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Type studies of Rossbeevera bispora, and a new species of Rossbeevera from south China
expand article infoMd. Iqbal Hosen, Xiang-Jing Zhong§, Genevieve Gates|, Takamichi Orihara, Tai-Hui Li
‡ Guangdong Institute of Microbiology, Guangzhou, China
§ Administration of Guangdong Xiangtoushan National Nature Reserve, Huizhou, China
| Tasmanian Institute of Agriculture, Hobart, Australia
¶ Kanagawa Prefectural Museum of Natural History, Odawarashi, Japan
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Abstract

The type of Rossbeevera bispora and additional collections from the type location and adjacent areas were studied. Molecular data for R. bispora derived from the new collections are provided. In addition, R. griseobrunnea, a new species of Rossbeevera, is described from Xiangtoushan National Nature Reserve, Guangdong Province of China. The new species is characterized by its globose to subglobose sequestrate basidiomata with grayish white to grayish brown pileus, pale bluish discoloration in some parts of the hymenophore when injured becoming rusty brown to dark brown after being exposed to the air, fusoid (star-shaped in cross section) basidiospores 17–20 × 9–12 μm, and subcutis elements in the pileus. Based on multi-locus (ITS+nrLSU+tef1-α+rpb2) molecular phylogenetic analyses, both species appear as sister to R. paracyanea. We present color photos, macro- and micro-description, SEM basidiospores, molecular affinities of the species and compare them with morphologically similar taxa within the genus. A key to the species known from northern and southern hemispheres is provided.

Keywords

Boletaceae, East Asia, multi-locus phylogeny, new taxon, taxonomy

Introduction

Rossbeevera T. Lebel & Orihara, a sequestrate ectomycorrhizal genus of Boletaceae, was erected in 2012 to accommodate Chamonixia pachydermis Zeller & C.W. Dodge as the type of the genus (Lebel et al. 2012). The genus represents a distinct monophyletic group in the subfamily Leccinoideae, and is strongly supported as a sister to another sequestrate genus Turmalinea Orihara & N. Maek. (Orihara et al. 2016). Species of Rossbeevera are easy to recognize in the field as they have rubbery sequestrate basidiomata, with or without bluish discoloration of either the pileus or the hymenophore and usually a thin pileipellis. Microscopically, they have a hymenium that is developed when the basidiomata is immature and collapses at maturity, and ornamented basidiospores with 3–5 longitudinal ridges (Lebel et al. 2012, Orihara et al. 2016).

Presently Rossbeevera includes 10 species (Lebel et al. 2012, Orihara et al. 2012, 2016), only known from Asia (China, Japan and Singapore/Malaysia) and Australasia (Australia and New Zealand). Prior to this study, two of them (R. bispora (B.C. Zhang & Y.N. Yu) T. Lebel & Orihara and R. yunnanensis Orihara & M.E. Sm.) were known from China (Zhang and Yu 1989, Orihara et al. 2012). In 2018, Orihara reported R. yunnanensis from Japan. Of the remaining eight species: four (R. cryptocyanea Orihara, R. eucyanea Orihara, R. griseovelutina Orihara and R. paracyanea Orihara) are known from Japan (Lebel et al. 2012; Orihara et al. 2016), two (R. vittatispora (G.W. Beaton, Pegler & T.W.K. Young) T. Lebel and R. westraliensis T. Lebel, Orihara & N. Maek) from Australia (Lebel et al. 2012), one (R. pachydermis (Zeller & C.W. Dodge) T. Lebel) (orthographic variant R. pachyderma) from New Zealand (Lebel et al. 2012) and one (R. mucosa (Petri) T. Lebel, Orihara & N. Maek.) from Singapore/Malaysia (Lebel et al. 2012, Orihara et al. 2016).

