Four New Species and a New Combination of Boletaceae (Boletales) from Subtropical and Tropical China

Previous studies have shown that boletes are abundant and diverse in China, especially in tropical and subtropical regions. In the present study, morphological, ecological, host relationship, and a four-locus (28S, tef1, rpb1, and rpb2) molecular phylogenetic analyses were used to study the family Boletaceae in subtropical and tropical China. Four new bluing species are described from three genera, viz. Boletellus verruculosus (Chinese name疣柄条孢牛肝菌), Xerocomellus tenuis (Chinese name细柄红绒盖牛肝菌), Xer. brunneus (Chinese name褐盖红绒盖牛肝菌), and Xerocomus zhangii (Chinese name张氏绒盖牛肝菌). Moreover, the genus Nigroboletus is treated as a synonym of Xerocomellus, and a new combination, namely Xer. roseonigrescens (Chinese name玫瑰红绒盖牛肝菌), is proposed.


Introduction
Boletaceae is an important group of Boletales (Basidiomycota).The majority of boletes are ectomycorrhizal fungi with favorable ecological and economic values, while a few are poisonous or bitter-tasting [1][2][3][4][5][6][7].Besides, the taxonomy of the family Boletaceae has been a hot topic for scholars both in China and abroad.
Currently, there are about 1200 species of boletes globally, one-third of which are confirmed to be distributed in China [37].The distribution of the family in China is territorial, mostly in subtropical and tropical areas, with obvious regional characteristics and a few species distributed across continents [4,42,44].However, China is a vast territory with a complex and diverse geography that harbors numerous unknown fungal species.This study aims to report new bluing boletes that are distributed in the subtropical and tropical regions of China, specifically Anhui and Yunnan Provinces.Meanwhile, the system position of Nigroboletus Gelardi, Vizzini, E. Horak, T.H. Li & Ming Zhang is rediscussed, then a synonym of Xerocomellus Šutara (Nigroboletus) and a new combination (Xer.roseonigrescens) are proposed.

Collection Sites and Sampling
Specimens were collected from Anhui Province and Yunnan Province in eastern and southwestern China, respectively.The fresh basidiomata were recorded and photographed in the field, then dried at about 50-60 • C for 12 h.The dried specimens were deposited at the Mycological Herbarium of Kunming Medical University (MHKMU).

Morphological Studies
Macroscopic descriptions were based on detailed notes and photographs taken of fresh basidiomata.Color codes followed Kornerup and Wanscher [45].For microscopic studies, 5% KOH and 1% Congo red solution (w/v) were used as mounting medium and staining agent, respectively.Microscopic structures were examined under a compound light microscope (DM2500, Leica Microsystems, Wetzlar, Germany).Basidiospores of dried specimens were examined using a ZEISS Sigma 300 scanning electron microscope (Carl Zeiss AG, Oberkochen, Germany).The following notations were used in this paper: [n/m/p] denotes 'n' basidiospores measured from 'm' basidiomata of 'p' collections; Q means the length/width ratio of a basidiospore in side view, and Qm is the average Q of all basidiospores ± standard deviation.

Dataset Assembly
Fifty-nine sequences (11 of ITS, 14 of 28S, 11 of tef1, 10 of rpb1, and 13 of rpb2) from 14 collections were newly generated.All assembled sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov,accessed on 6 May 2024) (Table 1).Due to the high variation among species, the ITS fragment is an ideal candidate for identifying the species of most fungal groups [7,46,[50][51][52][53][54][55].However, it is usually unsuitable for building a system tree, especially at high classification levels, due to alignment difficulties.Thus, in the present analysis, ITS is excluded from the concatenated dataset (28S + tef1 + rpb1 + rpb2) of the family Boletaceae.Sequences for the dataset were selected from previous studies or downloaded from GenBank using the BLAST option (Table 1).Genera phylogenetically closely related to our targeted genera (Boletellus Murrill, Xerocomellus, and Xerocomus Quél.) in Boletaceae were chosen as ingroups, and generic types in Paxillaceae were chosen as outgroups based on the analysis of Binder and Hibbett [21].All sequences were aligned using MUSCLE v3.6 [56] and then manually adjusted on BioEdit v7.0.9where necessary [57].SequenceMatrix 1.7.8 was used to concatenate the four gene fragments.The single-gene phylogenetic trees were analyzed separately and are shown in Supplementary Materials.

