Rubinosporusauriporus gen. et sp. nov. (Boletaceae: Xerocomoideae) from Tropical Forests of Thailand, Producing Unusual Dark Ruby Spore Deposits

Rubinosporus, a new bolete genus from tropical forests of Thailand is introduced with R. auriporus as the type species. The genus is unique among Xerocomoideae in producing dark ruby spore deposits. It can be differentiated from all other Boletaceae genera by the following combination of characters: pileus surface evenly covered with matted tomentum; stipe surface with evenly scattered minute squamules; golden yellow tubular hymenophore, which is relatively thin especially when young; unchanging surfaces and context when bruised or cut; smooth, broadly ellipsoid basidiospores; and dark ruby spore deposits. The Boletaceae-wide and Xerocomoideae-wide phylogenetic analyses based on four-gene data sets (atp6, cox3, rpb2, and tef1) support Rubinosporus as monophyletic and places it in Boletaceae subfamily Xerocomoideae. Full descriptions and illustrations of the new genus and species are presented.


Introduction
The family Boletaceae has been widely studied for over one hundred years. The former traditional taxonomy was based only on morphological characters. However, since molecular techniques and phylogenetic analyses have been developed and used as an advanced tool for the modern concepts in systematics and taxonomy, many genera, and species in Boletaceae have been recognized and described as new, e.g., [1][2][3]. In the last five years only, nine new Boletaceae genera have been described worldwide, namely  [4][5][6][7][8][9][10][11][12]. Five of those genera were described from tropical to subtropical Asia, where high fungal diversity has been reported, but yet, remains poorly known to science, e.g., [13,14].
Based on the current multiple gene phylogenies, the Boletaceae are classified into six sub-families and one phylogenetically unsupported group [2,3]. Xerocomoideae is one of the six sub-families, which consists of nine genera namely Alessioporus Gelardi [2,3]. Two additional genera, Corneroboletus and Sinoboletus, were also described in the subfamily but were later synonymized with Hemileccinum

Specimens Collecting
The specimens were collected during the rainy season, from May to June, between 2015 and 2017, in Chiang Mai Province, northern Thailand. The specimens were wrapped in aluminum foil and taken to the laboratory for morphological description. After the description of macroscopic characters, the specimens were dried in an electric drier at 45-50 • C for 24 h or until dried properly. Then, they were deposited in the following herbaria Chiang Mai University (CMUB), and Meise Botanic Garden (BR) [18].

Morphological Study
The macroscopic descriptions were made based on detailed field notes and photos of fresh basidiomata taken in the habitat and the laboratory. Color codes were given based on a Methuen Handbook of Colour [19]. Chemical solutions including 10% potassium hydroxide (KOH) and 28-30% ammonium hydroxide (NH 4 OH), were used to determine the chemical reactions (color reactions) of the pileus, pileus context, stipe, stipe context, and hymenophore. For the microscopical study, the dried specimens were observed using 5% KOH, NH 4 OH, Melzer's reagent, or stained with 1% ammoniacal Congo red. A minimum of 50 basidiospores, 20 basidia, and 20 cystidia were randomly measured under a Nikon Eclipse Ni microscope using the NIS-Elements D software. The notation '[m/n/p]' represents the number of basidiospores "m" measured from "n" basidiomata of "p" collections. Dimensions of microscopic structures are presented in the following format: (a-)b-c-d(-e), in which "c" represents the average, "b" the 5th percentile, "d" the 95th percentile, "a" the minimum, and "e" the maximum. Q, the length/width ratio, is presented in the same format. Section of the pileus surface was radially and perpendicularly cut to the surface at a point halfway between the center and margin of the pileus. Sections of stipitipellis were taken from halfway up the stipe and longitudinally cut perpendicularly to the surface. All microscopic features were drawn by freehand using an Olympus Camera Lucida model U−DA fitted to Olympus CX31 compound microscope. For scanning electron microscopy (SEM), a spore print was mounted onto an SEM stub with double-sided carbon tape. The sample was coated with gold, then examined and photographed with a JEOL JSM-5910 LV SEM (JEOL, Tokyo, Japan).