In this study, several collections of Rossbeevera resembling R. bispora have repeatedly been found in south China (Guangdong Province: Dinghushan Nature Reserve, a type locality of R. bispora; Baiyunshan Mountain, Tianluhu Park and Xiangtoushan National Nature Reserve). Among the collections examined in this study two of them appeared to be clearly different from R. bispora (although all collections were originally treated as R. bispora). Therefore, we studied the type material of R. bispora for comparison with the recent collections. A combination of morphological data and multi-locus phylogenetic analyses including sequences of the nuclear ribosomal internal transcribed spacer (ITS) region, nuclear ribosomal large subunit (nrLSU), translation elongation factor 1-α (tef1-α), and the second largest subunit of RNA polymerase II (rpb2) were used for the delimitation of a new species within the genus Rossbeevera.

Materials and methods

Sampling and morphological studies

The specimens were collected from south China (Guangdong Province: Dinghushan Nature Reserve, Tianluhu Park, Baiyunshan Mountain and Xiangtoushan National Nature Reserve). After being examined and described the dried specimens were deposited in the Fungal Herbarium of the Guangdong Institute of Microbiology, Guangzhou, China (GDGM).

Macromorphological descriptions were based on field notes and photographs. Micromorphological observations were made from small pieces of dried specimens mounted in H2O, 5% aqueous KOH (w/v), Congo Red and Melzer’s solution. In the description of the basidiospore measurements, the notation [n/m/p] is used, which means n basidiospores from m basidiomata of p collections. Dimensions for basidiospores are given as (a–)b–c(–d), in which ‘b–c’ contains a minimum of 90% of the measured values and extreme values ‘a’ and ‘d’ are given in parentheses, whenever necessary. Q denotes the length/width ratio of a measured basidiospore, Qm denotes the average of n basidiospores, and SD is their standard deviation. Results are presented as Qm ± SD. For describing the species, we used the taxonomic terminology pileus for ‘peridium’, hymenophore for ‘gleba’ and stipe for ‘columella’.

Molecular studies

Protocols for genomic DNA extraction, PCR amplification, and sequencing followed Hosen et al. (2013) and references therein. The ITS1-F/ITS4 (White et al. 1990), LROR/LR5 (Vilgalys and Hester 1990), ef1-983F/ef1-1567R (Rehner and Buckley 2005) and rpb2-B-F/rpb2-B-R (Wu et al. 2014) primer pairs were used for the amplification of ITS, nrLSU, tef1-α and rpb2 regions.

Currently molecular data are available for eight of the 10 reported species. The final dataset consisted of 10 species of Rossbeevera including R. bispora and a new species (see taxonomy). Representative sequences (ITS, nrLSU, tef1-α and rpb2) of Rossbeevera and its allied genera from the subfamily Leccinoideae were retrieved from GenBank. Individual gene fragments were aligned in MAFFT v.6.8 (Katoh et al. 2005), and manually edited in BioEdit v.7.0.9 (Hall 1999) using default settings. Prior to concatenating the multi-locus (ITS+nrLSU+tef1-α+rpb2) dataset, an individual aligned dataset was analyzed separately to detect the topologies (BS ≥70%). There was no significant incongruence detected while reconstructing ITS, nrLSU or ITS+nrLSU/ITS+nrLSU+tef1-α except for the individual dataset of tef1-α and rpb2 (because of the lack of representative tef1-α and rpb2 sequences for Rossbeevera species) phylogenetic trees. A multi-locus dataset was built using Phyutility (Smith and Dunn 2008) for further phylogenetic analyses, and the resulting dataset was deposited in TreeBASE (S23404). Maximum Likelihood (ML) was used to analyze the multi-locus dataset. ML was performed in RAxML v.7.2.6 (Stamatakis 2006) with default settings. Statistical support values were obtained using nonparametric bootstrapping (BS) with 1000 replicates.

