Type studies of Rossbeeverabispora, and a new species of Rossbeevera from south China

Abstract The type of Rossbeeverabispora 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.


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, 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 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.

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 depos-ited 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 H 2 O, 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, Q m denotes the average of n basidiospores, and SD is their standard deviation. Results are presented as Q m ± SD. For describing the species, we used the taxonomic terminology pileus for 'peridium', hymenophore for 'gleba' and stipe for 'columella'.
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.
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  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.
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.
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.   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.

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 ar-rangement 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), Q m = 1.66 ± 0.11], which is similar to that of the original description. The Q values [(1.63-1.83(-1.90), Q m = 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. 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 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)