A Contribution to Knowledge of Gyroporus (Gyroporaceae, Boletales) in China: Three New Taxa and Amended Descriptions of Two Previous Species

Some species of Gyroporus (Gyroporaceae, Boletales) in China are investigated on the basis of morphology and molecular phylogenetic analyses. Six Chinese species are recognized in the present study. Among them, G. memnonius, G. porphyreus and G. subglobosus are new to science; G. longicystidiatus and G. paramjitii are previously described; and one ambiguous taxon tentatively named G. cf. castaneus. A key to known species of Gyroporus in China is also provided. A preliminary biogeographical analysis shows that Gyroporus in East Asia and Southeast/South Asia are closely related. Gyroporus longicystidiatus and G. paramjitii are geographically widespread species occurring in East Asia and Southeast/South Asia; G. cf. castaneus is a shared species between East Asia and Europe. walled (up to 1 µm), light yellow in KOH hyphae 5–15 µm in width; terminal cells 30–80 × 10–13 µm, clavate or subcylindrical, with obtuse apex. Pileal trama made up of hyphae 5–17 µm in diam., slightly thick-walled (up to 1 µm), light yellow in KOH. Stipitipellis a trichoderm-like structure 60–90 µm in thickness, composed of thin- to slightly thick-walled (up to 1 µm), 5–12 µm wide, light yellow in KOH, emergent hyphae with clavate terminal cells (31–66 × 6–11 µm). Stipe trama composed of cylindrical, light yellow in KOH, slightly thick-walled (0.8–1 µm), interwoven hyphae 4–17 µm diam. Clamp connections present in all tissues. thin-walled, colorless in KOH, no encrustations. Pileipellis a trichoderm 70–100 µm in thickness, consisting of thin- to slightly thick-walled (up to 1 µm), light yellow in KOH, hyphae, 6–13 µm in width; terminal cells 41–69 × 9–11 µm, subfusiform, narrowly clavate or subcylindrical, with obtuse apex. Pileal trama made up of hyphae 4–18 µm in diameter, slightly thick-walled (0.8–1 µm), light yellow in KOH. Stipitipellis a trichoderm-like structure 60–100 µm in thickness, composed of thin- to slightly thick-walled (up to 1 µm), 5–11 µm wide, light yellow in KOH, emergent hyphae with subfusiform, fusiform or clavate terminal cells (25–40 × 7–14 µm). Stipe trama composed of cylindrical, light yellow in KOH, slightly thick-walled (0.8–1 µm), interwoven hyphae 3–15 µm wide. Clamp connections present in all tissues. Pleurocystidia 17–40 × 4–10 µm, subfusiform or fusiform, thin-walled, colorless in KOH, no encrustation. Pileipellis a trichoderm 60–100 µm in thickness, composed of thin- to slightly thick-walled (up to 1µm),light yellow in KOH, hyphae 5–13 µm in width; terminal cell 38–105 × 9–15 µm, clavate or subcylindrical, with obtuse apex. Pileal trama made up of hyphae 4–15 µm in diameter, slightly thick-walled (1 µm), light yellow in KOH. Stipitipellis a trichoderm-like structure 40–85 µm in thickness, composed of thin- to slightly thick-walled (up to 1µm), 4–12 µm wide, light yellow in KOH, emergent hyphae with subfusiform, fusiform or clavate terminal cells (25–53 × 6–14 µm). Stipe trama composed of cylindrical, light yellow in KOH, slightly thick-walled (up to 1 µm), interwoven hyphae 4–15 µm wide. Clamp connections present in all tissues. slightly thick-walled (up to 1 µm), light yellow in KOH. Stipitipellis a trichoderm-like structure about 100 µm in thickness, composed of thin- to slightly thick-walled (up to 1 µm), 5–12 µm wide, light yellow in KOH, emergent hyphae with subfusiform, fusiform or clavate terminal cells (26–100 × 9–11 µm). Stipe trama composed of cylindrical, light yellow in KOH, slightly thick-walled (0.8–1 µm), interwoven hyphae, 5–17 µm wide. Clamp connections present in all tissues. thick-walled (up to 1 µm), light yellow in KOH. Stipitipellis a trichoderm-like structure 50–100 µm in thickness, composed of thin- to slightly thick-walled (up to 1 µm), 5–10 µm wide, light yellow in KOH, emergent hyphae with clavate, broadly clavate or subfusiform terminal cells (20–55 × 7–12 µm). Stipe trama composed of cylindrical, light yellow in KOH, thin-to slightly thick-walled (up to 1 µm), interwoven hyphae, 5–18 µm wide. Clamp connections present in all tissues.


