An Unexpectedly High Number of New Sutorius (Boletaceae) Species From Northern and Northeastern Thailand

Sutorius is a poroid genus in Boletaceae that typically has chocolate brown to reddish brown or purplish brown basidiomata with a finely scaly stipe and produces a reddish brown spore deposit. During the survey on diversity of boletes in Northern and Northeastern Thailand, several Sutorius collections were obtained. Combined evidence from morphology and phylogenetic analyses of a combined three-gene data set (atp6, tef1 and rpb2) of the Sutorius collections along with selected Boletaceae in the Pulveroboletus group indicated that Thai collections represent seven new Sutorius species. The analyses also indicated that Tylopilus maculatoides belongs in Sutorius. Therefore, the transfer of T. maculatoides to Sutorius is proposed. Full descriptions and illustrations of the seven new species and S. maculatoides are presented in this study. With the seven new species and the new combination, eight of the eleven described Sutorius species are known to occur in Northern and Northeastern Thailand, whereas only one species is known from each of two continents, the Americas and Australia.


INTRODUCTION
. Typical characteristics of Sutorius are stipitate-pileate basidiomata with poroid hymenophore, chocolate to reddish brown or purplish brown basidiomata with finely scaly stipe, and a reddish brown spore deposit. The original publication of Sutorius also reported two Sutorius specimens originating from Chiang Mai Province, in Northern Thailand. One of them was morphologically identified as S. eximius, whereas another was phylogenetically recognized as a different species from S. eximius and S. australiensis. However, the latter was not described as a new species at that time (Halling et al., 2012). In 2016, Wu et al., recombined several bolete species (e.g., Boletus brunneissimus W.F. Chiu; B. hainanensis T.H. Li and M. Zang; B. luridiformis Rostk.; B. obscureumbrinus Hongo; B. tomentulosus M. Zang W.P. Liu and M.R. Hu) into the genus Sutorius based on their phylogenetic inferences. They also described several new Sutorius species from China (e.g., S. ferrugineus G. Wu, F. Li and Zhu L. Yang; S. ferrugineus G. Wu, F. Li and Zhu L. Yang; S. rubriporus G. Wu and Zhu L. Yang). Later, Chai et al. (2019) restudied the phylogenetic relationships of several Boletaceae specimens from subtropical and tropical China, reinstated the genus Neoboletus Gelardi, Simonini and Vizzini and recombined all Sutorius species described in Wu et al. (2016) to be into this latter. Chai et al. (2019) also newly described a third species of Sutorius, Sutorius subrufus N.K. Zeng, H. Chai and S. Jiang from Southern China. Moreover, the phylogeny in that study suggested an additional five undescribed Sutorius species clustering in 10 independent linages (three described species included). That inference also indicated the exemplars identified as S. eximius, originating from several different countries (China, Costa Rica, Indonesia, and United States), were in a polytomic lineage (Chai et al., 2019). So far, the distribution of Sutorius has been recorded to occur in several regions including the Americas, Africa, Asia, and Australia (Singer, 1947;Smith and Thiers, 1971;Fulgenzi et al., 2007;Chandrasrikul et al., 2011;Halling et al., 2012;Chai et al., 2019). At present, only three species have been placed in the genus (Halling et al., 2012;Chai et al., 2019).
In Thailand, there were a few reports of Sutorius species, originally identified as Tylopilus eximius (Peck) Singer (current name = S. eximius) (Chantorn et al., 2007;Chandrasrikul et al., 2008;Vadthanarat, 2009;Vadthanarat et al., 2010). However, the identifications in those reports were based on morphological evidence only, and further phylogenetic studies are needed to confirm those identifications. One Asian species of Boletaceae, Tylopilus maculatoides E. Horak, also has similar morphological characters as Sutorius. That name was proposed for Boletus maculatus Corner non-Raddi based on morphological evidence (Horak, 2011). However, the species has not been included in a phylogenetic context until now (see below).
