New and noteworthy boletes from subtropical and tropical China

Abstract The morphology, ecology, and phylogenetic relationships of specimens of the family Boletaceae from subtropical and tropical China were investigated. Four species, Butyriboletushuangnianlaii, Lanmaoamacrocarpa, Neoboletusmultipunctatus, and Sutoriussubrufus, are new to science. Chalciporusradiatus and Caloboletusxiangtoushanensis are redescribed. Caloboletusguanyui is proposed to replace Boletusquercinus Hongo, an illegitimate later homonym. The recently described Tylopiluscallainus is synonymized with the Japanese Boletusvirescens, and the new combination T.virescens (Har. Takah. & Taneyama) N.K. Zeng et al. is proposed. Moreover, Neoboletus is treated as an independent genus based on evidence from morphology and molecular phylogenetic data in the present study, and many previously described taxa of Sutorius are recombined into Neoboletus: N.ferrugineus (G. Wu et al.) N.K. Zeng et al., N.flavidus (G. Wu & Zhu L. Yang) N.K. Zeng et al., N.hainanensis (T.H. Li & M. Zang) N.K. Zeng et al., N.obscureumbrinus (Hongo) N.K. Zeng et al., N.rubriporus (G. Wu & Zhu L. Yang) N.K. Zeng et al., N.sanguineoides (G. Wu & Zhu L. Yang) N.K. Zeng et al. , N.sanguineus (G. Wu & Zhu L. Yang) N.K. Zeng et al., and N.tomentulosus (M. Zang et al.) N.K. Zeng et al.


Introduction
margin of the pileus. Sections of the stipitipellis were taken from the middle part along the longitudinal axis of the stipe. Five percent KOH was used as a mounting medium for microscopic studies. All microscopic structures were drawn by freehand from rehydrated material. The number of measured basidiospores is given as n/m/p, where n represent the total number of basidiospores measured from m basidiomata of p collections. Dimensions of basidiospores are given as (a)b -c(d), where the range b -c represents a minimum of 90% of the measured values (5 th to 95 th percentile), and extreme values (a and d), whenever present (a < 5 th percentile, d > 95 th percentile), are in parentheses. Q refers to the length/width ratio of basidiospores; Q m refers to the average Q of basidiospores and is given with a sample standard deviation.

DNA extraction, primers, PCR and sequencing
Total genomic DNA was obtained with Plant Genomic DNA Kit (TIANGEN Company, China) from materials dried with silica gel according to the manufacturer's instructions. The primers used for amplifying the nuclear ribosomal large subunit RNA (28S) were LROR/LR5 (Vilgalys and Hester 1990;James et al. 2006), ITS5/ITS4 (White et al. 1990) for the nuclear rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA (ITS), the translation elongation factor 1-α gene (tef1) with 983F/1567R (Rehner and Buckley 2005) and the RNA polymerase II second largest subunit gene (rpb2) with RPB2-B-F1/RPB2-B-R (Wu et al. 2014). PCR products were checked in 1% (w/v) agarose gels, and positive reactions with a bright single band were purified and directly sequenced using an ABI 3730xl DNA Analyzer (Guangzhou Branch of BGI, China) with the same primers used for PCR amplifications. Assembled sequences were deposited in GenBank (Table 1).