Phylogenetic Analyses
The combined dataset was analyzed using maximum likelihood (ML) and Bayesian inference (BI).Maximum likelihood tree generation and bootstrap analysis were performed using the program RAxML7.2.6, running 1000 replicates combined with an ML search [58].Bayesian analyses were performed using MrBayes v3.2 on the CIPRES portal [59,60].Mr-Modeltest 2.3 was used to estimate the optimal evolution models for each subset using the Akaike information criterion (AlC) [61].For the combined dataset, the best-fit likelihood models of 28S, tef1, rpb1, and rpb2 were GTR + I + G, GTR + I + G, HKY + I + G, and K80 + I + G, respectively.Bayesian analysis of the combined nuclear dataset was repeated for 4 million generations and sampled at intervals of 1000.Once the average standard deviation of split frequencies went below 0.01, the run was terminated.Trees sampled from the first 25% of the generations were discarded as burn-in, and Bayesian posterior probabilities (PP) were then calculated for a majority consensus tree of the retained Bayesian trees.Notes: C = Central, E = Eastern, N = Northern, S = Southern, SE = Southeastern, SW = Southwestern; * holotype; newly obtained sequences are in boldface.

Molecular Data
The combined dataset (28S + tef1 + rpb1 + rpb2) includes 112 taxa with 3601 nucleotide sites, and the alignment is available at TreeBase (Accession 31120).The tree topologies generated by the BI and ML analyses are almost identical, while the statistical support for certain relationships is slightly different.The ML tree inferred using RAxML is shown, together with the support values (Figure 1).On the basis of the molecular tree, our specimens form species-level lineages, which belong to three genera.In the Boletellus clade, the first new species, Bol.verruculosus, is strongly supported (BS = 100, PP = 1) as an independent branch, with Bol.putuoensis N.K. Zeng, Yi Li, Chang Xu, Xu Zhang & J.R. Wang as its sister group (BS = 100, PP = 1).In the Xerocomellus group, our specimens form two lineages.The second new taxon, Xer.brunneus, including HKAS56311 and our collection (MHKMU L.P. Tang 3774), forms a separate branch with strong statistical support (BS = 100, PP = 1), and clusters as a sister clade to Xer. bolinii J.A. Bolin, A.E. Bessette, A.R. Bessette, L.V. Kudzma, J.L. Frank & A. Farid with weakly statistical support (BS = 64).The third new taxon, Xer.tenuis, including two collections (MHKMU R. Xue 100 and MHKMU R. Xue 94), is strongly supported as an independent branch with strong statistical support (BS = 100, PP = 1) (Figure 1).Unexpectedly, the type species of the genus Nigroboletus forms a lineage within the species of Xerocomus, with strong statistical support (BS = 100, PP = 1).In the Xerocomus clade, the fourth new taxon, X. zhangii, including two collections (MHKMU L.J. Su 225 and 225-1), forms a separate branch with strong statistical support (BS = 100, PP = 1) and clusters with X. fulvipes Xue T. Zhu & Zhu L. Yang and X. galbanus L. Fan, N. Mao & T.Y.Zhao (BS = 100, PP = 1).

Xerocomellus roseonigrescens
Holotype: GDGM43238 (Guangdong Province, China).Notes: Nigroboletus roseonigrescens was originally proposed as the type species of the monotypic genus Nigroboletus [78].However, our molecular phylogeny reveals that this species should be a member of Xerocomellus, although no close relatives have been found yet (Figure 1).Morphologically, N. roseonigrescens also shares the discoloration-prone basidiomata, velvety pileus surface, yellow-tinted pores, and context with Xerocomellus species.Thus, Nigroboletus should be treated as a synonym of the genus Xerocomellus, and the type species of this genus should be treated as a new combination, namely Xer.roseonigrescens.This taxon can be easily separated from other species in the pastel pink pileus, the dull grayish to blackish discoloration of the basidiomata tissues when injured [78].
Xerocomellus tenuis L.P. Tang & R. Xue sp.nov.(Figure 3).MycoBank: MB 851815.Chinese Name: 细柄红绒盖牛肝菌 Etymology: Latin "tenuis", refers to the slender stipe.Diagnosis: Differs from other species by a very small-sized basidioma, a cracked pileus covered with reddish appressed scales, a grayish yellow to brick red hymenophore, a yellowish brown to reddish brown stipe, and a pileipellis composed of inflated hyphae.
Morphologically, the species is similar to Xerocomellus corneri Xue T. Zhu & Zhu L. Yang and Xer.carmeniae Garza-Ocañas, J. García & de la Fuente in the size and the color of the pileus.However, Xer. corneri has a larger basidioma, a red brown to dull brown pileus, and a purple-toned stipe [36]; Xer.carmeniae has a yellowish to grayish green hymenophore, wider basidiospores measuring 10.5-13.6 × 5.7-7.8µm, and a distribution in northeastern Mexico [83].
Xerocomus zhangii L.P. Tang, R. Xue & L.J. Su sp.nov.(Figure 5).MycoBank: MB 851817.Chinese Name: 张氏绒盖牛肝菌 Etymology: Latin "zhangii"; is named after the family name of the selfless senior M Chengyu Zhang (张澄宇), in appreciation of his help in collecting specimens in An Province, as well as his contributions to and efforts in the development of the wild mu Notes: Molecularly, Xerocomellus brunneus is closely related to Xer. bolinii and Xer.tenuis.However, Xer. bolinii has a pinkish brown pileus and stipe and is distributed in southeastern USA [77]; Xer.tenuis has a very small basidioma with reddish brown-toned pileus and stipe, and a pileipellis with inflated hyphae (See above).