In this study, morphological and phylogenetic analyses of several Sutorius collections, including T. maculatoides collections, inferred seven new Sutorius species among the Thai collections. Moreover, the phylogram also showed that T. maculatoides clustered within the Sutorius clade with high support. Consequently, the new combination Sutorius maculatoides is proposed. Their phylogenetic affinities and reports of other Sutorius species from Thailand or elsewhere are critically discussed.

Specimen Collection
Fresh basidiomata of Sutorius were collected in Chiang Mai Province in the North and Ubon Ratchathani Province, Northeastern Thailand, in 2012 to 2018. They were photographed in the field and then wrapped in aluminum foil for later description in the laboratory. The specimens were then dried after the morphological description in an electric drier at 45 • C-50 • C. Examined specimens were deposited at Department of National Parks, Wildlife and Plant Conservation, Bangkok, Thailand (BKF), Chiang Mai University, Chiang Mai, Thailand (CMUB), and Mae Fah Luang University, Chiang Rai, Thailand (MFLU) with duplicates in Botanic Garden Meise, Belgium (BR).

Morphological Studies
Macroscopic descriptions were made based on the detailed field notes and photographs of fresh basidiomata. Color codes are based on Kornerup and Wanscher (1978). Macrochemical reactions (color reactions) were observed using aqueous solutions of 10% KOH, 28-30% NH 4 OH, and Melzer reagent. Microscopic structures were observed from dried specimens and rehydrated in 5% potassium hydroxide or 1% ammoniacal Congo red. For dimensions of microscopic features, a minimum of 50 basidiospores (20 for other structures) were randomly measured at 1,000 × using a calibrated ocular micrometer on an Olympus CX31 compound microscope. The notations "[n/m/p]" show the number of basidiospores "n" measured from the number of basidiomata "m" of the number of collections "p." Dimensions of microscopic structure are presented in the following (a-) b-c-d (-e), in which "c" contains an average, "b" is the 5 th percentile, "d" is the 95 th percentile, and extreme values "a" and "e" are shown in parentheses. Q is the length/width ratio. Sections of the pileipellis were cut radially, perpendicularly to the surface halfway between the disc and margin of pileus. Sections of stipitipellis or the squamules on the stipe were taken halfway along the stipe length (Li et al., 2011;Hosen et al., 2013;Li et al., 2014;Zhu et al., 2015). All microscopic features were drawn free hand using an Olympus Camera Lucida model U-DA.

DNA Extraction, Polymerase Chain Reaction Amplification, and DNA Sequencing
Genomic DNA was extracted from fresh tissue preserved in CTAB or approximately 10-15 mg of dried tissue using a CTAB isolation procedure adapted from Doyle and Doyle (1990). Parts of three protein-coding genes, atp6, tef 1, and rpb2, were amplified by polymerase chain reaction (PCR) and sequenced. ITS-5.8 region of the nuclear ribosomal DNA was not sequenced because in Boletaceae, ITS commonly shows high levels of intraindividual polymorphisms for indels, thus mostly preventing straightforward phylogenetic analysis and moreover often making cloning necessary to obtain a sequence. The selected protein-coding genes have shown multiple times their usefulness for investigating phylogenetic relationships, at both infrageneric and suprageneric levels (e.g., Wu et al., 2016;Vadthanarat et al., 2019). For the amplification of atp6, ATP6-1M40F and ATP6-2M primers were used, with following the protocol and PCR program in Raspé et al. (2016). The primers EF1-983F and EF1-2218R (Rehner and Buckley, 2005) were used to amplify tef 1 gene, and bRPB2-6F and bRPB2-7.1R primers (Matheny, 2005) were used to amplify rpb2 gene. PCR products were purified by adding 1 U of exonuclease I and 0.5 U FastAP alkaline phosphatase (Thermo Scientific, St. Leon-Rot, Germany) and incubating at 37 • C for 1 h, followed by inactivation at 80 • C for 15 min. Sequencing was performed by Macrogen Inc. (Korea and the Netherlands) with PCR primers, except for atp6, for which universal primers M13F-pUC (-40) and M13F(-20) were used; for tef 1, additional sequencing was performed with the two internal primers, EF1-1577F and EF1-1567R (Rehner and Buckley, 2005).