Dataset assembly
For the concatenated multilocus dataset of Butyriboletus, 14 sequences (four of 28S, four of ITS, four of tef1, and two of rpb2) from four collections were newly generated (Table 1) and then combined with selected sequences from previous studies (Table 1). Rugiboletus extremiorientalis (Lj.N. Vassiljeva) G. Wu & Zhu L. Yang was chosen as outgroup on the basis of the phylogeny in Wu et al. (2016a). For the concatenated multilocus dataset of Caloboletus, Neoboletus, and Sutorius, 68 sequences (21 of 28S, 16 of ITS, 20 of tef1, 11 of rpb2) from 23 collections were newly generated and deposited in GenBank (Table 1) and then combined with selected sequences from previous studies (Table 1). Crocinoboletus laetissimus (Hongo) N.K. Zeng et al. and Cr. rufoaureus (Massee) N.K. Zeng et al. were chosen as outgroup based on the phylogeny in Wu et al. (2016a). For the concatenated multilocus dataset of Lanmaoa, eight sequences (three of 28S, two of ITS, and three of tef1) from three collections were newly generated and deposited in GenBank (Table 1), and then combined with selected sequences from previous studies (Table 1). Rugiboletus brunneiporus G. Wu & Zhu L. Yang was chosen as outgroup on the basis of the phylogeny in Wu et al. (2016a). To test for phylogenetic conflict among the different genes in three combined datasets (Butyriboletus, Caloboletus + Neoboletus + Sutorius, Lanmaoa), the partition homogeneity (PH) or incongruence length difference (ILD) test was performed with 1000 randomized replicates, using heuristic searches with simple addition of sequences in PAUP* 4.0b10 (Swofford 2002). The results of the partition homogeneity test showed that the phylogenetic signals present in the different gene fragments were not in conflict. Then the sequences of different genes in three combined datasets (Butyriboletus, Caloboletus + Neoboletus + Sutorius, Lanmaoa) were aligned with MAFFT v. 6.8 using algorithm E-INS-i (Katoh et al. 2005) and manually optimized on BioEdit v. 7.0.9 (Hall 1999). The sequences of the different genes were concatenated in three combined datasets (Butyriboletus, Caloboletus + Neoboletus + Sutorius, Lanmaoa) using Phyutility v. 2.2 for further analyses (Smith and Dunn 2008).

Phylogenetic analyses
The three combined datasets (Butyriboletus, Caloboletus + Neoboletus + Sutorius, Lanmaoa) were all analyzed by using maximum likelihood (ML) and Bayesian inference (BI). Maximum likelihood tree generation and bootstrap analyses were performed with the program RAxML 7.2.6 (Stamatakis 2006) running 1000 replicates combined with an ML search. Bayesian analysis with MrBayes 3.1 (Huelsenbeck and Ronquist 2005) implementing the Markov Chain Monte Carlo (MCMC) technique and parameters predetermined with MrModeltest 2.3 (Nylander 2004) was performed. The model of evolution used in the Bayesian analysis was determined with MrModeltest 2.3 (Nylander 2004). For the combined dataset of Butyriboletus, the best-fit likelihood models of 28S, ITS1+ITS2, 5.8S, tef1 and rpb2 were GTR+I+G, HKY+I+G, K80, SYM+I+G and K80+I+G, respectively; for the combined dataset of Caloboletus, Neoboletus, and Sutorius, the best-fit likelihood models of 28S, ITS1+ITS2, 5.8S, tef1 and rpb2 were GTR+I+G, HKY+I+G, K80, SYM+I+G and SYM+I+G, respectively; for the combined dataset of Lanmaoa, the best-fit likelihood models of 28S, ITS1+ITS2, 5.8S and tef1 were GTR+I+G, GTR+I, K80 and SYM+G, respectively. Bayesian analysis was run with one cold and three heated chains and sampled every 100 generations; trees sampled from the first 25% of the generations were discarded as burn-in; the average standard deviation of split frequencies was restricted to be below 0.01, and Bayesian posterior probabilities (PP) were then calculated for a majority consensus tree of the retained Bayesian trees.

Butyriboletus D. Arora & J.L. Frank
Butyriboletus, typified by But. appendiculatus (Schaeff.) D. Arora & J.L. Frank, was erected to accommodate the "butter boletes", which are mainly characterized by yellow hymenophore and context staining blue when injured and stipe surface usually covered with reticulations (Arora and Frank 2014;Zhao et al. 2015). Until now, six species, including But. hainanensis N.K. Zeng  Etymology. Latin, "huangnianlaii" is named after Chinese mycologist Nian-Lai Huang, in honor of his contribution to mycology.

