New insights into the genus Gyroporus (Gyroporaceae, Boletales), with establishment of four new sections and description of five new species from China

ABSTRACT Species of Gyroporus from southern China were studied in this study. Based on morphology and molecular phylogenetic analyses of DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS), the nuclear ribosomal large subunit (nrLSU), and the mitochondrial adenosine triphosphate ATP synthase subunit 6 (atp6), Gyroporus was divided into four main branches in the phylogenetic tree, and four sections were firstly proposed i.e. Gyroporus sect. Castaneus, G. sect. Cyanescens, G. sect. Longicystidiatus and G. sect. Pallidus. Five new species, i.e. G. alboluteus, G. atrocyanescens, G. pseudolongicystidiatus, G. pallidus and G. subcaerulescens, were revealed from China, and their phylogenetic positions were also analysed. Among them, G. alboluteus and G. pallidus were nested into the sect. Pallidus, although morphologically similar to G. castaneus; G. atrocyanescens and G. subcaerulescens, with obvious cyanescent oxidation reactions, were nested into the sect. Cyanescens; and G. pseudolongicystidiatus characterised by its long cystidia and was nested into the sect. Longicystidiatus. The new species were formally described and illustrated in the present study, and a key to the sections and species of Gyroporus in China was provided.


Introduction
Gyroporus Quél., typified by G. cyanescens (Bull.) Quél., is a small but poorly understood bolete genus in the family Gyroporaceae of Boletales. Members of Gyroporus are widely scattered throughout temperate, subtropical and tropical regions of the world, and strongly implicated as symbionts with an array of ectotrophic plants, such as Fabaceae, Fagaceae, Myrtaceae, Pinaceae, Phyllanthaceae, etc. (Singer et al. 1983;Agerer 1999;Raidl et al. 2006;Watling 2006Watling , 2008Wilson et al. 2012). Species in Gyroporus can be easily identified by the brittle and hollow stipe, the white to yellowish white hymenophore unchanging or changing to blue when bruised, white spore print, ellipsoid basidiospores and the presence of clamp connections (Singer 1986;Watling 2008;Das et al. 2017;Magnago et al. 2018;Huang et al. 2021;Xie et al. 2022). However, it is extremely complicated to determine their taxonomic positions at the species level owing to the overlap of phenotypic variation among species. Recently, molecular phylogenetic studies have provided more effective and accurate evidences for species identification of Gyroporus, and some new species have been reported (Das et al. 2017;Magnago et al. 2018;Huang et al. 2021;Xie et al. 2022), while the gene of mitochondrial adenosine triphosphate ATP synthase subunit 6 (atp6) has been identified as a utility DNA barcoding marker to determine the infrageneric relationships of Gyroporus (Davoodian et al. 2018;Huang et al. 2021).
However, a recent study proved that G. pseudomicrosporus is a member of Gyrodon Opat. (Huang et al. 2021). Although G. castaneus (Bull.) Quél. and G. cyanescens, originally reported from Europe, have been widely reported in China (Chiu 1948(Chiu , 1957Zang 1986;Bi et al. 1994;Wu et al. 2013;Tang 2015;Xie et al. 2022), recent phylogenetic studies indicated that G. castaneus might be only distributed in northeastern China; and there were no conclusive specimens or molecular data to prove the natural distribution of G. cyanescens in China (Huang et al. 2021), instead, four similar species of G. memnonius, G. paramjitii K. Das, D. Chakraborty & Vizzini, G. porphyreus and G. subglobosus were identified from subtropical and tropical regions of China (Xie et al. 2022).
In recent years, some collections of Gyroporus were found in southern China, further study based on both morphological data and molecular sequences from the nuclear ribosomal internal transcribed spacer (ITS), the nuclear ribosomal large subunit (nrLSU) and the gene of atp6 proved that they represent five species new to science; and phylogenetic analyses using the molecular data from all species with known sequences worldwide revealed that the genus could be divided into four new sections. The result should contribute to further understanding the species diversity of Gyroporus in China and the taxonomic relationships of the infrageneric taxa.

