Cyanescent Gyroporus (Gyroporaceae, Boletales) from China

Abstract Gyroporus species with cyanescent oxidation reactions were investigated, based on morphology and phylogenetic analysis of DNA sequences from the nuclear ribosomal large subunit (nrLSU), the nuclear ribosomal internal transcribed spacer (ITS) and the mitochondrial adenosine triphosphate ATP synthase subunit 6 (atp6). Three species, including two new species, namely G. alpinus and G. flavocyanescens and one previously-described species, namely G. brunneofloccosus, are revealed from China. Collections formerly reported from China as “G. cyanescens” are either G. alpinus or G. flavocyanescens. The new species are documented and illustrated in detail, while the concept of G. brunneofloccosus is refined with additional recently-collected materials. Additionally, the cyanescent species G. pseudomicrosporus, previously described from China, is shown to be a member of the genus Gyrodon, based on re-examination of the type specimen. A key to the cyanescent Gyroporus species from China is provided.

In this study, we used both morphological data and molecular sequences from the nuclear ribosomal large subunit (nrLSU), the nuclear ribosomal internal transcribed spacer (ITS) and the mitochondrial adenosine triphosphate ATP synthase subunit 6 (atp6), together with ecological data to evaluate the phylogenetic relationships of the cyanescent species within Gyroporus and make morphological and ecological comparisons.

Sampling and morphological studies
The collections of cyanescent species in Gyroporus were collected from Guizhou, Yunnan and Guangdong Provinces, China, in forests dominated by plants of the family Fagaceae or in the mixed forests dominated by plants of the families Fagaceae and Pinaceae. Fresh basidiomata were photographed and macroscopic characteristics, habitat, colour change when bruised, odour and taste were recorded. Basidiomata were then dried and deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN) and the Herbarium of the Guangdong Institute of Microbiology (GDGM). Macroscopic descriptions and microscopic studies followed Naseer et al. (2020), Zhang et al. (2019 and references therein. Colour description was according to Kornerup and Wanscher (1981). The notations and statistics of basidiospores followed Liu et al. (2020). Line drawings were prepared by free hand.

DNA extraction, PCR and sequencing
Genomic DNA was extracted from 100 mg of silica-gel dried samples or herbarium materials using the modified CTAB method (Doyle and Doyle 1987). PCR amplification primers ITS1 and ITS4 were used for the ITS region, LROR and LR5 were used for nrLSU and ATP6-F and ATP6-R were used for atp6 (White et al. 1990;Davoodian et al. 2018). PCR, amplification conditions, sequencing and sequence alignment followed those in Gelardi et al. (2019), Huang et al. (2021 and Gómez-Zapata et al. (2021).

Phylogenetic analysis
The phylogenetic analyses were based on three fragments (atp6, ITS and nrLSU). Two datasets, the atp6 dataset and the combined nrLSU and ITS dataset, were analysed using RAxML (Stamatakis et al. 2008). DNA sequences of the cyanescent species of Gyroporus from China and other continents (Crous et al. 2016(Crous et al. , 2017Das et al. 2017;Davoodian et al. 2018;Magnago et al. 2018) were used to infer the phylogenetic relationships between these species. Since seven cyanescent species have been reported from the Southern Hemisphere continent of Australia and their atp6 sequences are publicly available, the atp6 dataset was used to infer relationships of Australian cyanescent species with those from Europe, North America and East Asia in the Northern Hemisphere. The combined dataset was mainly used to infer relationships of species from East Asia, North America and Europe. In our preliminary analysis, the cyanescent species formed a monophyletic clade, thus, G. longicystidiatus Nagas. & Hongo without colour change when bruised was chosen as outgroup. For the combined dataset, Scleroderma areolatum Ehrenb., S. duckei B.D.B. Silva, M.P. Martín & Baseia and S. laeve Lloyd were selected as outgroup taxa.
The combined dataset was partitioned into four partitions (nrLSU, ITS1, 5.8S and ITS2). Statistical support for the phylogentic analyses was determined using a rapid bootstrapping with 1000 replicates in Maximum Likelihood (ML) analysis under the partitioned GTRGAMMA model. The scientific names, collection information and GenBank accession numbers for the specimens used in the phylogenetic analyses are presented in Table 1. Table 1. A tabulation of specimens used for molecular phylogenetic analyses in the present study. Sequences newly generated in this study are indicated in bold.