Caloboletus Vizzini
Caloboletus, typified by C. calopus (Pers.) Vizzini, is mainly characterized by yellow tubes, yellow or more rarely orange to red pores changing to blue when injured, bitter taste of the context due to the presence of calopin and cyclocalopin (Hellwig et al. 2002;Vizzini 2014;Zhao et al. 2014a;Wu et al. 2016a; illeg., later homonym) Etymology. Latin, "guanyui" is named for Guan Yu, a historic Chinese hero, said to have a reddish face, and thus sharing the same color of pores of the species when young.
Description. Basidiomata medium-sized to large. Pileus 5-10 cm in diameter, convex to applanate; surface dry, finely tomentose, dirty white to pale brown; context 0.5-1.8 cm thick in the center of the pileus, white, changing bluish quickly when injured, then back to white. Hymenophore poroid, depressed around apex of stipe; pores subround, 0.3-0.5 mm in diameter, reddish to reddish brown when young, then yellow or yellowish brown, changing bluish black when injured; tubes about 0.5-1 cm in length, yellowish, changing bluish quickly when injured. Stipe 5.5-9 × 0.7-1.5 cm, central, subcylindric, solid, usually flexuous; surface dry, densely covered with pale brown, brown to reddish brown, minute squamules; context white, sometimes tinged with pale red, unchanging in color when injured; basal mycelium white. Odor indistinct.
Habitat. Gregarious on the ground in forests dominated by Castanopsis kawakamii Hay. or Lithocarpus spp.
Distribution. Southeastern and southern China; Japan (Hongo 1967). Note. Caloboletus guanyui was originally described as B. quercinus from Japan (Hongo 1967). Nomenclaturally, the epithet quercinus of this species is an illegitimate name, because Schrader (1794) described a species using the same epithet before Hongo (1967). Therefore, the new epithet guanyui is proposed here for this species. Moreover, morphological and molecular evidence indicates the taxon is a member of the genus Caloboletus (Fig. 2), and is characterized by a dirty-white to pale-brown pileus, pores reddish to reddish brown when young, then yellow or yellowish brown, changing bluish black when injured, and a stipe densely covered with pale-brown, brown to reddish-brown squamules. Morphologically, C. taienus and C. xiangtoushanensis also have reddish pores (Bessette et al. 2016;, however, a dirty-white to pale-brown pileus easily distinguishes C. guanyui from the two taxa. Phylogenetically C. guanyui is closely related to C. firmus (Frost) Vizzini (Fig. 2), however, C. firmus has a stipe covered with whitish or reddish reticula, and it is restricted to North and Central America (Bessette et al. 2016). Description. Basidiomata medium-sized to large. Pileus 5.5-11 cm in diameter, convex to plane; surface dry, tomentose, yellowish brown, pale brown to brown; context 1-1.5 cm thick in the center of the pileus, yellowish, changing blue quickly when injured. Hymenophore poroid, adnate to depressed around apex of stipe; pores subround to angular, 0.5-1 mm in diameter, yellow, sometimes brownish red, changing blue quickly when injured; tubes 0.5-1.4 cm in length, yellowish, changing blue quickly when injured. Stipe 5-9 × 0.9-1.6 cm, central, subcylindric, solid, usually flexuous; surface dry, upper part covered with reddish brown, minute squamules, middle and lower part covered with brown minute squamules; context yellowish, changing blue quickly when injured; basal mycelium white. Odor indistinct.
Habitat Notes. Our recent collections and the holotype of C. xiangtoushanensis, a species originally described from Guangdong Province of southern China , phylogenetically group together with a strong statistical support (Fig. 2), which indicates that these specimens should be recognized as C. xiangtoushanensis. It is new to Fujian Province. Morphologically, several features of our collections also match well with the protologue of C. xiangtoushanensis ), but reticulations on the stipe were not observed in our specimens. Moreover, pores of our specimens are sometimes brownish red. In appearance, C. xiangtoushanensis is highly similar to Japanese B. bannaensis Har. Takah., which needs further confirmation for generic placement (Takahashi 2007). However, B. bannaensis has rufescent and faintly cyanescent context, small basidiospores measuring 6.5-9 × 3.5-4 μm, and narrower cystidia (Takahashi 2007). The molecular analyses also indicates that C. xiangtoushanensis is closely related to C. taienus (W.F. Chiu) Ming Zhang and T.H. Li (Fig. 2), a species originally described from Yunnan Province (Chiu 1948); their morphological differences have been elucidated in a previous study .
Habitat. Solitary, scattered or gregarious on the ground in forests of Pinus massoniana Lamb. or P. latteri Mason.
Distribution. Central (Zhang et al. 2015), southeastern, and southern China. Notes. Our molecular phylogenetic analyses indicate that the new collections and the holotype of Ch. radiatus, a species first described from Hunan Province of central China, group together with a strong statistical support based on a two-locus dataset (28S + tef1) (data not shown). This indicates that our specimens should be recognized as Ch. radiatus (Zhang et al. 2015). This species is new to Fujian and Hainan Province. Zhang et al. (2015) reported Ch. radiatus from under Cunninghamia lanceolata (Lamb.) Hook, Cyclobalanopsis spp. and Castanopsis spp. We found the species associated with Pinus spp.