Results

Molecular phylogenetic results

In this study, 15 new sequences were generated from the Chinese collections of Rossbeevera and deposited in GenBank (Table 1). The combined aligned dataset included 35 specimens from 24 species in the Boletaceae (10 of Rossbeevera, three of Turmalinea, two each of Leccinum Gray, Octaviania Vittad., Rhodactina Pegler & T.W.K. Young and Spongiforma Desjardin, Manfr. Binder, Roekring & Flegel, and one each of Borofutus Hosen & Zhu L. Yang, Leccinellum Bresinsky & Manfr. Binder and Retiboletus Manfr. Binder & Bresinsky). The combined alignment contained 3928 nucleotide sites (gaps included) for each sample, of which 1118 were ITS, 916 were nrLSU, 1128 were tef1-α and 766 were rpb2. Rossbeevera bispora is nested in a clade containing R. paracyanea and R. griseobrunnea with strong support (95% ML BS, Fig. 1). Two collections of R. griseobrunnea (GDGM 45266 and GDGM 45913) formed a monophyletic clade and sister to the Japanese R. paracyanea with moderate support (68% ML BS, Fig. 1). Interestingly, these three East Asian species formed a sister clade with the Australasian Rossbeevera species including R. westraliensis, R. vittatispora and R. pachydermis with moderate strong support (81% ML BS, Fig. 1). The summarized result of the phylogenetic analysis is presented in Fig. 1.

List of fungal taxa of Boletaceae with voucher number, country of origin and GenBank accession numbers used in the molecular phylogeny.

Name of the species Voucher/collection no. Country GenBank accession number
ITS nrLSU tef1 rpb2
Borofutus dhakanus HKAS 73785* Bangladesh JQ928605 JQ928615 JQ928577 JQ928596
Leccinellum sp. KPM-NC-0018041 Japan KC552053 KC552094
Leccinum scabrum KPM-NC-0017840 Japan KC552012 JN378515 JN378455
Leccinum versipelle KPM-NC-0017833 Japan JN378514 JN378454
Octaviania decimae KPM-NC-0017763 Japan JN257991 JN378465 JN378409
Octaviania tasmanica MEL2341996 Australia KC552004 JN378495 JN378436
Retiboletus sinensis HKAS 59832 China KT990633 KT990827 KT990464
Rhodactina himalayensis CMU25117 Thailand MG212603
Rhodactina rostratispora OR1055 Thailand MG212604 MG212644
Rossbeevera westraliensis Trappe14692 Australia HQ647131 HQ647153
Rossbeevera bispora GDGM 45612 China MK035705 MK036346 MK350308
Rossbeevera bispora GDGM 45639 China MK036347 MK350309
Rossbeevera bispora GDGM 46631 China MK035705 MK036348
Rossbeevera bispora GDGM 46638 China MK036349
Rossbeevera cryptocyanea KPM-NC0023387 Japan KP222893 KP222899 KP222913
Rossbeevera eucyanea KPM-NC-0018043 Japan KC551983 KC552029 KC552071
Rossbeevera eucyanea TNS-F-36986* Japan HQ693875 HQ693880 KC552068
Rossbeevera griseobrunnea GDGM 45266 China MH532533 MH537792 MK350310
Rossbeevera griseobrunnea GDGM 45913* China MH532534 MH537793 MK350307 MK350311
Rossbeevera griseovelutina TNS-F-36989* Japan HQ693876 KC552031 KC552076
Rossbeevera griseovelutina TNS-F-36991 Japan KC551985 KC552032 KC552077
Rossbeevera pachydermis MEL2079350 New Zealand HQ647138 HQ647157
Rossbeevera pachydermis PDD:89084 New Zealand GU222301
Rossbeevera paracyanea KPM-NC-0018023 Japan KC551988 KC552035
Rossbeevera paracyanea KPM-NC0023940 Japan KP222894
Rossbeevera vittatispora MEL2329434 Australia KJ001084 KJ001097 KJ001075
Rossbeevera vittatispora TO-AUS-72 Australia KC551977 KC552025 KC552065
Rossbeevera westraliensis MEL2231712 Australia HQ647140 HQ647162
Rossbeevera yunnanensis HKAS 70689* China JN979437
Rossbeevera yunnanensis KPM-NC 23352 Japan MF357925 MF354015
Spongiforma squarepantsii LHFB14 Malaysia HQ724511 HQ724509
Spongiforma thailandica DED7873* Thailand EU685113 EU685108 KF030436 KF030387
Turmalinea chrysocarpa HKAS 70601* China KF112448 KF112729
Turmalinea mesomorpha subsp. sordida KPM-NC-0018015* Japan KC552001 KC552049 KC552092
Turmalinea persicina KPM-NC-0018001* Japan KC551991 KC552038 KC552082
Figure 1. 