Before combining the data partitions, topological incongruence between the datasets was assessed using maximum likelihood (ML) and Bayesian inference (BI) separately on each of the four character sets, atp6, tef 1 exons, rpb2 exons, and the three introns of tef 1 + the intron of rpb2. Paired trees were examined for conflicts involving only nodes with ML bootstrap (BS) > 75% and Bayesian posterior probabilities (PPs) > 0.95 (Mason-Gamer and Kellogg, 1996;Lutzoni et al., 2004;Reeb et al., 2004). A conflict was assumed to be significant if two different relationships for the same set of taxa (one being monophyletic and the other non-monophyletic) were observed in rival trees. For ML, phylogenetic inference was performed using RAxML (Stamatakis, 2006) on the CIPRES web portal (RAxML-HPC2 on XSEDE; Miller et al., 2009), using the GTRCAT model with 25 categories. Three Butyriboletus species were used as outgroup based on the phylogeny in Vadthanarat et al. (2019). Statistical support of clades was obtained with 1,000 rapid BS replicates. For BI, the best-fit model of substitution among those implementable in MrBayes was estimated separately for each character set using jModeltest (Darriba et al., 2012) on the CIPRES portal, based on the Bayesian information criterion. The selected models were HKY + I + G for atp6, SYM + I + G for tef 1 exons, and K80 + I + G for rpb2 exons and intron partition. The Bayesian analysis was performed with MrBayes 3.2.6 software for Windows (Ronquist et al., 2012).
Then, phylogenetic inference of a combined, partitioned dataset was performed, using ML under the same model mentioned above. The phylogenetic tree was inferred from a single analysis with four character sets: atp6, tef 1 exons, rpb2 exons and the three introns of tef 1 + the intron of rpb2. The same combined dataset was also analyzed by BI, based on the best-fit model mentioned previously. The two runs of five chains were run for 1,000,000 generations and sampled every 200 generations. At the end of the run, the average deviation of split frequencies was 0.008306, and the potential scale reduction factor values of all parameters were close to 1. The burn-in phase (25%) was estimated by checking the stationarity in the plot generated by the sump command. A total of 7,396 stationary trees were used to reconstruct a 50% majority rule consensus tree and calculate the Bayesian PPs.

Phylogenetic Analyses
Fifty-four sequences from Sutorius collections corresponding to 11 phylogenetic species were newly generated and deposited in GenBank. The combined alignment of the three loci studied contained 190 sequences (32 for atp6, 80 for tef 1, 78 for rpb2) from 80 specimens ( Table 1) and was 2,681 characters long (gaps included) (TreeBase no. 27286).
The single-gene trees were very similar in topology (Supplementary Figures 1-4), except for one supported conflict. In the tef 1 exons tree (Supplementary Figure 2), Sutorius pachypus formed a clade sister to S. australiensis with high support (BS = 75%, PP = 0.92). This is in conflict with the topology of the tree based on the introns character set (Supplementary Figure 4), in which S. pachypus formed a clade sister to Sutorius rubinus (BS = 87%, PP = 0.97). Partitioned analyses of a dataset combining atp6, tef 1 exons, and rpb2 exons sequences were also performed (Supplementary Figure 5). Its tree topology showed no conflict to the single partitions of atp6, tef 1 exons, and rpb2 exons trees. Consequently, the combination of four-partition analyses was used to infer the relationship between species in this study.
Distribution: Malaysia, Singapore, and Thailand.  (2011) gave the new epithet maculatoides. He also transferred the species to Tylopilus. Here, we present phylogenetic evidence supporting placement in Sutorius. In our phylogenetic inference (Figure 1), all S. maculatoides collections clustered together in a wellsupported terminal clade (clade 13), within the Sutorius clade. The macromorphological and micromorphological characters of S. maculatoides also support its position in Sutorius. Therefore, the new combination S. maculatoides is proposed for T. maculatoides. In our phylogeny, among the S. maculatoides specimens, the Thai collections (OR0626 and OR0758) were molecularly slightly divergent from the Malaysian specimen (WAT25793). Being the amount of divergence is lower than the one observed between sister species, we consider those differences in the range of intraspecific genetic variability and compatible with conspecific, geographically distant populations. Sutorius maculatoides is characterized by the following characteristics: dark lilac pileus with lilac gray pores at first, with age becoming reddish brown and reddish pale brown, respectively; presence of cheilocystidia and infrequent pleurocystidia; pileipellis a sub-ixotrichoderm composed of moderately interwoven hyphae, with fusiform terminal cells that slightly taper at the apex.