Lanmaoa G. Wu & Zhu L. Yang
Lanmaoa, typified by L. asiatica G. Wu & Zhu L. Yang, was erected recently. However, Lanmaoa and its closely related genus Cyanoboletus share overlapping morphological features and the most important diagnostic feature of Lanmaoa defined by Wu et al. (2016a) is not constant . Here, we treat Lanmaoa as an independent genus until the true taxonomic relationship between Lanmaoa and Cyanoboletus can be studied.
Habitat Note. Lanmaoa macrocarpa is characterized by its large basidioma, brownish red pileus and stipe, thickness of hymenophore 3/5 times that of pileal context, and its association with Castanopsis spp. It is both morphologically similar and phylogenetically related to Chinese L. rubriceps N.K. Zeng & Hui Chai (Chai et al. 2018) and one collection tentatively named "Lanmaoa sp. HKAS 52518" (Fig. 3). However, L. rubriceps has a red to crimson, orange-red pileus, pores stuffed when young, sometimes tinged with reddish when old, and smaller basidiospores measuring 8-11 × 4-5 μm ; careful examinations showed that Lanmaoa sp. HKAS 52518 has a smaller basidioma, a reddish to red or blackish-red pileus, and surface of stipe turning blue when injured.

Neoboletus Gelardi, Simonini & Vizzini
Neoboletus, typified by N. luridiformis (Rostk.) Gelardi et al., is characterized by stipitate-pileate or sequestrate; when basidiomata stipitate-pileate, pores brown, dark brown to reddish brown when young, becoming yellow when old (Fig. 6c, d, f ), tubes always yellow (Figs 5f, l, 6e, h), hymenophore and context staining blue, and stipe usually covered with punctuations (Vizzini 2014;Wu et al. 2016a). The monophyly of Neoboletus has been assessed, and many species of the genus were described (Wu et al. 2014(Wu et al. , 2016b. Astonishingly, the same authors recombined Neoboletus species in the genus Sutorius after a short time (Wu et al. 2016a). As a matter of fact, the stipe ornamentation pattern, spore print color, and colors of pores and tubes are fully different between the two genera (Halling et al. 2012;Vizzini 2014;Gelardi 2017). Furthermore, with more sequences added, our molecular data infers that Neoboletus forms an independent clade with strong support, and the genus Sutorius is sister to Costatisporus T.W. Henkel & M.E. Sm. (Smith et al. 2015) (Fig. 2). Thus, we recognize Neoboletus as an independent genus.  (Zang et al. 2001). It was later also reported from Yunnan Province of southwestern China (Wu et al. 2016a) and was transferred to the genus Sutorius. It is called the "Black bolete" in Yunnan Province, and largely traded in local mushroom markets (Wang et al. 2004).
Habitat. Solitary on the ground in forests dominated by fagaceous trees including Lithocarpus spp.

Neoboletus obscureumbrinus
Distribution. Southern and southwestern China; Japan (Hongo 1968). Note. Boletus obscureumbrinus Hongo was originally described from Japan (Hongo 1968) and later reported from Guangdong Province of southern China and Yunnan Province of southwestern China (Wu et al. 2016a). It was transferred to the genus Sutorius by Wu et al. (2016a); in the present study, we place the species in Neoboletus according to the evidence referred to above (Fig. 2). It is new to Hainan Province. The fruit body of this species is eaten by the Li people who live in the region (our own investigations).
Etymology. Latin, "subrufus" refers to the stipe surface and context of the species turning reddish when injured.
Habitat. Scattered, gregarious or caespitose on the ground in forests dominated by fagaceous trees, including Lithocarpus spp. Note. Sutorius subrufus is characterized by a brown to pale reddish-brown pileus, stipe surface and context turning reddish when injured, relatively smaller basidi-ospores, and it is restricted in tropical China. It is both morphologically similar and phylogenetically related to S. eximius (Peck) Halling et al. and S. australiensis (Bougher & Thiers) Halling and N.A. Fechner. However, stipe surface and context of S. eximius does not change when injured. Moreover, S. eximius has larger basidiospores, and a distribution in North and Central America (Singer 1947;Smith and Thiers 1971;Halling et al. 2012); S. australiensis has relatively larger basidiospores, a distribution in Australia, and is associated with Myrtaceae and Casuarinaceae (Halling et al. 2012).
Tylopilus, typified by T. felleus (Bull.) P. Karst., is characterized by the pallid, pinkish, vinaceous and pinkish-brown hymenophore, white to pallid context without color change, but some species becoming rufescent or sea-green when injured, and the bitter taste of the context (Baroni and Both 1998;Henkel 1999;Fulgenzi et al. 2007;Osmundson and Halling 2010;Wu et al. 2016a;Magnago et al. 2017;Liang et al. 2018). In China, although lots of species of the genus have been previously discovered (Li et al. 2002;Fu et al. 2006;Gelardi et al. 2015;Wu et al. 2016a;Liang et al. 2018), still there are a large number of undescribed taxa in this region.  ). This taxon was previously thought to be different from B. virescens Har. Takah. & Taneyama, a species described from Japan (Terashima et al. 2016). After a careful re-evaluation of specimens, we now know that the two taxa are conspecific, and T. callainus is synonymized with B. virescens. Clarifying the taxonomic relationship between the two taxa also indicated that the B. virescens is a member of Tylopilus, and thus the new combination is proposed. Illustrations and a full description have been provided by Liang et al. (2018).