Phylogenetic relationships of Rossbeevera and its allied genera inferred from multi-locus (ITS+nrLSU+tef1-α+rpb2) analyses. Rossbeevera bispora and R. griseobrunnea are highlighted in bold on the tree. RAxML bootstrap (BS) support values (ML BS>50%) are indicated on the branches at nodes. Voucher number/collection number are provided after each species followed by country name.

Taxonomy

Rossbeevera bispora (B.C. Zhang & Y.N. Yu) T. Lebel & Orihara, Fungal Diversity 52(1): 58 (2012)

Figs 2, 3, 6a, b

Chamonixia bispora B.C. Zhang & Y.N. Yu, Mycotaxon 35(2): 278 (1989).

Description

Basidiomata hypogeous, 25–45 mm broad, 20–30 mm high, small, globose to subglobose, napiform, sometimes deformed or reniform, fleshy when fresh, firm when dry. Pileus thin, surface glabrous to slightly velvety in some cases, shiny, sometimes alveolate or cracking with age, dull white, grayish white to grayish brown, whitish at the lower portion, turning blue to deep blue when touched or injured or when mature, occasionally basal part covered with yellowish white mycelia. Hymenophore off-white, white to dull white when young, blue to dark blue immediately when cut or injured, fleshy, soft and smooth, composed of minute, irregular locules, becoming partially collapsed with many small empty chambers when dried. Stipe absent. Sterile base present but reduced, white, dull white to grayish white, somewhat dendroid or as a small basal pad or rhizomorph. Odor and taste not recorded.

Basidiospores [80/4/4] 16–21 × 9–11.5 μm [mean 18.55 × 10.58 μm, Q = 1.63–1.83(–1.90), Qm = 1.75 ± 0.11], fusoid, ornamented with 4-longitudinal ridges (star-shaped in cross section), inamyloid, brown to dark brown in KOH and H2O, thick-walled up to 2 μm thick, hilar appendages 1–3 μm long. Basidia 12–25 × 5–8 μm, narrowly clavate to cylindro- clavate, hyaline to pale yellow, usually 2-spored, occasionally 1-spored. Hymenial cystidia absent. Hymenium developed when immature but collapsed at maturity, hyaline to pale yellowish; subhymenium not developed. Hymenophoral trama 60–160 μm wide, subgelatinous, composed of hyaline, cylindrical, loosely interwoven to parallel, frequently branched, thin-walled, cylindrical hyphae 2–5 μm wide. Pileipellis a repent cutis, terminal cells short, clavate to cylindro-clavate, yellowish brown to brownish pigmented, smooth, thin-walled. Clamp connections absent in all tissues.

Habit and habitat

Solitary or in small groups beneath or on the ground, hypogeous or partially epigeous in a subtropical evergreen broad-leaved forest, putatively associated with Castanopsis fissa Rehder & E.H.Wilson, C. chinensis (Spreng.) Hance, C. fabri Hance and Schima superba Gardner & Champ.

Known distribution

Currently known from south China (Guangdong Province: (Baiyunshan Mountain, Dinghushan Nature Reserve and Tianluhu Park).

Specimens examined

CHINA. Guangdong Province, Zhaoqing City, Dinghushan Nature Reserve, 13 October 1982, You-Zao Wang, Wan-Ling Zhen, Jinag-Qing Li (GDGM 5688, holotype); 7 March 2013, Karl (GDGM 45612 and GDGM 45639); Baiyun Mountain, 7 April 2017, Yong He (GDGM 46631); Tianluhu Park, 21 March 2018, Tai-Hui Li, Chenghua Zhang, Xishen Liang (GDGM 46638).

Comments

Zhang and Yu (1989) described R. bispora as Chamonixia bispora B.C. Zhang & Y.N. Yu from south China (Dinghushan Nature Reserve, Guangdong Province) based on a single collection (Fig. 2). Lebel et al. (2012) transferred this species to Rossbeevera as it fits within the generic concept of Rossbeevera. This species is characterized by its whitish to grayish brown pileus turning bluish when injured, 2-spored basidia, and is associated with broad-leaved trees in south China.