In this study, S. maculatoides is redescribed based on specimens from Thailand (OR0626 and OR0758) and Malaysia (WAT25793), which were compared to the description of T. maculatoides E. Horak (Horak, 2011). Microcharacteristics of Watling's specimen were similar to Horak's description including size and shape of basidiospores, basidia, and pileipellis. However, the cheilocystidia in Watling's specimen (15-24 × 6-8 µm) are shorter than in Horak's description (35-45 × 6-11 µm), but Horak did not separately describe the length of cheilocystidia and pleurocystidia. He also did not mention the frequency of both types of cystidia. By comparison, microscopic characters between Watling's specimen and the Thai specimens were almost identical. However, the minimum value of the length of basidiospores in Watling's specimen (11.4-13.5 × 4.1-4.9 µm) is higher than for the Thai specimens (7.8-14.1 × 3-5.6 µm). The lower minimum value of length of basidiospores in Thai specimens could be explained by small quantitative differences between geographically distant populations or the less mature stage of some basidiomata from Thai collections. Moreover, according to our observations on Sutorius, this genus usually shows high within-species variation in spore size, which was also noted originally by Halling et al. (2012). Etymology: from Latin mucosus, means mucus, referring to a strongly gelatinized pileipellis.
Distribution: Chiang Mai Province, Northern Thailand. Notes: S. mucosus description is based on a single collection (OR0851); however, the strongly gelatinized pileipellis is a strikingly unique character, which, together with the phylogenetic evidence, which resolved this specimen as an isolated branch, within the Sutorius lineage, supports our interpretation of OR0851 as a new, distinct species. Other important characters that allow differentiating S. mucosus are the fusiform to utriform pileipellis terminal cells with rounded to subacute apex and the lack of cheilocystidia and pleurocystidia.
Phylogenetically, S. mucosus is resolved as an isolated branch, within a clade comprising three other distinct Sutorius species, including S. rubinus (clade 9), S. pachypus (clade 10), and S. australiensis (clade 11). However, they are morphologically different, and none of them has a strong gelatinized pileipellis, which is the most striking diagnostic character of S. mucosus. The other differences are as follows: S. pachypus has, on average, a wider stipe (2.5-4 cm wide) and paler stipe surface, presence of cheilocystidia and pleurocystidia, and palisadodermal pileipellis composed of subcylindrical terminal cells with rounded to subacute apex. S. australiensis has a trichoderm pileipellis with elongated to cylindrical hyphae with obtuse apex terminal cells. S. rubinus also has a trichoderm pileipellis but with different shapes of terminal cells, which are fusiform to broadly Etymology: from Latin obscurus and pellis, referring to the dark pileipellis.
Macrochemical reactions: KOH: dull yellow on pileus; yellow or pale yellow on pileus context, stipe, stipe context, and hymenophore. NH 4 OH: yellowish with only slight violet aura on pileus; yellowish on stipe and pileus context; negative on hymenophore and stipe context.
Habitat: On soil, gregarious to fasciculate in hill forest dominated by Castanopsis spp. Lithocarpus spp. mixed with some Dipterocarpus spp. and regularly subject to fire.
