016) found two clades of Austroboletus, Austroboletus. s.s. and a second clade where Austroboletus gracilis s.l. (strain, 112/96) is nested with Veloporphyrellus gracilioides, this species being separated from the Veloporphyrellus s.s. clade. Gelardi et al. (2020) also recovered Austroboletus as polyphyletic with Austroboletus s.s. containing most of the species and other samples divided into four more clades. Particularly, in their analyses, most A. gracilis samples nested close to Veloporphyrellus; this is the clade we are erecting now as Hemiaustroboletus.

Our analyses show that Hemiaustroboletus is related to Veloporphyrellus (Fig. 1). This is supported by previous analyses (Gelardi et al. 2020; Kuo and Ortiz-Santana, 2020); indeed, they differ in several morphological characteristics. Veloporphyrellus has a veil which often embraces the apex of the stipe in younger basidiomata; hymenophoral surface white when young becoming pinkish to pink when mature; basidiospores smooth subfusiform to oblong. In contrast, Hemiaustroboletus has furfuraceous, tomentose to minutely areolate pileus surface; whitish, pink-purple, lilac, magenta-grey to brown-violet hymenophoral surface; and slightly verrucose, cracked to pitted ornamented basidiospores (Table 2). Even while the phylogenetic relations between both genera are not statistically supported, nucleotide similarity demonstrated that they are the closest genera within Austroboletoidеae. The overall nucleotide similarity between genera in Austroboletoidеae in RPB2 is 89.23%, in LSU it is 88.19%, and in ITS it is 72.55%. Between Hemiaustroboletus and Veloporphyrellus, the average nucleotide similarity is 93.45% in RPB2, 94.01% in LSU and 74.75 in ITS (Table 3). These amounts of variation in the three markers also support the conclusion of recognising both genera.

Table 2.
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Comparative table of Austroboletoidеae genera, based on Wolfe (1979) and Wu et al. (2016).

Genera Basidiomata Basidiospores Cystidia Pileipellis
Austroboletus Pileus margin which embraces the stipe when young. Stipe surface distinctly reticulate, alveolate-lacunose Ornamented, elongate to amygdaliform, with warts, reticulate ridges or shallow to irregularly furrowed pits Cylindrical, clavate, fusoid Trichoderm with filamentous interwoven hyphae, sometimes strongly gelatinous
Fistulinella Stipitate-pileate to occasionally sequestrate, with or without veil, usually viscid to strongly glutinous pileus Smooth, elongate fusoid, inamyloid to dextrinoid Fusiform to ventricose fusiform or lageniform Trichoderm, ixotrichoderm or ixocutis
Hemiaustrobo letus Pileus surface furfuraceous, tomentose, minutely areolate, stipe surface longitudinally fibrillose to striate Slightly verrucose, cracked to pitted Clavate, Ropedunculate, subfusoid Ixotrichoderm or trichoderm, terminal cells cylindrical, fusoid, ventricose-rostrate
Mucilopilus Viscid pileus, stipe without colour change, white to pinkish or pink hymenophore Smooth, subfusiform to oblong Fusoid, ventricose to subfusiform Ixotrichoderm, composed of strongly gelatinous filamentous hyphae
Veloporphyrellus Pileus margin with distinct membranous veil or appendiculate, stipe nearly glabrous or fibrillose Smooth, subfusiform to oblong Subfusiform to ventricose Trichoderm composed of filamentous interwoven hyphae

Hemiaustroboletus gen. nov. accomplishes the guidelines for the establishment of new genera proposed by Vellinga et al. (2015). It is a monophyletic group supported by morphological data and phylogenetic analyses (BPP = 0.98) (Fig. 1). When Hemiaustroboletus is recognised, the related clade Austroboletus s.s. (the clade including A. dictyotus, the genus type) becomes monophyletic. Additionally, the DNA sequence sampling is broad in taxonomic and geographic terms and uses ribosomal markers and protein coding genes. Indeed, holotypes for both species described are represented with the three markers included in the phylogenetic analyses.

Table 3.
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Average nucleotide similarity amongst genera of Austroboletoidеae.