Alignment and Phylogeny Inference
The two reads of newly generated sequences were assembled in GENEIOUS Pro v. 6.0.6 (Biomatters). A Boletaceae-wide sequence dataset, including selected sequences representative of the whole family, downloaded from GenBank, was aligned using MAFFT [24] on the server accessed at http://mafft.cbrc.jp/alignment/server/ (accessed on 19 December 2021). For this dataset, the introns in rpb2 and tef 1 were removed based on the amino acid sequence of previously published sequences. Maximum likelihood (ML) phylogenetic inference was performed using RAxML on the CIPRES web portal (RAxML-HPC2 on XSEDE) [25,26]. The phylogenetic tree was inferred by a single analysis with four partitions (one for each gene), using the general time reversible computerized adaptive testing (GTRCAT) model with 25 categories. The outgroup consisted of two Buchwaldoboletus and seven Chalciporus species from sub-family Chalciporoideae, based on previous phylogenies e.g., [1][2][3]11]. Statistical support of clades was obtained with 1000 rapid bootstrap replicates. For Bayesian Inference (BI), the best-fit model of substitution among those implementable in MrBayes was estimated separately for each region using jModeltest [27] on the CIPRES portal, based on the Bayesian Information Criterion (BIC). The selected models were HKY + I + G for atp6, GTR+I+G for cox3 and tef1 exons, and K80 + I + G for rpb2 exons. Partitioned Bayesian analysis was performed with MrBayes 3.2.6 software for Windows [28]. Two runs of five chains were run for 11,000,000 generations and sampled every 1000 generations. The chain temperature was decreased to 0.02 to improve convergence. At the end of the run, the average deviation of split frequencies was 0.008614. A total of 8252 trees were used to construct a 50% majority rule consensus tree and calculate the Bayesian posterior probabilities (BPP).
For a subfamily Xerocomoideae-wide tree, all selected taxa in Xerocomoideae were aligned using the MAFFT online software (introns included). ML phylogenetic tree was inferred by a single analysis with five partitions (atp6, cox3, rpb2 exons, tef1 exons, and intron of rpb2 + introns of tef 1) (one for each gene), outgroup were four Butyriboletus species in Pulveroboletus group, using the same analytical software and model used for family Boletaceae-wide tree. For BI, the same analytical software for family Boletaceae-wide tree was used. However, the selected models were GTR+I+G for atp6, cox3, and intron of rpb2 + introns of tef 1, K80 + I + G for rpb2 exons, and SYM+I+G for tef 1 exons. Two runs of five chains were sampled every 200 generations and stopped after 800,000 generations. At the end of the run, the average deviation of split frequencies was 0.007928. A total of 2709 trees were used to construct a 50% majority rule consensus tree and calculate the BPPs.
Diagnosis-Distinguished from the other genera in Boletaceae by the following combination of characters: pileus surface even, with matted, cracked tomentum; stipe surface even, scattered with minute squamules, golden yellow tubular hymenophore which is relatively thin, especially when young; unchanging surfaces and context when touched or cut; smooth, broadly ellipsoid basidiospores; dark ruby spore deposit.    Description-Basidiomata stipitate-pileate with tubular hymenophore, medium-sized. Pileus hemispherical at first then convex to plano-convex or applanate in age; margin inflexed to deflexed, exact to slightly exceeding; surface even to subrugulose at places, dull, greyish red to pastel red to reddish brown, with greyish yellow, greyish orange to brownish orange to brown matted, cracked tomentum; context firm, off-white to yellowish white, unchanging when cut. Stipe central, terete, or sometimes slightly compressed, cylindrical or subcylindrical with slightly wider base; surface topography even, yellowish white to pinkish white at places, with scattered yellowish white to orange to light brown minute squamules, to bright yellow near the top; basal mycelium yellowish white; context solid, offwhite to yellowish white, unchanging when cut. Hymenophore tubulate, narrowly adnate, mostly segmentiform to subventricose. Tubes relatively thin, especially when young, golden yellow becoming orange-yellow, separable from the pileus context, unchanging when bruised. Pores topography subirregular, irregularly arranged, roundish to slightly angular composite pores; golden yellow at first, golden yellow to greyish yellow with irregularly reddish brown at places in age, unchanging when touched. Odor mild fungoid. Taste mild to slightly sweet. Spore print dark ruby in mass. Basidiospores broadly ellipsoid, thin-walled, smooth, yellowish to brownish hyaline in water, yellowish hyaline in KOH or NH 4 OH, yellowish to reddish in Melzer's reagent (weakly dextrinoid). Basidia 4-spored, clavate without basal clamp connection. Cheilocystidia clavate with rounded apex or fusiform to broadly fusiform or utriform, thin-walled, hyaline to yellowish hyaline in KOH or NH 4 OH. Pleurocystidia fusiform with narrower apex, thin-walled, hyaline to yellowish hyaline in KOH or NH 4 OH. Pileipellis a tomentum to intricate trichoderm, composed of moderately interwoven thin-walled hyphae; terminal cells cylindrical with obtuse apex, hyaline to yellowish at places in KOH. Stipitipellis a tomentum composed of loosely to moderately interwoven cylindrical hyphae, anastomosing at places, scattered with groups of rising cells to clusters of basidiole-like cells mixed with caulocystidia, and rarely with caulobasidia, hyaline to yellowish hyaline in KOH or NH 4 OH. Clamp connections were not seen in any tissue.