Species
Voucher Locality GenBank Accession No. ITS LSU atp6

Molecular analysis
In this study, sixteen new sequences of Gyroporus (six for ITS, six for nrLSU and four for atp6) were generated. Two datasets were analysed: the combined nuclear ribosomal DNA dataset (nrLSU + ITS) consists of 31 sequences and is 1720 bp long; the mitochondrial atp6 dataset consists of 23 sequences and is 596 bp long. The alignments were submitted to TreeBASE (27864). Phylograms inferred with RAxML, including the support values, are illustrated (Figs 1, 2). In both of our analyses, species with cyanescent colour changes when bruised cluster together with high support (100% in Fig. 1 and 99% in Fig. 2). The phylogenetic analysis of atp6 data indicates that the Australian cyanescent Gyroporus species form an independent lineage, while the other cyanescent species from the Northern Hemisphere form another lineage ( Fig. 1). It should be noted that the Northern Hemisphere lineage has relatively low bootstrap support (59%) in the atp6 analysis; however, its two main constituent sub-lineages have high support (70% and 100%) ( Yang, and one previously-described species, namely G. brunneofloccosus. The phylogenetic analysis of the combined (nrLSU + ITS) dataset also indicates that the Australian cyanescent Gyroporus species form an independent Southern Hemisphere lineage, while the other cyanescent species from the Northern Hemisphere form another lineage, yet without statistical bootstrap support, but its two main constituent sub-lineages also have high and moderate support (100% and 70%) (Fig. 2)   Diagnosis. This species differs from other cyanescent species of Gyroporus in its initially ivory yellow to greyish-yellow and then grey-orange to brownish-yellow pileus, scaly to floccose pileal surface, distribution in alpine forests with altitude up to 3800 m, broad basidiospores (5.5-8.5 µm wide) and long and slender basidia measuring 35-55 × 7-12 µm.
Habitat and distribution. Scattered on soil in alpine mixed forests dominated by Abies and Picea (Pinaceae) and Quercus (Fagaceae). Currently known from southwestern China.
Note. Gyroporus alpinus is characterised by the initially ivory yellow to greyishyellow and then grey-orange to brownish-yellow pileus with scaly to floccose squamules, the slightly extended pileal margin, the white pileal context staining cerulean blue to dull blue when bruised, the white to cream or yellowish hymenophore staining dull blue when bruised, the white to yellowish-white stipe, the spongy and then hollow context in the stipe, the frequent clamp connections in all tissues, the ellipsoid to somewhat broadly ellipsoid basidiospores and the distribution in alpine forests dominated by plants of the families Pinaceae and Fagaceae. In China, specimens of G. alpinus have been identified as G. cyanescens (Ying and Zang 1994;Zang 2013). Indeed, G. alpinus is closely related to G. cyanescens (Figs 1, 2). However, G. cyanescens, originally described from Europe, can be distinguished from G. alpinus by its relatively large basidiomata which are measuring 5.1-12.7 cm in diam., pale straw-coloured pileus, relatively narrow basidiospores measuring (7) 9-11 × 4.5-6 µm and distribution in forests dominated by Pinus sylvestris or Fagus sylvatia (Fries 1821;Watling 1970;Vizzini et al. 2015). In our analysis of the atp6 dataset, sequences of G. alpinus cluster together with sequences labelled as G. cyanescens from South Korea and Japan without statistical support (Fig. 1). Nagasawa (2001) treated the Japanese cyanescent taxon as G. cyanescens var. violaceotinctus Watling, because of the similar colours of their basidiomata and the similar-sized basidiospores. However, G. cyanescens var. violaceotinctus, originally described from Michigan, USA, is characterised by the white to tan context staining lilaceous and then indigo when bruised, the small basidia measuring 18-23.5 × 8-9 µm, the small cheilocystidia measuring 22.5-27.5 × 4.5-7.5 µm and the distribution in forests dominated by Acer (Aceraceae) and Betula (Betulaceae) (Watling 1969). These traits are greatly different from those of G. cyanescens and, therefore, Blanco-Dios (2018) treated G. cyanescens var. violaceotinctus as a novel species G. violaceotinctus (Watling) Blanco-Dios, while the Japanese taxon differs from G. violaceotinctus in its white context staining greyishblue at first and then blackish-blue when bruised without any lilaceous or violaceous tint, relatively large basidia measuring 24-42 × 9-11 µm, two types of cheilocystidia with the slender type measuring 30-64 × 6-12 µm and the voluminious type measuring 18-55 × 15-20 µm and distributions in mixed forest dominated by Fagus (Fagaceae), Quercus (Fagaceae), Betula (Betulaceae), Carpinus (Betulaceae) and Acer (Aceraceae) (Nagasawa 2001). The Chinese G. alpinus can be distinguished from G. violaceotinctus and the Japanese taxon by the dimensions of its basidiospores and basidia, morphology of cheilocystidia and host plants.
Gyroporus alpinus is phylogenetically related and morphologically similar to G. pseudocyanescens originally described from Spain in Crous et al. (2017) in our analysis of the combined dataset (Fig. 2). However, G. pseudocyanescens has a strawish-cream to yellow cream and then more or less brownish to yellowish-brown pileus, a velutinous pileal surface often cracking at maturity, relatively narrow basidiospores measuring 8-11 × 4.5-6 (6.5) µm, short terminal cells of the hyphae on the surface of the pileus measuring 50-80 × 9-15 µm and a distribution in forests dominated by Pinus spp. or Quercus spp. (Crous et al. 2017).
Discussion. Gyroporus brunneofloccosus, originally described from southern China, is characterised by the initially dark brown to brown and then brown to light redbrown pileus with concolorous floccose-scaly to coarsely tomentose squamules, the extended pileal margin, the white pileal context staining cerulean blue or greenish-blue to dark blue or deep blue when bruised, the initially yellowish to pale yellow and then greenish-yellow hymenophore staining cerulean blue to greenish-blue when bruised, the brownish to brown or light red-brown stipe, the spongy and then hollow context in the stipe, the frequent clamp connections in all tissues, the ellipsoid basidiospores and the distribution in tropical forests dominated by plants of the families Fagaceae and Pinaceae (Li et al. 2003).
Habitat and distribution. Scattered on soil in the tropical forests dominated by Castanea sp. (Fagaceae) and Quercus sp. (Fagaceae). Currently known from southwestern China.
Note. Gyroporus flavocyanescens is characterised by the flavous to dull yellow or grey-yellow and then grey-orange to greyish-orange pileus, the nearly glabrous to fibrillose to finely tomentose pileal surface, the slightly extended pileal margin, the white pileal context staining strong dark blue or indigo-blue when bruised, the white to grey or cream to yellowish hymenophore staining cyanine blue to porcelain blue when bruised, the white to cream and then pale yellow to flavous stipe, the spongy and then hollow context in the stipe, the frequent clamp connections in all tissues, the ellipsoid to somewhat broadly ellipsoid basidiospores and the distribution in tropical forests dominated by plants of the family Fagaceae.