Introduction
Dataset assembly Fifty-ve sequences (twenty-one of 28S, seventeen of ITS, sixteen of TEF1, one of RPB2) from seventeen collections were newly generated. Edited sequences were deposited in GenBank; the GenBank accession numbers of 28S, ITS, and TEF1 are listed in Table 1, and one of RPB2 from G. longicystidiatus Nagas. & Hongo is presented here [N.K. Zeng2974 (FHMU1935): MW354541]. For the combined dataset, the 28S, ITS and TEF1sequences were aligned with selected sequences from GenBank and previous studies (Table 1). Phlebopus marginatus Watling & N.M. Greg. and Phlebopus sp. (REH8795) were chosen as the outgroup inferred from Davoodian et al. (2018). Single-gene phylogenetic trees based on the 28S, ITS, and TEF1 fragments, respectively, were analyzed to test for phylogenetic con ict. The topologies of the phylogenetic trees based on a single gene were almost identical, indicating that the phylogenetic signals present in the different gene fragments were not in con ict. Then, the sequences of the different genes were aligned using MUSCLE (Edgar 2004), and alignments were purged from unreliably aligned positions and gaps using Gblocks (Castresana 2000). The sequences of the different genes were concatenated using Phyutility v. 2.2 for further analyses (Smith and Dunn 2008).  (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, the best-t likelihood models of 28S, ITS and TEF1 were GTR + I + G, HKY + I + G, SYM + I + G, respectively. Bayesian analysis was repeated for ve point seven million generations and sampled every 100 generations; trees sampled from the rst 25% of the generations were abandoned as burn-in; the average standard deviation of split frequencies was restricted to be below 0.01, and Bayesian Posterior Probabilities (BPP) were then calculated for a majority consensus tree of the retained Bayesian trees.

Results
The combined Gyroporus dataset (28S + ITS + TEF1) consisted of 107 taxa and 2566 nucleotide sites, and the alignment was deposited in TreeBASE (S27451). The phylogram with branch lengths generated from BI, including the support values is shown in Fig. 1. The topologies of the phylogenetic trees based on the combined dataset generated from ML and BI analyses were almost identical, but statistical support showed slight differences.

Habitat
Solitary, scattered or gregarious on the ground in forests dominated by fagaceous trees.

Known distribution
Eastern (Chang et al. 2001;Chou et al. 2005), southwestern (Li 2007), southeastern and southern China; Japan Notes: Gyroporus longicystidiatus, a species originally described from Japan (Nagasawa 2001) and later reported in China (Chang et al. 2001;Chou et al. 2005;Li 2007;Davoodian et al. 2018), is well characterized by a drab pileus, long and wide cystidia, and elliptical basidiospores with Qm = 1.56 ± 0.24. Our new collections match well with the protologue of G. longicystidiatus, except that the pleurocystidia were described as "absent" by Nagasawa (2001) whereas they are present in our specimens. Moreover, one new collection (FHMU1935) and true G. longicystidiatus de ned by Davoodian et al. (2018) grouped together with strong statistical support based on RPB2 sequences (data not shown), which further proved that our specimens are conspeci c. Besides Chinese collections, a single accession labeled Gyroporus sp.

