A New Species and a New Combination of Caloboletus from China
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Phytotaxa 309 (2): 118–126 ISSN 1179-3155 (print edition) http://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2017 Magnolia Press Article ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.309.2.2 A new species and a new combination of Caloboletus from China MING ZHANG1, TAI HUI LI1*, MATTEO GELARDI2, BIN SONG1 & XIANG JING ZHONG3 1State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collec- tion and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China. 2Via Angelo Custode 4a, Anguillara Sabazia 00061, Italy. 3Management Bureau of Xiangtoushan National Nature Reserve of Guangdong, Huizhou 516001, China. * Corresponding author’s e-mail: [email protected] Abstract A new species, Caloboletus xiangtoushanensis sp. nov., is reported from China. This medium-sized bolete is distinguished by a brownish orange to greyish yellow pileus, greyish yellow to olive yellow hymenophore that quickly stains blue when bruised, reticulate stipe, bitter taste and oblong to subfusoid basidiospores 9–13(–13.5) × 4–5 μm. On the basis of its mor- phological and molecular characters (ITS, nrLSU, tef1-α and rpb1), this novel taxon can be assigned to the genus Calobo- letus, sister to Boletus taienus. Accordingly, a new combination C. taienus comb. nov. is also proposed in this study based on both morphological and phylogenetic evidence. Keywords: Boletes, Phylogeny, Taxonomy Introduction Caloboletus Vizzini has recently been erected as a new genus within Boletaceae to accommodate Boletus calopus Pers. and its allies, which “corresponds quite well to the section Calopodes Fr. emend. Lannoy & Estadès of the genus Boletus L.” (Vizzini 2014). The genus is characterized by the combination of the yellow then olive yellow tubes, yellow or more rarely orange to red pores changing to blue when injured, distinctly bitter taste of the context due to the presence of particular chemical compounds, namely calopin and cyclocalopin, smooth basidiospores that are olive brown in deposit, interwoven pileipellis of filamentous hyphae, “Boletus-type” hymenophoral trama and absence of clamp connections (Hellwig et al. 2002; Zhao et al. 2014). Our molecular phylogenetic analyses based on internal transcribed spacer (ITS) region, nuclear ribosomal large subunit (nrLSU), translation elongation factor 1α (tef1-α) and the gene encoding the largest subunit of RNA polymerase II (rpb1) in this study revealed an undescribed species from southern China. Furthermore, a previously described species, Boletus taienus W.F.Chiu should be taxonomically transferred to Caloboletus, both taxa nesting into the well supported Caloboletus clade. Morphological analysis of both species also showed that their characters fit the genus concept. Thus, a new species and a new combination are proposed herein. Materials and methods Morphological studies:—Photographs of the basidiomes were taken in the type locality when collected. Specimens were dried and then deposited in the Fungal Herbarium of Guangdong Institute of Microbiology (GDGM). Methods for morphological descriptions in the previous study by Zhang et al. (2015) were followed. Colours were recorded and described in general terms following Kornerup and Wanscher (1978). Notations “basidiospores (n/m/p)” indicate that the measurements were made on n basidiospores from m basidiomes of p collections. Microstructures were observed from rehydrated materials and line drawings were drawn by free hand. DNA extraction, PCR amplification and sequencing:—Total genomic DNA was extracted from silica-gel- dried materials of each voucher specimen using the Sangon Fungus Genomic DNA Extraction kit (Sangon Biotech, 118 Accepted by Masoomeh Ghobad-Nejhad: 17 May 2017; published: 13 Jun. 2017 Shanghai, China) according to the manufacturer’s instructions. Primer pairs ITS1/ITS4 (White et al. 1990), LR0R/LR5 or LR7 (Vilgalys and Hester 1990), EF1-B-F1/EF1-B-R, and RPB2-B-F1/RPB2-B-R (Wu et al. 2014) were used for the amplification of the internal transcribed spacer (ITS) region, the large subunit nuclear ribosomal RNA (nrLSU) region, the translation elongation factor 1-alpha subunit (tef1-α), and the largest subunit of RNA polymerase II (rpb1), respectively. PCR protocol and sequencing were conducted following the method described in an earlier study (Zhang et al. 2015). Newly generated sequences in this work have been submitted to GenBank. Phylogenetic analyses:—A total of 81 sequences, including 10 newly generated in this study and 71 retrieved from GenBank, were used in phylogenetic analyses. DNA sequences of 4 loci (ITS, nrLSU, tef1-α and rpb1) were aligned using Clustal X v1.81 (Thompson et al. 1997) and manually modified where necessary in