Figure 2. 

Type specimen of Rossbeevera bispora (as Chamonixia bispora, GDGM 5688, holotype) a dried basidiomata of Rossbeevera bispora b basidiospores.

Figure 3. 

Basidiomata of Rossbeevera bispora (new collections). a Dull white to grayish brown pileus with blue tinges in some portion (GDGM 45612) b Bluing hymenophore (after injured) and pileus surface (GDGM 46631) c Bluing pileus with reduced stipe (GDGM 46638) d Bluing hymenophore (after injured) and pileus surface (GDGM 46638).

Rossbeevera griseobrunnea Iqbal Hosen & T.H.Li, sp. nov.

MycoBank No: MB826880
Figs 4, 5, 6c, d

Diagnosis

Basidiomata hypogeous, small; pileus grayish white to grayish brown, surface bluing slightly when injured; hymenophore dull white to very pale blue in some portion when injured, finally rusty brown to dark brown at maturity; stipe absent; basidiospores 17–20 × (8–)9–12 μm, fusoid, ornamented with 4-longitudinal ridges (star-shaped in cross section), brown to dark brown; pileipellis a subcutis, with terminal elements short cylindro-clavate.

Typification

CHINA. Guangdong Province, Boluo County, Xiangtoushan National Nature Reserve, 19 November 2015, Tai-Hui Li, Ting Li, Hao Huang & Jun-Ping Zhou (GDGM 45913, holotype).

Etymology

The epithet name ‘griseobrunnea’ (Lat.) refers to the grayish brown pileus.

Description

Basidiomata hypogeous, 15–35 mm broad, 12–25 mm high, small, globose to subglobose, napiform, sometimes deformed or reniform, fleshy when fresh, firm when dry. Pileus very thin, surface glabrous to slightly velvety, shiny, grayish white to grayish brown, whitish at the lower portion, turning to pale blue when touched or injured. Hymenophore off-white, white to dull white when young, becoming pale blue to bluish in some parts/patches then rusty brown to dark brown when exposed to air for 3–5 minutes, often greenish brown around insect damage, firm, composed of minute, irregular locules, becoming partially collapsed with many small empty chambers when dried. Stipe absent. Stipe base present but reduced, white, dull white to grayish white, somewhat dendroid or as a small basal pad or rhizomorph. Odor and taste not recorded.

Basidiospores [50/2/2] 17–20(−21) × (8–)9–12(–13) μm [mean = 18.5 × 10.5 μm, Q = (1.52–)1.63–1.91(–2.1), Qm = 1.81 ± 0.18] fusoid, ornamented with 4-longitudinal ridges (star-shaped in cross section) (up to 2.5 μm high), inamyloid, non-dextrinoid, brown to dark brown in KOH and H2O, thick-walled up to 2 μm thick, hilar appendages 1.5–3 μm long. Basidia 15–27 × 5–9 μm, narrowly clavate to cylindro- clavate, hyaline to pale yellow, usually 2-spored, occasionally 1-spored; basidioles 18–25 × 8–10 μm, clavate to short clavate. Hymenial cystidia absent. Hymenium developed when immature but collapsed at maturity, hyaline to pale yellowish; subhymenium not developed. Hymenophoral trama 60–130 μm wide, subgelatinous, composed of hyaline, cylindrical, loosely interwoven to parallel, frequently branched, thin-walled, cylindrical hyphae 2–5 μm wide. Pileipellis a subcutis with terminal elements 15–20 × 7–9 μm, short clavate to cylindro-clavate, yellowish brown to brownish pigmented, smooth, thin-walled. Clamp connections absent in all tissues.

Additional specimen examined

CHINA, Guangdong Province, Boluo County, Xiangtoushan National Nature Reserve, 19 November 2015, Tai-Hui Li, Ting Li, Hao Huang & Jun-Ping Zhou (GDGM 45266).

