Mycoscience: Advance Publication doi: 10.47371/mycosci.2021.03.001 Short Communication (Received December 26, 2020; Accepted March 8, 2021) J-STAGE Advance Published Date: March 27, 2021 Short Communication Neoboletus infuscatus, a new tropical bolete from Hainan, southern China Shuai Jianga,b, Hong-Xu Mib , Hui-Jing Xiea , Xu Zhanga , Yun Chenb , Zhi-Qun Liangc, Nian-Kai Zenga,* a Key Laboratory of Tropical Translational Medicine of Ministry of Education, Hainan Key Laboratory for Research and Development of Tropical Herbs, School of Pharmacy, Hainan Medical University, Haikou 571199, China b Yinggeling Branch of Hainan Tropical Rainforest National Park, Baisha, Hainan 572800, China c College of Science, Hainan University, Haikou 570228, China * Corresponding author. Hainan Medical University, Xueyuan Road, Longhua District, Haikou, China. E-mail address: [email protected] (N. K. Zeng). Advance Publication - 1 - Mycoscience: Advance Publication ABSTRACT Neoboletus infuscatus (Boletaceae, Boletales) is described as a new species from Yinggeling of Hainan Tropical Rainforest National Park, southern China. It is morphologically characterized by a large basidioma with a nearly glabrous, brownish yellow, yellowish brown to pale brown pileus, pores orangish red when young, yellowish brown to brown when old, context and hymenophore staining blue when injured, a yellow stipe with red punctuations, surfaces of the pileus and the stipe usually covered with a thin layer of white pruina when young. Phylogenetic analyses of DNA sequences from part of the 28S gene, the nuclear rDNA internal transcribed spacer (ITS) region, and part of the translation elongation factor 1-α gene (TEF1) also confirm that N. infuscatus forms an independent lineage within Neoboletus. Detailed morphological description, color photos of fresh basidiomata and line-drawings of microstructures are provided. Keywords: molecular phylogeny, morphology, new taxon, taxonomy, tropical China Publication Advance - 2 - Neoboletus infuscatus, a new tropical bolete from Hainan, southern China Members of Boletaceae are diverse worldwide and attracted much attentions due to their ectomycorrhizal, edibility, toxicity and pharmaceutical potentialities (Roman, Claveria, & Miguel, 2005; Sitta & Floriani, 2008; Dentinger et al., 2010; Feng et al., 2012; Wu et al., 2013; Chen, Yang, Tolgor, & Li, 2016). With the development of molecular techniques, several new species were discovered. In addition, some species were reassessed and new genera were established to accommodate certain taxa in Boletaceae (Li, Feng, & Yang, 2011; Arora & Frank, 2014; Zeng, Wu, Li, Liang, & Yang, 2014; Gelardi, Simonini, Ercole, Davoli, & Vizzini, 2015; Farid et al., 2018). Neoboletus Gelardi, Simonini & Vizzini was newly segregated from Boletus L. and typified by N. luridiformis (Rostk.) Gelardi, Simonini & Vizzini (Vizzini, 2014). This treatment was phylogenetically supported (Binder & Hibbett, 2006; Nuhn, Binder, Taylor, Halling, & Hibbett, 2013; Arora & Frank, 2014; Urban & Klofac, 2015) and accepted by more and more researchers (Urban & Klofac, 2015; Sarwar, Jabeen, Khalid, & Dentinger, 2016; Gelardi, 2017; Chai et al., 2019; Gelardi et al., 2019; Vadthanarat, Lumyong, & Raspé, 2019; Kuo & Ortiz-Santana, 2020). The genus is characterized by stipitate-pileate or sequestrate basidiomata; when basidiomata stipitate-pileate, pores brown, dark brown toPublication reddish brown when young, becoming yellow when old, tubes always yellow, hymenophore and context staining blue, and stipe usually covered with punctuations (Vizzini, 2014; Wu et al., 2016b; Chai et al., 2019). Yinggeling of Hainan Tropical Rainforest National Park, southern China is considered a hotspot of biodiversity (Jiang et al., 2013). In the past decade, thirteen new species of macrofungi including boletes were described in the region (Liang, An, Jiang, Su, & Zeng, 2016; Liang, Chai, Jiang, Ye, & Zeng, 2017a; Liang, Su, Jiang, & Zeng, 2017b; An, Liang, Jiang, Su, & Zeng, 2017; Zeng, Liang, Tang, Li, & Yang, 2017Advance; Zeng et al., 2018; Chai, Liang, Jiang, Fu, & Zeng, 2018; Chai et al., 2019; Xue et al., 2019; Wang et al., 2020; Xie et al., 2020). With further field investigations, more new species are expected to be discovered. Recently, we encountered some specimens in Yinggeling, which of the genus proved to be a new species of genus Neoboletus