Four New Species of Strobilomyces (Boletaceae, Boletales) from Hainan Island, Tropical China

Strobilomyces, one of the most noticeable genera of Boletaceae (Boletales), is both ecologically and economically important. Although many studies have focused on Strobilomyces in China, the diversity still remains incompletely understood. In the present study, several collections of Strobilomyces from Hainan Island, tropical China were studied based on morphology and molecular phylogenetic analyses. Four species are described as new, viz. S. baozhengii, S. conicus, S. hainanensis, and S. pachycystidiatus. Detailed descriptions, color photos of fresh basidiomata, and line drawings of microstructures of the four species are presented.

Strobilomyces plays an important role in maintaining the biodiversity of forest ecosystems, for species of the genus are usually symbiotic with many plants including Dipterocarpaceae, Fabaceae, Fagaceae, Myrtaceae, and Pinaceae [3,[5][6][7].Besides ecological value, the edible and medicinal values of the genus were also noted, as for example, S. confusus Singer, S. glabriceps W.F. Chiu, and S. strobilaceus (Scop.)Berk.were put on the Chinese edible mushroom list; S. strobilaceus was believed to have anticancer activity [16,17].
In China, species of Strobilomyces are also abundant.Currently, approximately 32 species of the genus have been uncovered [3,[5][6][7]13,[18][19][20][21][22].Among them, most are from temperate and subtropical China, while a few species are from tropical regions of China.It is well known, tropical China especially Hainan Island is a biodiversity hotspot [23,24].With more field investigations in the region, more species of the genus are expected to be revealed.During field investigations of Hainan Island, several collections of Strobilomyces were compiled; further morphology and molecular phylogenetic analyses confirm that these collections represent four novel species.They are described in an effort to further demonstrate the species diversity of Boletaceae in tropical China.

Morphological Studies
The fresh basidiomata were described and photographed in the field in daylight, then dried at 60 • C for 12 h.Dried specimens were deposited in the Fungal Herbarium of Hainan Medical University, Haikou City, Hainan Province of China (FHMU).Color codes comply with Kornerup and Wanscher [25].The pileipellis sections were cut between the center and the margin of the pileus and the stipitipellis sections were taken from the middle part along the longitudinal axis of the stipe [26].Samples were rehydrated using 5% KOH.All microscopic structures were drawn by freehand from rehydrated material.For basidiospores, the notation 'n/m/p' indicates 'n' basidiospores from 'm' basidiomata of 'p' collections.Dimensions of basidiospores are expressed 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 expressed in parentheses.Q indicates the length/width ratio of basidiospores; Qm indicates the average Q of basidiospores and is given with standard deviation.The basidiospores were examined by Zeiss Sigma 300 scanning electron microscopy (SEM) using a small piece of hymenophore (2-5 mm diameter) from one dried specimen, which was sprayed with gold by ion sputtering.

Dataset Assembly
A total of forty-five sequences (thirteen of 28S, thirteen of ITS, thirteen of tef1, and six of rpb2) from fifteen specimens were newly generated and edited sequences were deposited in GenBank, and accession numbers of 28S, ITS, tef1, and rpb2 are provided in Table 1.The sequences were aligned with selected sequences from previous studies and GenBank (Table 1).Anthracoporus holophaeus (Corner) Yan C. Li & Zhu L. Yang (HKAS50508 and HKAS59407) was chosen as outgroup following Wu et al. [20].Sequences of 28S, ITS, tef1, and rpb2 were aligned, using MUSCLE (Edgar 2004) separately to test for phylogenetic conflict.There were no conflicts in the topologies of these trees, indicating that phylogenetic signals from different gene fragments were congruent.The sequences of the different genes were concatenated using Phyutility v. 2.2 for further analyses [32].

Phylogenetic Analyses
Maximum likelihood (ML) and Bayesian inference (BI) were used to reconstruct phylogenetic trees based on the combined nuclear dataset (28S + ITS + tef1 + rpb2).ML analysis was conducted with the program RAxML 7.2.6 running 1000 replicates combined with an ML search.Bayesian analysis was conducted with MrBayes 3.1 using the Markov Chain Monte Carlo technique and parameters predetermined with MrModeltest 2.3 [41].The best-fit likelihood models for the combined dataset were GTR + I + G, GTR + G, SYM + I + G, and K80 + I + G for 28S, ITS, tef1, and rpb2, respectively.Bayesian analysis of the combined nuclear dataset (28S + ITS + tef1 + rpb2) was repeated for 30 million generations.Trees sampled from the first 25% of the generations were discarded as burnin.The average SD of split frequencies was constrained to be below 0.01, and Bayesian posterior probabilities (PP) were then calculated for a majority consensus tree of the retained Bayesian trees.

MycoBank no: MB 850251
Etymology: "baozhengii" is named for Bao Zheng, a historic Chinese righteous officer, said to have a dark face.
Description: Pileus 2.7-7.5 cm diameter, subhemispherical when young, then convex to applanate; surface dirty white, densely covered with blackish-brown (7F1) to black (11F1), conical scales; margin usually appendiculate with thick and triangular or irregular lacy veil remnants concolorous with pileal surface; context 0.6-1.4cm in thickness in the center of the pileus, white (2A1) to greyish-white (5A1), turning reddish-brown (5B3) then black (7F1) when injured.Hymenophore tubulate, slightly depressed around the apex  Habitat: Solitary or scattered on the ground in forests dominated by fagaceous trees.Known distribution: Southern China (Hainan Province).

Discussion
Although the diagnosis of Strobilomyces is relatively easy, species within the genus are difficult to identify because of an absence of molecular data as well as morphological convergence documented in this group in previous studies [11,12].With the development of molecular phylogenetic analyses, many previously described taxa have been re-evaluated, which enhanced our understanding of Strobilomyces diversity worldwide [7,8,14].For example, S. confusus and S. strobilaceus, two taxa originally described from North America and Europe, respectively [45], were previously thought to be widely distributed species.However, recent studies have indicated that S. confusus and S. strobilaceus actually represent species complexes [7,8,14].Our molecular data also showed that specimens identified as S. confusus or S. strobilaceus were present in different parts of the tree (Figure 1).

Conclusions
Although Strobilomyces is widely distributed in the world, the diversity of this genus has not been completely resolved.In this work, four new species of Strobilomyces, viz.S. baozhengii, S. conicus, S. hainanensis, and S. pachycystidiatus are described based on morphological and molecular phylogenetic analysis, which provides us with further understanding of this genus diversity in tropical China.In the future, we hope that more and more species of the genus will be uncovered.

Table 1 .
Taxa, vouchers, locations, and GenBank accession numbers of DNA sequences used in phylogenetic analyses.The new sequences are in bold.