Habit and habitat

Solitary or in small groups beneath or on the ground, hypogeous or partially epigeous in a subtropical evergreen broad-leaved forest, putatively associated with Castanopsis fissa, C. chinensis, C. fabri and Schima superba.

Known distribution

Currently known only from south China (Guangdong Province, Xiangtoushan National Nature Reserve).

Comments

The voucher specimen (GDGM 45266) was cited as Rossbeevera sp. by Hosen et al. (2018), while studying boletes from the Xiangtoushan National Nature Reserve, Guangdong Province, China. After morphological and molecular comparisons with other known species of Rossbeevera the voucher specimen (GDGM 45266) is described here as R. griseobrunnea. It is characterized by its dull white, grayish white to grayish brown basidiomata, with hymenophore that discolors very slowly in some portions (pale blue in some patches becoming rusty brown to dark brown) after being cut or injured, fusoid ornamented basidiospores with mostly 4-longitudinal ridges and subcutis elements (short clavate terminal cells) in the pileus.

Figure 4. 

Basidiomata of Rossbeevera griseobrunnea. a Unchanged pileus surface with reduced stipe (GDGM 45266) b Unchanged pileus surface and pale blue (in some patches) hymenophore when injured (GDGM 45266) c Pale bluing hymenophore (after injured) with reduced stipe (GDGM 45913, holotype) d Dark brownish hymenophore after exposed to air (GDGM 45913, holotype).

Figure 5. 

Microscopic features of Rossbeevera griseobrunnea. a Basidia and basidioles development at different stages (GDGM 45266) b Basidiospores in different views (star-shaped in side view and quadrangular in polar view) (GDGM 45913, holotype).

Figure 6. 

SEM of basidiospores of Rossbeevera species. a, b Rossbeevera bispora (a GDGM 45612 b GDGM 45639) c, d Rossbeevera griseobrunnea (c GDGM 45266 d GDGM 45913, holotype).

Discussion

Most of the species within Rossbeevera share common features like globose to subglobose sequestrate basidiomata with bluish discoloration (due to oxidation of pulvinic acid) when injured (either pileus or hymenophore), usually 1–2-spored but sometimes 4-spored basidia, ornamented basidiospores with 4–5 longitudinal ridges (star-shaped in cross section), absence of hymenial cystidia (except R. griseovelutina), and loose arrangement of hymenophoral trama with 2–5 μm wide hyphae. However, continental distance, habitat with different hosts, molecular data or genetic distance and some macro- and micro-morphological differences make them distinct species within Rossbeevera.

In the protologue, the basidiospore size of R. bispora is 15–21 × 10–12 μm (Zhang and Yu 1989). Our re-examination of the type material of R. bispora (GDGM 5688) showed that the basidiospore size is 16–21 × 10–12 μm [mean 18.35 × 11.02 μm, Q = [1.56–1.72(–1.81), Qm = 1.66 ± 0.11], which is similar to that of the original description. The Q values [(1.63–1.83(–1.90), Qm = 1.75 ± 0.11] of basidiospores derived from the new collections are slightly higher than the type material of R. bispora. However, the color changes of the pileus and hymenophore, 2-spored basidia and their association with broad-leaved trees suggest that the modern collections (including those from the type locality, Dinghushan Nature Reserve) are conspecific with R. bispora. Extraction of DNA sequences from the type material of R. bispora (GDGM 5688) was not successful due to the poor quality of the DNA from the aged specimen (collection date: October 13, 1982; Zhang and Yu 1989). For examination of evolutionary relationships within Rossbeevera and phylogenetic stability of this species we provide DNA sequences derived from the new collections. Prior to this study, R. bispora was known only from the type locality (Guangdong Province: Dinghushan Biosphere Reserve Forest), but we demonstrate it has a wide geographic distribution in south China (Guangdong Province: Baiyun Mountain and Tianluhu Park).