Discussion
Molecular phylogenetic analyses have been used widely to define the genera of boletes, and as a result, many genera were erected or merged (Zeng et al. 2012(Zeng et al. , 2014bNuhn et al. 2013;Wu et al. 2014Wu et al. , 2016a. Recently, the genus Neoboletus was synonymized with Sutorius solely based on the evidence of molecular data (Wu et al. 2016a). Our molecular phylogenetic analyses based on a four-locus dataset (28S + ITS + tef1 + rpb2) with sequences from taxa of Neoboletus, Sutorius, Costatisporus, and Caloboletus (Fig. 2) indicate those species that morphologically match the concept of genus Neoboletus do not belong in Sutorius; instead, they form an independent clade with strong support (Fig. 2). At the same time, the morphological features including the stipe ornamentation pattern, spore print color, and color change of tissues are different between the two genera and has been noted in previous studies (Halling et al. 2012;Gelardi 2017). It is noteworthy that the color of tubes of Neoboletus is always yellow (Figs 5f, l, 6e, h), and in this genus the pores usually become yellow when old (Fig. 6d, f ), whereas the color of tubes and pores of Sutorius are always tinged with reddish at different growth stages (Fig. 6i-k).
The present study further shows that the most important diagnostic feature of the genus Lanmaoa, viz. "short hymenophoral tubes (thickness of hymenophore 1/3-1/5 times that of pileal context at the position halfway to the pileus center) and a slow color change when injured" defined by Wu et al. (2016b) is not constant , for the thickness of hymenophore is about 3/5 times that of pileal context in our newly described L. macrocarpa. Additionally, context and hymenophore of our new species turn quickly and strongly when injured (Fig. 5c).
According to current molecular data, 10 lineages (lineages 1-10) of Sutorius were found (Fig. 2). Lineages 4 and 6 were identified as S. australiensis and S. eximius respectively in a previous study (Halling et al. 2012). Lineages 1, 2, 3, 5, 7 and 9 may have not diverged enough (Fig. 2) and are treated here as a series of closely related taxa or disjunct populations of previously described entities; these will be assessed in the future with more DNA sequences and more collections. As to lineages 8 and 10, they should be treated as independent taxa due to their high degree divergence. Moreover, morphological and ecological features (described above) of specimens (FHMU 2004, FHMU 2006, FHMU 2101 in lineage 8 from Hainan Province are also different from the described taxa of Sutorius, and thus, the new taxon S. subrufus was proposed. Lineage 10 was not described due to the paucity of the materials (Halling et al. 2012).
Subtropical and tropical China is believed to be a biodiversity hotspot. Mycologists have paid much attention to boletes of the region in the past decade, and many taxa have been discovered (Bi et al. 1997;Zeng and Yang 2011;Zeng et al. 2012Zeng et al. , 2013Zeng et al. , 2014aZeng et al. , b, 2015aZeng et al. , b, 2016Zeng et al. , 2017Zeng et al. , 2018Zang 2013;Liang et al.2016Liang et al. , 2017Liang et al. , 2018Chai et al. 2018;Xue et al. 2018). Among of them, many have been found to be as North American or European species (Bi et al. 1997;Zang 2013), and recent studies have shown that species shared between subtropical/tropical China and North America/Europe are rare but that there are many common species between Japan and subtropical/tropical China . Our study now reveals that the geographic distributions of the Japanese C. guanyui, N. obscureumbrinus, and T. virescens extend into subtropical or tropical China.