Morphological studies
Photographs of the fresh basidiomata were taken in the field. Specimens were dried and deposited in the Fungarium of Guangdong Institute of Microbiology (GDGM). Descriptions of macro-morphological characters and habitats were obtained from photographs and field notes. Colour codes followed Kornerup and Wanscher (1981). Microscopic observations were carried out on tissue sections stained with 5% KOH and 1% aqueous Congo red under a light microscope (Olympus BX51, Tokyo) with magnification up to 1000 × . All measurements were made in 5% KOH. For basidiospore descriptions, the notation (a-)b-c (-d) describes basidiospore dimensions, where the range b-c represented 90% or more of the measured values and "a" and "d" were the extreme values; Q referred to the length/width ratio of an individual basidiospore and Q m referred to the average Q value of all basidiospores ± sample standard deviation. All line-drawings of microstructures were made based on rehydrated materials.

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from the voucher specimens using the Sangon Fungus Genomic DNA Extraction kit (Sangon Biotech Co. Ltd., Shanghai, China), according to the manufacturer's instructions. Primer pairs ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Vilgalys and Hester 1990), and atp6-2/atp6-3 (Kretzer and Bruns 1999) were used for amplifying ITS, nrLSU and atp6, respectively. PCR reactions was performed in a total volume of 25 μl containing 0.5 μl template DNA, 11 μl distilled water, 0.5 μl of each primer and 12.5 μl 2 × PCR mix (DreamTaq tm Green PCR Master Mix, Fermentas). Amplification reactions were performed in a Tprofessional Standard Thermocycler (Biometra, Göttingen, Germany) under the following conditions: 95°C for 4 min; then 35 cycles of denaturation at 94°C for 60s, annealing at 53°C for 60s, and extension at 72°C for 60s; with a final extension at 72°C for 8 min. The PCR products were electrophoresed on 1% agarose gels and sequencing was performed on an ABI Prism® 3730 Genetic Analyser (PE Applied Biosystems, Foster, CA, USA) at the Beijing Genomic Institute (BGI) using the same PCR primers. The raw sequences were assembled and checked with SeqMan implemented in Lasergene v7.1 (DNASTAR Inc., USA). The newly generated sequences in this study were submitted to GenBank.

Phylogenetic analyses
Sequences generated in this study and those downloaded from GenBank were combined and used for phylogenetic reconstruction. Detailed information of specimens included in this study was given in Table 1. Sequence matrix of ITS, nrLSU and atp6 were separately aligned with software MAFFT v7 using the E-INS-i strategy (Katoh and Standley 2013) and manually adjusted in MEGA 6 (Tamura et al. 2013).  Phylogenetic analyses were performed in the software of PhyloSuite (Zhang et al. 2020). Maximum likelihood phylogenies were inferred using IQ-TREE (Nguyen et al. 2015) under the TPM2u+R3 + F model for 5000 ultrafast bootstraps, as well as the Shimodaira-Hasegawa-like approximate likelihood-ratio test. Bayesian Inference (BI) phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al. 2012), the best models for the combined datasets ITS-nrLSU were searched via PartitionFinder 2 (Lanfear et al. 2017), and for atp6 region was searched via ModelFinder (Kalyaanamoorthy et al. 2017). BI analysis using 4 chains were conducted by setting generations to 20 million and stoprul command with the value of stopval set to 0.01, trees were sampled every 1000 generations, the first 25% generations were discarded as burn-ins and posterior probabilities (PP) were then calculated from the posterior distribution of the retained Bayesian trees. The phylogenetic trees were visualised using FigTree v1.4.23.