MEGA5 (Tamura et al. 2011). Then 4 single-gene datasets were concatenated using MEGA5 (Tamura et al. 2011). The intron regions of protein-coding genes were retained in the final analyses, and the ambiguously aligned regions were detected with Gblocks (Castresana 2000) and excluded. The full alignment of the combined data was submitted to the TreeBASE (20725).The partitioned alignment was then analyzed using RAxML v7.2.6 (Stamatakis 2006) and MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003) for Maximum Likelihood (ML) and Bayesian Inference (BI), respectively. For both BI and ML analyses, the substitution model suitable for the 4 gene partitions were individually determined using the Akaike Information Criterion (AIC) complemented in MrModeltest v2.3 (Nylander 2004). HKY+G, GTR+I+G, SYM+I and SYM+G were chosen as the best model for ITS, nrLSU, tef1-α and rpb1, respectively. All parameters in the ML analysis were kept at their default values except for the model choosing as GTRGAMMAI, and statistical support was obtained using rapid nonparametric bootstrapping with 1000 replicates. BI analysis using selected models and 4 chains were conducted by setting generations to 3 million and stoprul command with the value of stopval set to 0.01. Trees were sampled every 100 generations. The trees were summarized and statistical values were obtained using the sump and sumt commands with the first 25% generations were discarded as burn-ins. Rubroboletus sinicus (W.F. Chiu) Kuan Zhao & Zhu L. Yang and Butyriboletus appendiculatus (Schaeff.) D. Arora & J.L. Frank were selected as outgroups based on recent studies (Zhao et al. 2014). Results Molecular phylogenetic results The multi-locus (ITS+nrLSU+tef1-α+rpb1) dataset included sequences from 23 fungal samples representing 10 taxa. The dataset had an aligned length of 3144 nucleotide sites, 2860 were retained using Gblocks for the final analyses, consisting of 598, 831, 665 and 766 sites (including gaps) for ITS, nrLSU, tef1-α and rpb1, respectively. Phylogenetic trees generated from ML and BI analyses shared the same topology, with only minimal differences in statistical support values, thus, only the ML tree topology is shown (Fig. 1). Our results show that the samples newly collected from China clustered in two independent lineages in the well supported Caloboletus clade (Wu et al. 2014, Zhao et al. 2014) with high statistical support (BT = 100%, PP = 1), which morphologically represent two different species. Taxonomy Caloboletus xiangtoushanensis Ming Zhang, T.H. Li & X.J. Zhong, sp. nov. Mycobank: MB 820363 Etymology: The epithet xiangtoushanensis refers to the type locality Xiangtoushan. Type:—CHINA, Guangdong Province, Huizhou County, Xiangtoushan National Nature Reserve, 23°18′ N, 114°23′ E, 500 m, 7 July 2015, Hao Huang & Ting Li (GDGM 44725, holotype!). Macroscopic characters:—Basidiomes small to medium-sized. Pileus 4–9 cm in diam, subhemispherical to convex or nearly plane; margin usually slightly inrolled, especially when young; surface dry, covered with tomentose or with short fibrils to tomentose squamules, often slightly areolate or cracking from margin to disc, dull red to brownish orange at first (8C3–5C3), gradually fading to greyish yellow to pallid yellow (4C3–2C3) when mature, often with greyish ting. Context 1–1.5 cm thick at center of pileus, white to yellowish (2A2–4A2) with a faint pinkish ting, pink to purplish pink (12A4–14A4) beneath the pileipellis, staining light blue quickly when exposed, more obviously near hymenophore and inconspicuously under pileipellis. Hymenophore 5–8 mm deep, depressed around stipe, yellow, A NEW SPECIES AND A NEW combination OF CALOBOLETUS Phytotaxa 309 (2) © 2017 Magnolia Press • 119 FIGURE1. Phylogenetic relationships among representative specimens of Caloboletus inferred from a multigene (ITS, LSU, tef1-α and rpb1) dataset using the Maximum Likelihood and Bayesian Inference methods (only the ML tree is shown). The bootstrap frequencies (BT ≥ 70 %) and posterior probabilities (PP ≥ 0.90) are shown on the supported branches. greyish yellow to olive yellow (2B8–3B8, 2C8–3C8) when mature, bluing quickly when injured; pores angular, 2–3 per mm, deep orange to reddish orange when young, greenish yellow to olive yellow (2B8–3B8, 2C8–3C8) when mature, staining dark blue quickly when injured. Stipe 3–8 × 1–1.5 cm, central, subcylindrical or clavate, solid, equal to slightly enlarged downwards, yellowish white to pale yellow (2A2–3A2, 2A3–3A3), covered with yellowish red to vivid red (8A8–9A8) reticulation or longitudinal striated ornamentation at first, then fading to greyish yellow with age upwards from the base but often retaining some reddish hues at apex in mature specimens, unchanging to very weakly staining blue when injured; basal mycelium