The rusty brown to dark brown or chocolate brown hymenophore (after exposure to air for 3–5 minutes or at maturity) in R. griseobrunnea occurs also in R. vittatispora, R. pachydermis and R. griseovelutina Orihara. However, R. vittatispora, originally described from Australia has a white to pale grayish to buff pileus staining greenish blue or indigo blue in some patches on the surface and shorter and narrower basidiospores measuring 9–12(–13) × 4–5.5(−6) μm (Lebel et al. 2012). Rossbeevera pachydermis, originally described from New Zealand, has a restricted distribution to that country and differs from the new species in having large basidiomata (up to 50 mm broad), relatively smaller basidiospores measuring 11–14 × 8–10 µm, and is mainly associated with Nothofagus (Lebel et al. 2012). The East Asian R. griseovelutina is distinctive on account of its velvety basidiomata, abundant hymenial cystidia, trichodermal elements in the pileus, and relatively longer basidiospores 14.4–31.9 × 6.7–10.4 μm (Lebel et al. 2012, Orihara et al. 2012). Phylogenetically, R. paracyanea, originally described from Japan, is a close sister species to R. griseobrunnea with moderate support value (68% ML BS, Fig. 1), but significantly differs from the latter species in having white to grayish basidiomata when young, becoming blue-gray to dark gray with age, an off-white hymenophore when young that turns indigo blue very quickly and strongly when touched or exposed to air, relatively narrower basidiospores (14–19.3 × 6.0–9.2 μm), and it occurs with Quercus gilva Blume and Castanopsis cuspidata Schottky (Orihara et al. 2016).

Besides the comparisons with the closely related species of Rossbeevera, two known Chinese species, R. bispora and R. yunnanensis can be compared with R. griseobrunnea. Both R. griseobrunnea and R. bispora share 2-spored basidia, brown to dark brown hymenophore at maturity, and are putatively associated with broad-leaved trees. However, R. bispora can be differentiated from R. griseobrunnea by the deep bluing reaction of the pileus and hymenophore when bruised or exposed to air (Zhang and Yu 1989) and it is also a phylogenetically distinct species (Fig. 1). Rossbeevera yunnanensis, known as the earliest divergence lineage within Rossbeevera, is distinguished from R. griseobrunnea in having a very thin, whitish pileus which becomes blue-green when injured and a reddish brown to blackish brown hymenophore at maturity (Orihara et al. 2012, Orihara 2018). Apart from China, R. yunnanensis is known also from Japan which is about 3150 km from the type locality (Chuxiong, Yunnan Province, China vs Hiroshima Prefecture, Japan) (Orihara 2018), suggesting that the species has a wide geographic distribution.

Key to the taxa Rossbeevera known from Northern Hemisphere (China, Japan and Singapore/Malaysia) and Southern Hemisphere (Australia and New Zealand)

1 Geographical distribution- Southern Hemisphere (Australasia) 2
Geographical distribution- Northern Hemisphere (Asia) 4
2 Distributed in Australia, basidiospores within 9–15 × 3–6 μm 3
Distributed in New Zealand, basidiospores 11–14 × 8–10 μm, associated with mainly Nothofagus spp., no grayish tints on the surface R. pachydermis
3 Basidiospores 12–14 × 3–4.5 μm, basidiomata turn deep blue on bruising, restricted to western Australia R. westraliensis
Basidiospores 9–12 × 4–5.5 μm, basidiomata turn blue to deep blue in some patches, widespread in eastern Australia R. vittatispora
4 Distributed in East Asia 5
Distributed in Southeast Asia (Singapore/Malaysia), (13–)15–17 × (7–)8–9 μm, with Q values = 1.76–2.05 R. mucosa
5 Distributed in both Japan and China R. yunnanensis
Distributed either in Japan or China 6
6 Basidia constantly 1 or 2-spored, either dark/strong or partial bluing pileus and hymenophore 7
Basidia 2-, 3-and 4-spored, bluing pileus and hymenophore 8
7 Pileus and hymenophore not bluing or partially pale bluing when injured or exposed to air, turn rusty brown to dark brown after exposure to air for a long time, found in China R. griseobrunnea
Pileus and hymenophore bluing, basidiospores 15–21 × 10–12 μm, found in China R. bispora
8 Strong bluing reaction, basidiospores mean 5–18 × 6.5–9.4 μm 9
Strong bluing reaction, velvety pileus, cystidia present, basidiospores mean >22.2 × 8.7 μm, found in Japan R. griseovelutina
9 Hilar appendages (HA) 1.6–3.2 μm, basidiospores Qm = 1.9, found in Japan R. eucyanea
Hilar appendages 2.1–5.9 μm, basidiospores Qm = 2.1–2.4 10
10 Hilar appendages 2.1–4.5 μm, basidiospores 14–19.3 × 6.9–9.2 μm (mean 16.7 × 8 μm), found in Japan R. paracyanea
Hilar appendages 2.4–5.9 μm, basidiospores 13.4–18.3 × 5.8–7.3 μm (mean 15.8 × 6.5), found in Japan R. cryptocyanea