by morphological and molecular phylogenetic analyses. Therefore, a detailed description of the new species was presented. - 3 - Mycoscience: Advance Publication Fresh specimens were photographed in the field, a collection number was given for each specimen, and a detailed record of morphological characters of fresh fruit body was also made, then the specimens were dried with air flow dryer and deposited in the Fungal Herbarium of Hainan Medical University (FHMU), Haikou City, Hainan Province of China. Color codes are from Kornerup and Wanscher (1981). Sections of the pileipellis on the pileus were cut perpendicularly and halfway between the center and margin of the pileus. Sections of the stipitipellis on the stipe were taken from the middle part along the longitudinal axis of the stipe. KOH (5%) was used as a mounting medium for microscopic studies. All the microscopic structures were observed and measured with an optical light microscope (CX23, Olympus, Tokyo, Japan), then drawn freehand from rehydrated material. The number of measured basidiospores is given as n/m/p, where n represents 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 (5th to 95th percentile), and extreme values (a and d), whenever present (a < 5th percentile, d > 95th percentile), are in parentheses. Q refers to the length/width ratio of basidiospores; Qm refers to the averagePublication Q of basidiospores and is given with standard deviation. Total genomic DNA was obtained with Plant Genomic DNA Kit (TIANGEN Company, Beijing, China) from materials dried with silica gel according to the manufacturer’s instructions. Primer pairs used for amplifying the nuclear ribosomal large subunit RNA (28S) were LROR/LR5 (Vilgalys & Hester, 1990; James et al., 2006), ITS5/ITS4 (White, Bruns, Lee, & Taylor, 1990) for the nuclear rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA (ITS),Advance and EF1-α-F/EF1-α-R (Mikheyev, Mueller, & Abbot, 2006) for the translation elongation factor 1-α gene (TEF1). PCR was performed in a total volume of 25 μL containing 13 μL GoTaq® Green Master Mix (Promega, Madison, USA), 2 μL per primer (10 mM), 2 μL DNA template and 8 μL nuclease-free water. PCR reactions were performed with 4 min initial denaturation at 95 °C, followed by 34 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C (28S and ITS) or 53 °C (TEF1) for - 4 - Neoboletus infuscatus, a new tropical bolete from Hainan, southern China 30 s and extension at 72 °C for 120 s, a final extension at 72 °C for 7 min. 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, Guangzhou, China) with the same primers used for PCR amplifications. Forward or reverse sequences were compiled with BioEdit (Hall, 1999). Assembled sequences were deposited in GenBank (Table 1). Nine sequences (four of 28S, one of ITS, four of TEF1) from four collections were newly generated and deposited in GenBank (Table 1). For the concatenated data set, the 28S, ITS, and TEF1 sequences were aligned with selected sequences from GenBank and previous studies (Table 1). Sutorius subrufus N.K. Zeng, H. Chai & S. Jiang was chosen as outgroup based on the phylogeny in Chai et al. (2019). Single- gene phylogenetic trees based on the 28S, ITS, and TEF1 fragments, respectively, were analyzed to test for phylogenetic conflict. 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 conflict. Then, the sequences of the different genes were aligned using MUSCLE (Edgar, 2004), and alignments were purged from unreliably alignedPublication positions and gaps using Gblocks (Castresana, 2000). The sequences of the different genes were concatenated using Phyutility v2.2 for further analyses (Smith & Dunn, 2008). The combined nuclear data set (28S + ITS + TEF1) was 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 1,000 replicates combined with a ML search. A bayesian analysis with MrBayes 3.1 (Huelsenbeck & Ronquist, 2005) implementing the MarkovAdvance 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 three-gene combined data set of Neoboletus, the best-fit likelihood models of 28S, ITS and TEF1 were GTR+I+G, HKY+G and SYM +G, respectively. Bayesian analysis was repeated for 5.4 million generations and sampled every 100 generations; - 5 - Mycoscience: Advance Publication trees sampled from the first 25% of the generations were discarded as burn-in; the average standard deviation of split frequencies was restricted