Distribution: Chiang Mai Province, Northern Thailand. Notes: Based on a single collection (OR0949), S. obscuripellis is characterized by the following combination of characteristics: basidiomata small-to medium-sized; pale violet-gray pores color; lack of pleurocystidia; Pileipellis an intricate trichoderm composed of brown to dark hyphae with cylindrical terminal cells with subacute to rounded apex, showing scattered small parietal brownish yellow to reddish pale to dark brown encrustations. Phylogenetically, S. obscuripellis formed a clade (clade 8) sister to Sutorius ubonensis (clade 7) and S. subrufus (clade 6). However, the latter two species are morphologically different from S. obscuripellis as follows: S. ubonensis has larger basidiomata, darker pores, and basidiomata especially when young, pleurocystidia, and a slightly gelatinized tomentose pileipellis, and has so far only been found in dry dipterocarp forest in Ubon Ratchathani Province, in northeastern Thailand. S. subrufus has larger basidiomata, a stipe surface and context turning more reddish when injured, presence of pleurocystidia, and a trichoderm pileipellis composed of cylindrical hyphae and clavate or subclavate terminal cells with obtuse apex, colorless to yellowish in KOH (Chai et al., 2019), whereas S. obscuripellis has the pileipellis composed of brown to dark brown hyphae, colorless to brownish yellow to pale brown in KOH. Etymology: from Greek means "thick" referring to the wide stipe.
Macrochemical reactions: KOH: yellowish brown on pileus and stipe; NH 4 OH: yellowish brown with greenish to pinkish aura on pileus; orange with blue green aura on stipe.
Habitat  Notes: S. pachypus is characterized by the following combination of characteristics: medium-sized basidiomata; wide cylindrical stipe with granulose squamules on the stipe surface that mostly are pale violet gray to violet brown at places; pileipellis is a palisadoderm to trichoderm, subcylindrical terminal cells with rounded to subacute apex; presence of cheilocystidia and pleurocystidia. The holotype (OR0411) has oblong to ellipsoid basidiospores because it is immature. However, a mature specimen (TWO1171) has the typical shape of basidiospores, which are narrowly ellipsoid to subcylindrical with suprahilar depression. The specimen (TWO1171) was originally morphologically identified as S. eximius (Halling et al., 2012), but our more detailed examination and phylogenetic analyses indicate a new species, S. pachypus. Phylogenetically, S. pachypus formed a clade (clade 10) closely related to S. rubinus (clade 9), S. australiensis (clade 11), and S. mucosus (clade 12). However, all of those differ from S. pachypus mainly in having a narrower stipe with darker granulose squamules. The other differences are as follows: S. rubinus has redder basidiomata, lacks pleurocystidia, and has a trichoderm pileipellis with fusiform to broadly fusiform terminal cells with acuminate apex. S. australiensis has darker violet-brown pores (when young), and it has been found so far only in Australia. S. mucosus lacks both cheilocystidia and pleurocystidia and has a waxy to subviscid pileus, an ixotrichoderm pileipellis with fusiform to utriform terminal cells with rounded to tapering apex. Etymology: From Greek pseudo-, false, and Tylopilus refers to the macromorphological similarity to the genus Tylopilus.
Macrochemical reactions: KOH: yellowish then brownish on the cap, stipe, and pileus context; yellowish or greenish on stipe context; yellowish on hymenium. NH 4 OH: yellowish red on the pileus; yellowish to brownish on pileus context, stipe, stipe context, and hymenium.
Habitat: Solitary or fasciculate on soil, in dipterocarp forest dominated by D. obtusifolius, D. tuberculatus, Sh. obtusa, and Sh. siamensis and a few Hopea sp., as well as Castanopsis spp.
Distribution: Chiang Mai Province, Northern Thailand. Notes: Sutorius pseudotylopilus is characterized by the following combination of characteristics: medium-sized basidiomata, purplish to reddish brown basidiomata when young; lack of pleurocystidia; pileipellis constituted by an intricate to flattened trichoderm with cylindrical, slightly curly terminal cells with rounded apex. Phylogenetically, the three S. pseudotylopilus specimens formed a clade (clade 4) sister to three undescribed Sutorius species: clade 1 contains three specimens from China (HKAS59657, HKAS52672, and HKAS56291); Sutorius clade 2 contains another specimen from China (HKAS56291), and Sutorius clade 3 contains one specimen (TWO986) from Costa Rica in Central America. The closest described species is S. eximius (American species) in clade 5. However, S. eximius morphologically differs from S. pseudotylopilus by the following characteristics: stipe context unchanging when injured; presence of pleurocystidia (Singer, 1947;Smith and Thiers, 1971;Halling et al., 2012). Moreover, based on our latest phylogenetics analyses with more Sutorius exemplars, we hypothesize that S. eximius is restricted to North America (clade 5). Etymology: From Latin rubinus, red, referring to basidiomata color.