Molecular phylogeny
In the concatenated (nrLSU + ITS) dataset, 188 sequences (95 for nrLSU and 88 for ITS) from 121 fungal collections were included, including 39 sequences newly generated in this study. The alignment length was 1816 characters including gaps (888 characters for ITS, and 928 characters for nrLSU), TVM + I + G and TIM + I + G were selected for ITS and nrLSU respectively for the BI analysis. In the atp6 dataset, 89 sequences were included, including 23 sequences newly generated in this study. The alignment length was 616 characters. GTR + F + I + G4 was selected as the best models for Bayesian inference. Phlebopus spongiosus Pham & Har. Takah. was selected as outgroup based on recently studies (Davoodian et al. 2018;Xie et al. 2022). The tree topologies obtained by ML and Bayesian analyses were similar; thus, only the ML topology was shown in Figures 1 and 2. Phylogenetic analyses showed that Gyroporus was supported as a monophyletic group, and five new lineages were discovered in present study (Figures 1  and 2). Species of Gyroporus formed four main branches, and four new sections were firstly proposed herein, i.e. "G. sect. Castaneus", "G. sect. Cyanescens", "G. sect. Longicystidiatus" and "G. sect. Pallidus". Gyroporus sect. Castaneus was well-supported as a monophyletic group, and located at the base of the phylogenetic trees. Gyroporus sect. Cyanescens formed an independent branch, but with moderate support in the atp6 tree, and weak support in the ITS-nrLSU tree. Two new species G. atrocyanescens and G. subcaerulescens nested into the sect. Cyanescens. Gyroporus atrocyanescens was well-supported as an  independent clade in the phylogenetic trees ( Figures  1 and 2), and formed sister relationship with G. flavocyanescens. Gyroporus subcaerulescens formed an independent clade in Figure 1, while clustered together with G. atrocyanescens and G. flavocyanescens in Figure 2. The sect. Longicystidiatus was well-supported in the phylogenetic trees, and three species were included, containing a new species discovered in present study. The sect. Pallidus was well-supported as an independent branch in the trees (Figures 1 and 2), and different from the sect. Castaneus. Two new species G. alboluteus and G. pallidus nested into the branch, and close to "G. cf. castaneus" and G. memnonius.  (1886) Etymology: "castaneus" refers to the species in this section similar to G. castaneus.
Notes: Gyroporus sect. Castaneus as a monophyletic branch is strongly supported in our phylogenetic analyses (Figures 1 and 2, BS/BPP = 96%/0.98; BS/ BPP = 100%/1). Species in this section are mainly characterised by their brown to yellowish brown pileus, usually with red or purple tinge, white context unchanging when bruised, oval to elliptical basidiospores, and a trichoderm pileipellis composed of clavate to subcylindrical hyphae. Seven species,  (1886) Etymology: "cyanescens" refers to the species in this section usually with cyanescent oxidation reactions similar to that of G. cyanescens.
Basidiomata medium to large-sized. Pileus hemispherical to convex, dry, greyish yellow, greyish orange, brown or red-brown, covered with floccose-scaly to coarsely tomentose squamules; context white, becoming bluish, greenish blue or dark blue or deep blue when bruised. Hymenophore poroid, white, yellowish, to greenish-yellow, becoming bluish, greenish blue or dark blue when bruised. Stipe central, dry, covered with tomentose to fibrillose squamules, unchanging or changing to blue when handled; basal mycelium white; annulus indistinct to as a weak annular zone. Basidiospores ellipsoid to broadly ellipsoid, smooth, yellowish in 5% KOH. Basidia clavate, 4-spored, hyaline in 5% KOH. Cheilocystidia clavate to subfusiform, thin-walled, yellowish to hyaline in 5% KOH. Pleurocystidia absent or present. Pileipellis a trichodermium, composed of elongated or somewhat clumped, parallel to slightly interwoven, thin to thick-walled hyphae, colourless or yellowish in 5% KOH. Stipitipellis composed of thinto thick-walled hyphae, colourless to yellowish. Clamp connections frequently present in all tissues.