Diagnosis
Characterized by a small to medium-sized basidioma, a dark brown pileus, a yellowish brown stipe, elliptical basidiospores with Qm = 2.07 ± 0.23, and a distribution in subtropical area of Asia.
Phylogenetically, G. memnonius is genetically distant from G. ammophilus and G. paramjitii; it is closely related to collections named G. castaneus from northeastern China, Europe, USA, respectively (Fig. 1). Phylogenetic relationships of G. memnonius to G. punctatus and G. tuberculatosporus are unknown due to a lack of DNA sequences from the latter two.
Gyroporus paramjitii K. Das,D. Chakraborty & Vizzini,Nordic J. Bot. 35 (6) Notes: Our molecular phylogenetic analyses indicate that Chinese collections and the holotype of G. paramjitii, a species originally described from India (Das et al. 2017), grouped together with strong statistical support (Fig. 1), which indicates that our new specimens should be recognized as G. paramjitii. Morphologically, our new collections match well with the protologue of G. paramjitii (Das et al. 2017), except that the size of cheilocystidia is slightly different, those from Indian collections being 27-45 × 5-7 µm (Das et al. 2017), whereas cheilocystidia from Chinese specimens are 20-35 × 7-13 µm. The diagnostic morphological features of G. paramjitii are summarized as follows: a small basidioma, a pileus covered with red-brown to dark red-brown squamules, oval to ellipsoid basidiospores with Qm = 1.59 ± 0.13, relatively short hymenial cystidia, and the hyphae in the pileipellis usually with cystidioid terminal cells. Besides the aforementioned collections, two vouchers labeled G. castanaeus (RWH8804) from Thailand and G. sp. (HKAS63505) from southwestern China, respectively, should also be identi ed as G. paramjitii as inferred from our phylogenetic tree (Fig. 1).  Characterized by a small to medium-sized basidioma, a pileus colored with yellow-brown, brown to red-brown when young, then purple, a brown, pale red-brown to red-brown stipe, oval to ellipsoid basidiospores with Qm = 1.67 ± 0.34, relatively short hymenial cystidia, and a distribution in subtropical region of China.

Etymology
Latin "porphyreus" refers to the pileus of the new species colored with purple when old.

Notes
The pileus of G. atroviolaceus and G. purpurinus is also tinged with purple, however, G. atroviolaceus, a species rstly described from Indonesia, has a pileus colored with black besides purple, wider basidiospores measuring 8.5-10 × 6-6.5 µm, and longer cystidia (Corner 1972;Horak 2011); G. purpurinus has larger basidiospores measuring 8-11(-12 smithii has narrower cystidia measuring 24-36 × 4-8 µm, thicker pileipellis hyphae 6-20 µm broad and distributing in USA (Davoodian et al. 2020). Phylogenetically, G. porphyreus is distant from G. purpurinus. Phylogenetic relationships of G. porphyreus with G. atroviolaceus is unknown due to a lack of DNA sequences from the latter. Moreover, we also noted that G. porphyreus is phylogenetically related to the Europen G. ammophilus (Fig. 1). However, G. ammophilus has a large basidioma [up to 15 (-20) cm], surfaces of the pileus and the stipe colored with salmon to brown, tubes and pores salmon to straw-color, becoming brown with age, a context salmon-color to pinkish cream and nally bluish, longer basidiospores measuring 8.5-12 × 4-5.5 (-6.5) µm, and a distribution in coniferous forests (Castro and Freire 1995). In addition, our molecular data also indicate that one collection labeled G. castaneus from Japan and specimens of G. porphyreus grouped together with strong statistical support (Fig. 1), indicating that the Japanese material should be identi ed as G. porphyreus.  Basidiomata small-sized. Pileus 2.5-3 cm diam., convex when young, then applanate; margin uplifted when old; surface dry, subtomentose, sometimes slightly rimose in age, yellow-brown (4B7), red-brown (7D6) to dark brown (5C7); context about 0.2 cm in thickness in the center of the pileus, white (1A1), unchanging in color when injured. Hymenophore poroid, depressed around apex of stipe; pores subrounded to angular, 0.5-1 mm diam, white when young, then yellow (1A2), unchanging in color when bruised; tubes 0. 5-0.8 cm in length, yellowish (1A4), unchanging in color when injured. central,subcylindrical, (Magnago et al. 2018); G. castanaeus has a larger basidioma (more than 10 cm), longer basidiospores measuring 8-12 (-14) × 4.5-6 (-7) µm, and a distribution in Europe (Heinemann and Rammeloo 1979;Moser 1983;Castro and Freire 1995); Malaysian G. malesicus has a very small basidioma, narrower basidiospores measuring 9-10× 5-5.5 µm, and absence of clamp connections (Corner 1972;Horak 2011); G. mcnabbii has a larger basidioma (pileus up to 9 cm), longer basidiospores measuring (7.6-)8.4-10.7(-11.9) ×(4.9-)5.5-6.7(-7) µm, and a distribution in Australia (Davoodian et al. 2018); G. paramjitii has a dark brown stipe, longer basidiospores measuring (7.5-) 8-11.6 (-13) × (4.8-) 5-6.6 (-7) µm, and longer cheilocystidia (Das et al. 2017); G. madagascariensis has shorter and narrower basidiospores measuring 6.7-8.4(-9.1) × 4.1-5.2 µm, tubes that some areas discoloring to orange but lacking for cheilocystidia and pleurocystidia (Davoodian et al. 2020 (Corner 1972;Singer et al. 1983;Castro and Freire 1995;Bougher and Syme 1998). With the rapid development of molecular phylogenetic analyses, many previously described taxa have been re-evaluated, providing a better understanding of the diversity of Gyroporus worldwide (Vizzini et al. 2015;Das et al. 2017;Davoodian et al. 2018;Magnago et al. 2018;Huang et al. 2021). For example, G. castaneus, originally described from Europe, was believed to be a widely distributed species in the world. Conversely, recent studies indicated that G. castaneus represents a species complex rather than a single widespread species. Our molecular data also show that collections named G. castaneus resolved in several different parts of the tree (Fig. 1). Interestingly, specimens of G. castaneus from Europe resolved in more than one part of the tree (Fig. 1). The true G. castaneus may just be restricted to a wide area comprising Europe, temperate and subtropical regions of Asia (Fig. 2), a hypothesis that should be con rmed with more collections and DNA sequences from the holotype locality of the species. In China, G. castaneus s. l. likely occurs in temperate and subtropical regions of China, collections identi ed as G. castaneus from tropical areas of the country are certainly to be referred to other species (Davoodian and Halling 2013). As for G. cyanescens, it also resolved in several different parts of the tree (Fig. 1), which indicated G. cyanescens also represents a species complex rather than a single widespread species.