Discussion
Boletes are widely distributed in the subtropical and tropical regions of China.In present study, we propose four new species, a new combination, and treat Nigroboletus a synonym for Xerocomellus based on a comprehensive analysis of morphology, molecu biology, habitat, and host relationships.
Xerocomus fulvipes (Holotype HKAS68246) was originally described from Yunn Province and also reported from Henan Province by Wu et al. in 2016 [36].Howev MycoBank: MB 851817.Chinese Name: 张氏绒盖牛肝菌 Etymology: Latin "zhangii"; is named after the family name of the selfless senior Mr. Chengyu Zhang (张澄宇), in appreciation of his help in collecting specimens in An-hui Province, as well as his contributions to and efforts in the development of the wild mushroom industry in Anhui and Yunnan Provinces.
Diagnosis: Differs from other species by a light brown to yellowish brown pileus, cyanescent context and hymenophore, a pale yellow to reddish stipe covered with longitudinal stripes.
Known distribution: Currently only known in Anhui Province (elevation about 200 m), eastern China.

Discussion
Boletes are widely distributed in the subtropical and tropical regions of China.In the present study, we propose four new species, a new combination, and treat Nigroboletus as a synonym for Xerocomellus based on a comprehensive analysis of morphology, molecular biology, habitat, and host relationships.
Xerocomus fulvipes (Holotype HKAS68246) was originally described from Yunnan Province and also reported from Henan Province by Wu et al. in 2016 [36].However, HKAS52556 from Yunnan Province was mislabeled as the holotype of this species in the molecular tree [36], and no sequence of the type specimen for this species could be found in GenBank.Recently, a new species, X. galbanus, described from Shanxi Province, was found to be closely related to X. fulvipes (HKAS52556) [79].Interestingly, the specimen (HKAS76666, Henan Province) previously identified as X. fulvipes by Wu et al. in 2016 [36] clusters with the type specimen of X. galbanus in our molecular tree.Therefore, according to the current data, X. fulvipes is distributed only in Yunnan Province, southwestern China, while X. galbanus is distributed in Henan and Shanxi Provinces, central and northern China.
Xerocomellus was originally erected to accommodate Xer.chrysenteron and its relatives in 2008 [86].The majority of species in this genus are distributed in North America and Europe [26,[87][88][89], whereas just two species have been identified in China, viz.Xer.communis Xue T. Zhu & Zhu L. Yang and Xer.corneri [36].In the present study, two new species of Xerocomellus were discovered in Anhui and Yunnan Provinces, increasing the species diversity of the genus in China.
Nigroboletus was proposed to accommodate N. roseonigrescens, a species described from tropical China [78].Later, Farid et al. [77] built a relatively well-developed molecular tree comprising a wider range of species and showed that Nigroboletus is strongly supported as the base of all Xerocomellus sequences.Furthermore, our phylogenetic analysis indicates that Nigroboletus is embedded in the Xerocomellus lineage (Figure 1).Morphologically, Nigroboletus also shares some common characteristics with Xerocomellus, such as discoloration-prone basidiomata, velvety pileus surface, yellow-tinted pores and context, and the absence of reticulation on the stipe surface.Thus, we propose that Nigroboletus is improper as a separate genus and that it should be a synonym of Xerocomellus.
Sometimes, simple molecular phylogenetic analyses may reveal the wrong phylogenetic position of species, and similar faults have also occurred in other fungal groups.Shen et al. [90] conducted a systematic study of the genus Hyphodermella J. Erikss.& Ryvarden and suggested that as many integrated taxa as possible should be sampled for molecular phylogenetic analyses to avoid data limitations, especially when proposing new monotypic families or genera.
Figure S1.Phylogenetic tree of Boletaceae based on 28S dataset; Figure S2.Phylogenetic tree of Boletaceae based on tef1 dataset; Figure S3.Phylogenetic tree of Boletaceae based on rpb1 dataset; Figure S4.Phylogenetic tree of Boletaceae based on rpb2 dataset.nology in Yunnan Province under Grant No. 202005AE160004; and Research Project on Undergraduate Educational and Teaching Reforms in Yunnan Province (JG2023001).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 1 .
Species, isolates, locations, and GenBank accession numbers of the DNA sequences used in this study.