New species of Retiboletus (Boletales, Boletaceae) from China based on morphological and molecular data

Abstract Species of the genus Retiboletus in China were investigated based on morphology and phylogenetic analysis of DNA sequences from the nuclear ribosomal large subunit (nrLSU) and the translation elongation factor 1-α gene (TEF1-α). Nine species were recovered from China, including two new and seven known species. The new species, namely Retiboletus ater and R. sinogriseus, from southwestern and northeastern China respectively, are documented and illustrated in this paper. Retiboletus ater is morphologically characterized by its black to grayish black pileus, white to grayish hymenophore, black to blackish stipe and white to grayish white context. Retiboletus sinogriseus is morphologically characterized by its brown to grayish-brown pileus, yellow to grayish-yellow hymenophore, pale yellow to brownish stipe and yellow to brownish-yellow context. Descriptions and line drawings of these two novel species and their comparisons with allied taxa are presented.


Introduction
The genus Retiboletus Manfr. Binder & Bresinsky was erected to accommodate Boletus ornatipes Peck and allied species (Binder and Bresinsky 2002). The genus is morphologically characterized by the combination of the following characters: pileus convex to plane, dry, subtomentose, black, dark gray, mustard yellow or olive-brown; hymenophore pallid, grayish or yellow, unchanging or staining brown or orange-brown when bruised; hymenial cystidia present; stipe reticulate; context pallid, yellow or vivid yellow, unchanging or bruising orange-brown; clamp connections absent; spore deposit olive-brown to yellow-brown, basidiospores smooth, ellipsoid to subfusoid, inamyloid and partly dextrinoid (Binder and Bresinsky 2002;Zeng et al. 2016). The separation of Retiboletus from Boletus s. str. and its establishment at the generic rank is strongly supported Zeng et al. 2016Zeng et al. , 2018Badou et al. 2018). So far thirteen species of this genus have been described from North/Central America and East Asia, seven out of which have been reported from China (Binder and Bresinsky 2002;Zeng et al. 2016Zeng et al. , 2018. During field investigation of Boletaceae across China, we encountered two impressive Retiboletus species from southwestern and northeastern China, respectively. These species can be easily recognized by their conspicuous colors in the field. Molecular phylogenetic analysis of this genus based on the nuclear ribosomal large subunit (nrLSU) and the translation elongation factor 1-α gene (TEF1-α) indicated that they represent two distinct species. Combined with morphological characters, Retiboletus ater and R. sinogriseus, are proposed and described herein. It is noteworthy that an additional collection from northeastern China, labeled R. aff. kauffmanii (HY56), was included in our molecular phylogenetic analysis. But its classification can't be clarified due to its paucity of mature material. Further collections are needed to better estimate its taxonomic status.

Morphological studies
Specimens were described and photographed in the field and deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN) and Herbarium of Jilin Agriculture University (HMJAU). In the descriptions, macroscopic characters were based on field notes and color slides of the specimens. Color codes are from Kornerup and Wanscher (1981). Microscopic characters were from the observations of the specimens through light microscopy. For microscopic study, dried materials were sectioned and mounted in 5% KOH solution. Sections of the pileipellis were cut tangentially and halfway between the center and margin of the pileus. All measurements were made in KOH mounts and observed under the light phase. All line drawings of microstructures were made from rehydrated material. Melzer's reagent was used for testing color reactions of the tissue fragments to the solution. The notations "basidi-ospores (n/m/p)" indicate that the measurements were made on n basidiospores from m basidiomata of p collections. The expressions (a)b-c(d) stand for the dimensions of basidiospores; the range b-c contains a minimum of 90% of the measured values, a and d in the brackets stand for the extreme values. The following abbreviations are used: Q (length/width ration of basidiospores) and Q m (average Q ± standard deviation).

DNA extraction, PCR and DNA sequencing
Protocols for DNA extraction, PCR, sequencing and sequence alignment followed those in Vadthanarat et al. (2019), Gelardi et al. (2019), Zhang et al. (2019) and references therein. The primer pair used for amplifying the nrLSU region was LROR and LR5 (Vilgalys and Hester 1990). DNA sequences were compiled with SeqMan (DNASTAR Lasergene 9). Sequences were aligned with MUSCLE 3.6 (Edgar 2004) and manually adjusted where necessary. Edited sequences were deposited in GenBank (Table 1).

Phylogenetic analysis
19 sequences (10 of nrLSU and 9 of TEF1-α) from 10 collections were newly generated in this study and aligned with selected sequences from GenBank and previous studies (Binder and Bresinsky 2002;Ortiz-Santana et al. 2007;Zeng et al. 2016Zeng et al. , 2018 (Table  1)
Habitat Discussion. Retiboletus ater is characterized by the black to blackish or gray to yellowish-gray pileus, the white to yellowish hymenophore, the gray to brownish-gray stipe, the prominent and coarse reticulum over the upper 1/3 of the stipe and the trichoderm pileipellis with hyphae 9-15 μm wide. It generally shares the same colored pileus and hymenophore with R. fuscus (Hongo) N.K. Zeng & Zhu L. Yang, R. griseus (Frost) Manfr. Binder & Bresinsky, R. nigrogriseus N.K. Zeng, S. Jiang & Zhi Q. Liang, and R. pseudogriseus N.K. Zeng & Zhu L. Yang. However, R. fuscus is characterized by an overall reticulate stipe, slight longer basidiospores (9-12 × 3.5-4.5 μm) and narrower pileipellis hyphae (4-8 μm wide) (Zeng et al. 2016). Retiboletus griseus has a reticulum over the upper 2/3 of the stipe, a cream or grayish-brown stipe often with orange-yellow stains when hurt, and a distribution in North/Central America (Smith and Thiers 1971;Ortiz-Santana et al. 2007). Retiboletus nigrogriseus is characterized by the white to olivaceous contex in the stipe, the entirely reticulate stipe and the cutis pileipellis with hyphae 4-10 μm wide. Retiboletus pseudogriseus has a grayish white pileus which is covered with brown to blackish brown squamules, white context becoming brown when injured, and a slender and completely reticulate stipe.
Habitat Discussion. Retiboletus sinogriseus has a grayish-brown to brown pileus, a pale yellow to blackish-yellow stipe. Such traits are very similar to those of R. griseus. Interestingly, R. sinogriseus clusters with R. griseus with strong statistical support (Fig. 1). However, R. griseus, originally described from North America but not found in China yet, has a distinctly pallid hymenohore and broad pileipellis hyphae which are up to 17 μm wide (Singer 1947;Smith and Thiers 1971;Ortiz-Santana et al. 2007). Additionally, the R. sinogriseus/R. griseus clade is clustered with R. zhangfeii N.K. Zeng & Zhu L. Yang, R. nigrogriseus and R. nigerrimus (R. Heim) Manfr. Binder & Bresinsky (however without bootstrap support). In this assemblage, R. zhangfeii differs significantly from R. sinogriseus by its differently colored pileus, hymenophore, stipe and context (Zeng et al. 2016). Retiboletus nigrogriseus has a black to gray pileus, white to grayish white hymenophore, white to olivaceous contex in the stipe and much smaller basidiospores 8-10.5 × 3.5-4.5 μm. Retiboletus nigerrimus, originally described from Papua New Guinea, has a pileus with a distinctive blue tinge, a context lemon yellow in pileus and orange in the base of stipe and longer and narrower basidiospores 11.5-14.5 × 3.6-4.6 μm (Heim 1963).
Nine species of Retiboletus were recorded from China, including two new species described herein. For the convenience of identification, a key to the species in China is given below.