Acknowledgements

This work was supported by the 11th Special Fund of the China Postdoctoral Science Foundation (No. 2018T110854), the GDAS’ Special Project of Science and Technology Development (2019GDASYL-0104011), the China Postdoctoral Science Foundation (No. 2017M610514, 61st phase) and the NSFC Research Fund for International Young Scientists (No. 31750110476) to the first author (MIH). Thanks to Mr. Xishen Liang (Guangdong Institute of Microbiology, Guangzhou, China) for conducting partial molecular experiment. Mr. Ting Li, Mr. Huang Hao, Mr. Jun-ping Zhou and Yong He (Guangdong Institute of Microbiology, Guangzhou, China) are acknowledged for providing the voucher specimens. The authors also thank the reviewers for the suggestions and corrections to improve this work.

References

  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
  • Hosen MI, Feng B, Wu G, Zhu XT, Li YC, Yang ZL (2013) Borofutus, a new genus of Boletaceae from tropical Asia: phylogeny, morphology and taxonomy. Fungal Diversity 58: 215–226. https://doi.org/10.1007/s13225-012-0211-8
  • Hosen MI, Zhong XJ, Li T, Zhang M, Li TH (2018) Exploration of boletes from Xiangtoushan National Nature Reserve, Guangdong Province, China. Open Access Journal of Mycology & Mycological Sciences 1: 000101.
  • Katoh K, Kuma K, Toh H, Miyata T (2005) MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research 33: 511–518. https://doi.org/10.1093/nar/gki198
  • Lebel T, Orihara T, Maekawa N (2012) The sequestrate genus Rossbeevera T. Lebel & Orihara gen. nov. (Boletaceae) from Australasia and Japan: new species and new combinations. Fungal Diversity 52: 49–71. https://doi.org/10.1007/s13225-011-0109-x
  • Orihara T (2018) First report of a rare sequestrate fungus, Rossbeevera yunnanensis (Boletaceae, Boletales) from Japan. Truffology 1: 5–8.
  • Orihara T, Lebel T, Ge ZW, Smith ME, Maekawa N (2016) Evolutionary history of the sequestrate genus Rossbeevera (Boletaceae) reveals a new genus Turmalinea and highlights the utility of ITS minisatellite-like insertions for molecular identification. Persoonia 37: 173–198. https://doi.org/10.3767/003158516X691212
  • Orihara T, Smith ME, Ge ZW, Maekawa N (2012) Rossbeevera yunnanensis (Boletaceae, Boletales), a new sequestrate species from southern China. Mycotaxon 120: 139–147. https://doi.org/10.5248/120.139
  • Rehner SA, Buckley EP (2005) A Beauveria phylogeny inferred from nuclear ITS and EF1-α sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97: 84–98.
  • Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990
  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR Protocols: a guide to methods and applications. Academic Press, San Diego, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
  • Wu G, Feng B, Xu J, Zhu XT, Li YC, Zeng NK, Hosen MI, Yang ZL (2014) Molecular phylogenetic analyses redefine seven major clades and reveal 25 new generic lineages in the fungal family Boletaceae. Fungal Diversity 69: 93–115. https://doi.org/10.1007/s13225-014-0283-8
  • Zhang BC, Yu YN (1989) Chamonixia bispora sp. nov. (Boletales) from China. Mycotaxon 35: 277–281.