Genus 1 Genus 2 Average nucleotide similarity (ITS) % Average nucleotide similarity (LSU) % Average nucleotide similarity (RPB2) %
Hemiaustroboletus Hemiaustroboletus 95.49 98.93 97.96
Hemiaustroboletus Mucilopilus 92.51 91.25
Hemiaustroboletus Austroboletus 71.27 85.94 87.75
Hemiaustroboletus Fistulinella 88.58 89.76
Hemiaustroboletus Veloporphyrellus 74.75 94.01 93.45
Veloporphyrellus Veloporphyrellus 95.49 100
Veloporphyrellus Austroboletus 85.64 86.66
Veloporphyrellus Mucilopilus 91.45 89.73
Veloporphyrellus Fistulinella 88.06 89.5
Fistulinella Fistulinella 90.48 89.5
Fistulinella Mucilopilus 87.61 89.5
Fistulinella Austroboletus 83.03 86.87
Austroboletus Austroboletus 86 92.06
Austroboletus Mucilopilus 85.05 87.88
Mucilopilus Mucilopilus 98.5 99.4

Hemiaustroboletus is proposed as a new genus with two species H. vinaceobrunneus and H. vinaceus, including several of the revised material being previously identified as A. gracilis by Singer et al. (1991), Ayala-Vásquez et al. (2018) and Saldivar et al. (2021). The genus has at least one more known clade (Fig. 1) containing samples originally identified as A. gracilis (TM03-434) from Canada, A. gracilis var. gracilis (CFMR BOS-547) and A. gracilis var. flavipes (CFMR BOS-562) from USA. As found in our analyses and previous works (Wu et al. 2016; Gelardi et al. 2020; Kuo and Ortiz-Santana 2020), A. gracilis is a name widely applied to several clades. In our analysis, the sample A. gracilis 112/96 belongs to Austroboletus (maybe because it lacks RPB2 locus), while the rest of the sequences with this epithet belong to Hemiaustroboletus. As this species is polyphyletic, establishing the true identity of A. gracilis s.s. requires the sequencing of its type specimen, a task beyond the objectives of this study.

Hemiaustroboletus differs morphologically from Austroboletus sect. Austroboletus sensu Wu et al. (2016) (Austroboletus s.s. in this study) because the species of the latter have clearly reticulated to costate stipe, elongate, fusoid or amygdaliform basidiospores with warts, reticulate ridges, irregularly furrowed pits or shallow ornamentation and a subrepent to trichoderm pileipellis, composed of filamentous interwoven hyphae, sometimes strongly gelatinous. In contrast, Hemiaustroboletus is characterised by a subclavate, tomentose, pruinose, granular furfuraceous, striate surface, longitudinally fibrous, very finely reticulated stipe, oval-elliptical, cylindrical to subfusoid, oblong, ovoid-oblong basidiospores with slightly verrucose, cracked to pitted surface, its pilleipellis is an ixotrichoderm or trichoderm with terminal cells cylindrical, fusoid or ventricose-rostrate with or without incrustations in the wall.

Finally, A. gracilis, described by Ortiz-Santana et al. (2007) from Central America, is probably Hemiaustroboletus vinaceus or a close species, because they match the description presented here. Further analysis of these collections and others, labelled as A. gracilis in subtropical regions of Central America and eastern Asia, are needed to fully understand the diversity and distribution of Hemiaustroboletus.

Acknowledgements

Ayala-Vásquez acknowledges financial support from the Mexican Council of Science and Technology CONACYT 449637 for financial support (Scholarship); the MEXBOL network project CONACYT 280896, the CONACYT-PRONACES FOP07-2021-03 Project 316198; Javier Isaac de la Fuente, César Martínez-González for technical support, Laura Margarita Marquez Valdelamar, Head of the Sequencing facility at IB-UNAM; Lidia Irene Cabrera Martínez Head of the Molecular Biology Laboratory of the Botany Department of IB-UNAM; María Berenit Mendoza-Garfias, Head of the Laboratory of Scanning Electron Microscopy facility at IB-UNAM; García-Jiménez thanks CONACYT for financial support and the Technological Institute of Mexico.

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