Macrochemical reactions: KOH: dark brown to black on the stipe, reddish on the hymenophore, negative on the pileus, pileus context, and stipe context.
Distribution: Chiang Mai Province, Northern Thailand Notes: S. rubinus is characterized by the following combination of characteristics: medium-sized basidiomata, dark reddish brown to reddish brown; lack of pleurocystidia; pileipellis a trichoderm with fusiform to broadly fusiform terminal cells with acuminate apex.
Phylogenetically, all S. rubinus specimens formed a clade (clade 9) close to three Sutorius species including S. pachypus (clade 10), S. australiensis (clade 11), and S. mucosus (clade 12). Nevertheless, they are morphologically different from S. rubinus as follows: S. pachypus has on average wider (2.5-4 cm wide) stipe and has pleurocystidia, and the pileipellis is a palisadoderm composed of subcylindrical with rounded to subacute apex. S. australiensis has dark violet brown pores when young, a trichoderm pileipellis composed of elongated to cylindrical elements with obtuse apex and is found only in Australia. S. mucosus has a waxy to subviscid pileus with an ixotrichoderm forming the pileipellis composed of gelatinized hyphae, with fusiform to utriform terminal cells with rounded to subacute apex.
Additional collections examined: THAILAND: Chiang Mai Province: Mae On District, Ban Huay Keaw community forest, Etymology: ubonensis refers to Ubon Ratchathani Province where the species was found.
Notes: S. ubonensis is characterized by the combination of following characteristics: medium sized basidiomata, purplish gray when young becoming purplish to reddish brown with age; unchanged context; pileipellis a tomentum to slightly gelatinized tomentum, with cylindrical terminal elements with rounded apex; found in dipterocarp forest in Northeastern Thailand. Morphologically, S. ubonensis is superficially similar to S. eximius in macro-characters especially when young. Both species are also similar in some microscopic characters including the presence and shape of cheilocystidia, pleurocystidia, and caulocystidia, as well as the shape of elements on pileipellis. However, they are different in pileipellis structure, with S. eximius having a trichoderm, whereas S. ubonensis has a tomentum to slightly gelatinized tomentum. The two species also occur on different continents, with S. ubonensis being found in Ubon Ratchathani Province, Northeastern Thailand, Southeast Asia, whereas S. eximius is found in North America (Singer, 1947;Smith and Thiers, 1971;Halling et al., 2012).
Phylogenetically, S. ubonensis (clade 7) was clusters in a poorly supported, long-branch, sister to S. obscuripellis (clade 8) from Chiang Mai Province, Thailand, and S. subrufus (clade 6) from China. However, these two species differ from S. ubonensis as follows: S. obscuripellis has smaller basidiomata and is also paler, especially when young; lacks pleurocystidia; and has a trichoderm pileipellis composed of slightly dark to dark hyphae, with cylindrical terminal cells with subacute to rounded apex. So far, S. obscuripellis has been found only in Chiang Mai Province. S. subrufus also has paler basidiomata; paler pores, especially when young, which are pale brown to brown to pale reddish brown; stipe surface and context turn reddish when injured; a trichoderm pileipellis, composed of rather vertically arranged, with clavate or subclavate terminal cells, with obtuse apex; found in the forests dominated by Fagaceae trees, including Lithocarpus spp. (Chai et al., 2019). So far, S. ubonensis is the only Sutorius species found in Ubon Ratchathani Province, Northeastern Thailand. It occurs in dry dipterocarp forest at the lowest elevation (about 150-175 m) compared to the other Sutorius species.
Additional Etymology: Named after Else C. Vellinga, who collected the type specimen.
Taste not distinctive. Spore print not observed.
for the permit number 0907.4/4769 granted by the Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment for collecting in Doi Suthep-Pui National Park. The publication of this work was financially supported by Mae Fah Luang University.