Notes: Gyroporus sect. cyanescens formed an independent branch in the phylogenetic trees (Figures 1  and 2), but with moderate support at atp6 tree and weak supported at ITS-nrLSU tree. Morphologically, species in this section all can produce cyanescent oxidation reactions, and pileus surface always covered with elongated and somewhat clumped tomentum. Fourteen species were proved to belong to this section, including two new species G. atrocyanescens and G. subcaerulescens discovered in present study. Fungal Name: FN570980 Etymology: "atro-" means black, "cyanescens" means becoming blue, "atrocyanescens" refers to the basidiomata instantly changing to blackish blue when bruised.
Diagnosis: This species is characterised by its white to greyish yellow pileus densely covered with greyish yellow floccose squamules, white to yellowish white hymenophore, broadly elliptical basidiospores (7.5-10 × 4.8-6 μm), and the whole basidiomata immediately staining dull blue, deep blue to dark blue when bruised.
In morphology, G. occidentalis Davoodian, Bougher & Halling resembles G. atrocyanescens in the rapidly bluing oxidation reaction. However, G. occidentalis, reported from Western Australia, differs in its larger basidiomata, yellow-white to yellow buff to dirty yellow pileus, and smaller and narrower basidiospores 7.7-8.4(9.1) × 3.5-4.2 µm (Davoodian et al. 2018). The bluing species G. alpinus, G. brunneofloccosus and G. cyanescens are also similar to G. atrocyanescens in the discolouration. However, G. alpinus recently reported from southwestern China, differs in its ivory yellow to brownish-yellow pileus covered with concolourous appressed scaly to floccose squamules, broader basidiospores (5.5-8.5 µm broad), and distribution in alpine forests dominated by Abies, Picea and Quercus (Huang et al. 2021); G. brunneofloccosus, reported from subtropical regions of southern China, differs in its dark brown to light red brown pileus covered with concolourous floccose-scaly to coarsely tomentose squamules, yellowish to greenish-yellow hymenophore staining cerulean blue to greenishblue when bruised, brownish to light red-brown stipe, and clavate to subfusiform cheilocystidia hyaline in 5% KOH Huang et al. 2021); while G. cyanescens, originally described from Europe, differs in its larger basidiomata, pale straw, buff to ivory pileus covered with obviously fibrillose tomentum, more robust stipe with a pseudo-annular zone and horizontal fissures at stipe apex, and distribution in forests dominated by Pinus sylvestris or Fagus sylvatia (Watling 1970;Vizzini et al. 2015).
Diagnosis: This species is characterised by its white to orange white pileus covered with orange white to reddish white coarsely tomentose squamules, white hymenophore and pileus context slowly changing to pastel blue when bruised, elliptical basidiospores (6.5) 8-10 × 5.5-6.5 (7.0) µm.
Habitat and distribution: Solitary or scattered on soil in subtropical mixed forest mainly dominated by Fagaceae trees, with a few pine trees (Cunninghamia sp.). Currently known from Hunan Province, China.
Notes: Phylogenetic analyses shown that G. subcaerulescens was well nested into the sect. Cyanescens, and closely related to G. alpinus, G.
cyanescens, G. flavocyanescens and G. atrocyanescens. However, they can be separated from each other by the genetic distance. Additionally, G. alpinus, recently reported from alpine forests of China, differs in its ivory yellow to brownish-yellow pileus densely covered with concolourous appressed floccose squamules, and broader basidiospores (6.5) 7-10 × 5.5-7.5 (8.5) µm (Huang et al. 2021); G. cyanescens, originally reported from Europe, differs in its larger basidiomata can up to 12 cm broad, pale straw pileus, larger but narrow basidiospores (9-11 × 4.5-6 µm) (Watling 1970;Vizzini et al. 2015;Huang et al. 2021); G. flavocyanescens, recently reported from tropical forests of China, differs in its larger basidiomata can up to 10 cm broad, nearly glabrous and flavous to greyishorange pileus, white context staining strong dark blue when bruised, white to yellowish hymenophore staining cyanine blue to porcelain blue when bruised (Huang et al. 2021); G. atrocyanescens, newly described in this study, can be easily distinguished by its strongly cyanescent oxidation reactions.