Species diversity of Gyroporus from China
In the present study, unexpected abundant species diversity was revealed in China, and ten lineages of Gyroporus were uncovered (Fig. 1). Three (lineages 4, 8 and 9) are described as new: Gyroporus memnonius, G. subglobosus and G. porphyreus; ve (lineages 1, 2, 3, 6 and 10) are previously described taxa: Gyroporus avocyanescens, Gyroporus alpinus, Gyroporus brunneo occosus, G. longicystidiatus and G. paramjitii; one (lineage 5) is tentatively named G. cf. castaneus; and one (lineage 7) is not described due to the paucity of material. Besides the aforementioned species, there are two additional taxa described from China, viz. G. pseudomicrosporus and G. tuberculatosporus. It is worth noting that G. pseudomicrosporus is not a true Gyroporus (Dr. N. Davoodian, private communications). At the same time, we also noted that basidiospores of G. tuberculatosporus were described as "tuberculatae" (Zang et al. 1996), but they are smooth according to our re-examinations of the holotype, moreover, the species is a member of G. cyanescens complex (Dr. N. Davoodian, private communications). In the future, more collections from a wide area (including the type locality), and more DNA sequences are expected for elucidating their true taxonomic relationships to other Gyroporus species. In previous studies, G. atroviolaceus, G. cyanescens, G. malesicus and G. purpurinus were also reported in China (Zang 1986;Zang et al. 1996;), yet their occurrence in the country remains to be con rmed.

Phylogenetic relationships and geographic divergence of Gyroporus
Recent phylogenetic studies have uncovered useful information concerning the phylogeny and geography of Gyroporus (Davoodian et al. 2018). Our molecular data based on three-locus DNA sequences with a number of additional collections from East Asia provide new insights. It is evident there are common or allied species shared between East Asia and Europe/North America as inferred from our data set (Fig. 1). For example, collections tentatively named G. cf. castaneus as occurring in temperate areas of East Asia and Europe was uncovered. Similar scenarios have been documented for many other fungi (Tang et al. 2013;Cui et al. 2016;Huang et al. 2020). The a nities of Gyroporus species between East Asia and Southeast/South Asia are evident (Fig. 1), both regions share two common taxa, i.e., G. longicystidiatus (East Asia-Southeast Asia) and G. paramjitii (East Asia-South Asia). In addition, G. longicystidiatus and G. porphyreus both occur in China and Japan.
Declarations Figure 1 Phylogram inferred from a combined (rDNA 28S, ITS and TEF1) dataset using BS/PP. BS ≥70% and PP ≥0.95 are indicated above or below the branches as RAxML BS/PP. SE = southeast; SW = southwest; NE = northeast.