Rubinosporus auriporus
Macrochemical reactions: KOH, yellow to orange on cap, stipe, and hymenium; none or yellowish on pileus context and stipe context; NH 4 OH, yellow to orange to brown on cap, stipe and hymenophore; none or yellowish on pileus context, stipe context and hymenium.

Discussion
The new genus Rubinosporus is distinguished from other Boletaceae by a combination of striking characters, i.e., a golden yellow tubular hymenophore that is relatively thin especially when young, and dark ruby spore deposits. The character of golden yellow tubular hymenophore is also found in Aureoboletus, Alessioporus, and Pulchroboletus, which also belong to the subfamily Xerocomoideae. However, Aureoboletus species differ from Notes-In the new species, the hymenophoral cystidia contained greenish yellow (1A8) pigments when fresh specimens were observed in water under a compound microscope. However, the pigment was discolored when the cystidia were observed in KOH or NH 4 OH, or after treatment of the specimen with heat (drying at 45-50 • C).
A macro-morphologically similar species, Butyriboletus roseoflavus (Hai B. Li & Hai L. Wei) D. Arora & J.L. Frank originally described from China, has a similar color tone of basidiomata with a light pink, light purplish red to rose-red pileus; and lemon-yellow, oliveyellow or honey-yellow hymenophore. However, it can be differentiated from R. auriporus by having a yellower and reticulated stipe which is lemon-yellow or light yellow with almost entirely reticulate stipe or at least in lower part; yellower context which is lemonyellow and also variable staining reaction in response to bruising, bruising blue slowly or unchanging; bruising blue promptly hymenophore; subfusiform basidiospores; olive brown spore deposit; and the habitat in Pinus or mixed forests dominated by Pinus [52,53].