Notes: Gyroporus sect. Longicystidiatus was wellsupported as a monophyletic clade in our phylogenetic analyses (Figures 1 and 2; BS/BPP = 99%/0.98; BS/BPP = 100%/1). Species in this section mainly characterised by their brownish orange to brownish yellow pileus, white context unchanging when bruised, longer cystidia can up to 100 μm, and trichoderm pileipellis. Three species were included in this section, containing a new species described as follow. Gyroporus pseudolongicystidiatus Ming Zhang, D.C. 7 Fungal Name: FN570981 Etymology: "pseudolongicystidiatus" refers to the species similar to G. longicystidiatus.
Habitat and distribution: Solitary or scattered on soil in mixed broadleaf-coniferous forests, mainly dominated by Cyclobalanopsis spp. and Castanopsis spp., alt. 900 m. Currently known from Hainan Province, China.
Notes: The combined morphological characters include the brownish orange to yellowish brown pileus covered with fibrillose or velvet-subtomentose when young and nearly smooth in age, the white context and tubes unchanging when bruised, the hollow and brittle stipe, elliptic to cylindrical basidiospores, and the longer cheilocystidia up to 100 μm; which allowed G. pseudolongicystidiatus to be easily separated from other species of the genus.
Basidiomata small. Pileus 2-3 cm broad, hemispheric, convex to plane, dry, fibrillose, velvet-subtomentose when young and nearly glabrous in age, white at first, pale yellow to pale orange at maturity, paler towards margin. Context 3-4 mm thick at centre, fleshy, white, unchanging when exposed. Tubes 3-4 mm deep, depressed or nearly free at stipe in age, white, unchanging when bruised. Pores 2-3 per mm, circular, white, unchanging when bruised. Stipe 30-50 × 6-10 mm, central, equal or slightly swollen downwards, brittle, stuffed with a soft pith, becoming hollow or developing several cavities in age, surface dry, glabrous or with white pruina, concolourous with pileus or paler, unchanging when handled, with white basal mycelium; stipe context white, unchanging when exposed. Odour none. Taste mild.
Habitat and distribution: Solitary or scattered on soil in mixed forest dominated by Fagaceae trees, and mixed with Pinus massoniana Lamb. Currently known from Guangdong Province, China.
Notes: The combined morphological features of the small basidiomata, the pale yellow to pale orange coloured pileus, the white hymenophore and context unchanging when bruised, and the elliptical to cylindrical basidiospores make G. alboluteus easily distinguished from other species of Gyroporus. Ecologically, G. alboluteus is distributed in subtropical mixed forests, which are dominated by Fagaceae trees and mixed with a small amount of Pinus massoniana.

Gyroporus pallidus
Diagnosis: This species is characterised by its small basidiomata, brownish orange to light brown pileus usually cracked into small scales on the surface, white to yellowish white context unchanging when bruised, and elliptical basidiospores 8-10 × 5-6 µm.
Basidiomata small to medium-sized. Pileus 3-5 cm broad, convex when young, then applanate with age; margin decurved at first, then slightly upward when old; surface dry, subtomentose, usually cracking into small scales when mature or in dry conditions, brownish orange, light brown to brown (5C4-5C6, 5D5-6D5); context 3-4 mm thick, white, unchanging in colour when injured. Hymenophore adnate to slightly depressed around stipe when mature, 3-5 mm long, white (1A1) when young, yellowish white (3A2) when mature, unchanging when bruised; pores angular to roundish, 2-3 per mm, white to yellowish white, unchanging when bruised. Stipe 4-6 × 0.6-1.2 cm, central, sub-cylindrical to clavate, concolourous with pileus, slightly paler to yellowish brown to yellowish downward the base; surface roughened, unchanging when bruised; context white to yellowish white, spongy when young and then hollow in age, unchanging when bruised. Odour none. Taste mild.