Discussion
Cyanescent Gyroporus species in the Southern Hemisphere form independent lineages in the analyses of atp6 and combined nrLSU + ITS datasets (Figs 1, 2) and mainly associate with plants of the family Myrtaceae, while the cyanescent species in the Northern Hemisphere also form independent lineages, but without or with low statistical bootstrap support in the analyses of the combined and atp6 datasets and mainly associate with plants of the families Fagaceae and Pinaceae. Davoodian et al. (2020) suggest that the Southern Hemisphere lineage is derived from the Northern Hemisphere lineage, within which the Southern Hemisphere lineage is embedded. Further field investigations, careful morphological observations and extensive molecular analysis using multiple genes should help better understand the geographical relationships amongst the cyanescent species. Sixteen cyanescent Gyroporus species were revealed, based on former and present studies, including nine distributed in the Northern Hemisphere and seven distributed in the Southern Hemisphere. Three cyanescent Gyroporus have been reported from China before our study, namely G. cyanescens, G. brunneofloccosus and G. pseudomicrosporus (Zang 1986(Zang , 2013Bi et al. 1990Bi et al. , 1994Ying and Zang 1994;Mao 2000;Li et al. 2003). Gyroporus cyanescens was regarded as geographically widespread in Europe, North America and East Asia in the past. Our study identified the disjunct populations of this taxon in Europe and North America, but its distribution in China has not been found yet. The specimens from China labelled "G. cyanescens" are either G. alpinus or G. flavocyanescens. Gyroporus pseudomicrosporus, originally described from China by Zang (1986), is characterised by the cyanescent discolouration when bruised, the decurrent hymenophore, the short tubes measuring 2-4 mm long, the eccentric stipe and the small ellipsoid to ovoid basidiospores. These traits match well with those of the genus Gyrodon Opat. In conclusion, there are still three cyanescent species in China, but they are G. alpinus, G. brunneofloccosus and G. flavocyanescens.