Discussion
The new genus Rubinosporus is distinguished from other Boletaceae by a combination of striking characters, i.e., a golden yellow tubular hymenophore that is relatively thin especially when young, and dark ruby spore deposits. The character of golden yellow tubular hymenophore is also found in Aureoboletus, Alessioporus, and Pulchroboletus, which also belong to the subfamily Xerocomoideae. However, Aureoboletus species differ from Rubinosporus in usually having a viscid pileus surface especially when moist, olive brown spore deposit, and subfusiform or oblong ovoid to subglobose basidiospores [3,16,31]. Alessioporus is clearly different by the reticulated stipe occasionally with a granular ring-like zone in the middle or lower half of the stipe; rapidly bluing hymenophore, stipe surface, and context when bruised or exposed; sub-ellipsoid to fusiform, ellipsoid to subcylindrical basidiospores; olive brown spore deposit; and distribution in Mediterranean Italy and subtropical USA [54,55]. Pulchroboletus differs by the stipe surface with scattered red to reddish brown punctae, occasionally with reticulum or longitudinal striations, and with a pseudo-annulus; hymenophore and context usually intensively staining blue when bruised or cut; ellipsoidal to ellipsoid-fusoid basidiospores; olive brown colored spore deposit; so far found in Mediterranean Europe and tropical to subtropical America [46,54,56].
The thin hymenophore is also present in Baorangia and Lanmaoa G. Wu & Zhu L. Yang, which both belong to the Pulveroboletus group. They have a very thin hymenophore, with a tube length 1/3 to 1/5 times the thickness of the pileus context. However, Baorangia and Lanmaoa differ from Rubinosporus by having yellow hymenium (not golden or bright yellow) that immediately turns light blue to greenish blue when touched; olive-brown spore deposit; and subfusiform to elongated subfusiform basidiospores [5,17,35].
Although the color of spore deposit in Rubinosporus is somewhat similar to the color tone in Austroboletus (Corner) Wolfe and Ionosporus Khmeln., Austroboletus has a rufous madder to chocolate to purplish vinaceous spore deposit which is browner than in Rubinosporus. Also, Austroboletus species produce basidiomata with pileipellis markedly exceeding pileus margin, embracing the stipe in young basidiomata, whitish to pinkish hymenium, and ornamented basidiospores [3,57,58]. Ionosporus has pale violet to reddish brown spore deposit. However, their basidiospores have an obvious reaction in potassium hydroxide solution, turning deep purple violet. The basidiospores also have granulose pitted surface under SEM. Moreover, Austroboletus and Ionosporus phylogenetically belong to different subfamilies, the Austroboletoideae and Leccinoideae, respectively [2,10]. The color of spore deposit is one of the important character used to differentiate mushroom genera, both in the Agaricales and Boletales. Several previous studies used this character to differentiate new genera. For example, Tylopilus eximius (Peck) Singer, which has a reddish-brown spore deposit, was separated from Tylopilus (having a pinkish spore deposit), and placed in a new genus, Sutorius [48]. Cacaoporus is distinguished from the most similar genus Sutorius by its dark brown spore deposit while the genus Sutorius has a reddish-brown spore deposit [11]. Moreover, they were all supported by the phylogenies.
In the Xerocomoideae-wide phylogeny obtained in this study, the monophyly of all genera was highly supported. However, no sequences of Alessioporus were added in the phylogeny because among the genes that were used to infer our phylogeny, only a partial tef1 sequence of A. ichnusanus was available in GenBank. However, in ITS and combined ITS+ LSU+tef 1 phylogenies of previous studies, Alessioporus was sister to Pulchroboletus [46,54,56]. In this study, Pulchroboletus was sister to Aureoboletus with high support, and distant from Rubinosporus. Moreover, Alessioporus is morphologically clearly different from Rubinosporus as discussed above. The relationship of Rubinosporus to the other genera within Xerocomoideae remains unclear. It formed a clade close to Hemileccinum with poor support. More species, genes, phylogenies are needed to reveal the sister relationship of Rubinosporus. In addition, the three genera Phylloporus, Hourangia, and Xerocomus formed a highly supported clade. Phylloporus formed a clade sister to Xerocomus, and both genera are sisters to Hourangia. The result is slightly different from Wu et al. [3] phylogeny (based on 28S, tef 1, rpb1, and rpb2), in which the three genera also formed a highly supported clade but Phylloporus was sister to Hourangia, not Xerocomus like in this study.
Most of the Boletaceae genera have been recognized as important ectomycorrhizal fungi in forest ecosystems [65,66]. Rubinosporus also presumably forms ectomycorrhizal relationships with either Dipterocarpaceae or Fagaceae, or both. These two tree families were dominant around the area where the genus was found. However, further research is needed to confirm the ectomycorrhizal host species of Rubinosporus.