Two new species G. porphyreus and G. subglobosus recently reported from China, also similar to G. pallidus. However, G. porphyreus differs in its yellow-brown, redbrown to purple pileus, brown to redbrown stipe, and narrower basidiospores (4-5.5 μm wide) with a relatively large Q value (1.4-2.56) (Xie et al. 2022); G. subglobosus differs in its yellowish brown, red-brown to dark brown pileus, brown to redbrown stipe, and subglobose basidiospores (6.5-9.5 × 5-7 μm) with a small Q value (1.1-1.5) In sect. Castaneus, G. castaneus has been widely reported in Europe, North America and eastern Asia. However, the reported collections of "G. castaneus" are actually a complex consisting of several different taxa (Das et al. 2017;Davoodian et al. 2018;Xie et al. 2022); for example, the specimens labelled as "G. castaneus" represent obviously more than one species in the phylogenetic tree (Figure 1). The specimens of this complex from subtropical and tropical regions of Asia or other continents represent different species, such as G. mcnabbii, G. memnonius, G. naranjus, G. paramjitii, G. pallidus etc. It is believed that further studies with more samples will contribute more to reveal the diversity of G. castaneus complex.
The sect. Cyanescens formed a monophyletic group in the phylogenetic tree (Figures 1 and 2), and consisted of species with cyanescent oxidation reactions. Gyroporus atrocyanescens and G. subcaerulescens are well nested into this section, and closely related to the Chinese species G. flavocyanescens, but they can be separated by the morphological features and the genetic distance. In this section, the species from Southern Hemisphere clustered together, while the species from Northern Hemisphere formed two well supported clades in the atp6 phylogenetic tree (Figure 2), which was consistent with the previous study by Davoodian et al. (2018). Gyroporus cyanescens was reported to be widely distributed in China in the past Zheng 1990, 1994;Ying and Zang 1994;Mao 2000;Li et al. 2015), and a recently study proved that the distribution of G. cyanescens in China is highly suspectable and specimens fully identical to the European species has not been found yet (Huang et al. 2021). Specimens from temperate regions of China labelled as "G. cyanescens" could be G. alpinus, and specimens from subtropical or tropical regions of China with obvious cyanescent oxidation reactions could be G. brunneofloccosus, G. flavocyanescens or G. atrocyanescens.
The sect. Longicystidiatus was well supported (Figures 1 and 2; BS/BPP = 99%/1; BS/ BPP = 100%/1), and formed a sister relationship with the sect. Cyanescens. However, species in this section lack of cyanescent oxidation reaction and can be easily distinguished from other species in Gyroporus by their very large and conspicuous cystidia, especially in G. longicystidiatus and G. pseudolongicystidiatus, the size of cheilocystidia can up to 100 µm in length.
The sect. Pallidus formed a monophyletic clade in the phylogenetic trees (Figures 1 and 2), and four species were included. Species in this section are difficult to distinguish from the species in sect. Castaneus in morphology, but they can be easily separated from each other in phylogeny. Besides, species in sect. Pallidus usually have paler pileus colour, and without red or purple tinge to compare with species in the sect. Castaneus. The species "G. cf. castaneus", described from China in Xie et al. (2022), has been proved to belong to the sect. Pallidus, and represents a different species from G. castaneus.
As noted in previous studies, species of Gyroporus are widely distributed in China and eastern Asia. Although several new species have been reported, there are still a larger number of unidentified specimens waiting to be studied, and numerous additional hidden species would be revealed based on more collections and DNA molecular evidences in the future. Pileus and stipe obviously brown, from brownish orange, light brown, light reddish brown to dark brown, with brown floccose-scales and long hairs or villi; context white, turning light turquoise at first, then quickly becoming dark turquise or dark blue when exposed; basidiospores 8.5-10 × 5-6 µm . .