Retiboletus Huanggangensis, A New Species of Retiboletus From China


 A new species of the genus Retiboletus, Retiboletus huanggangensis, collected from Huanggang mountain, Wuyishan Nature Reserve, Yanshan County, Shangrao City, Jiangxi Province, China, is described based on morphological and phylogenetic analysis, nuclear ribosomal large subunit (nrLSU), the translation elongation factor1-α gene (TEF1-α) and the RNA polymerase II second largest subunit gene (RPB2) were used for phylogenetic analysis. Retiboletus huanggangensis is morphologically characterized by its white to grayish black to black pileus, white to pale yellow to black stipe, white to pale yellowish white hymenophore, white to grayish white or pale yellow context, prominently and coarsely reticulate over the upper 3/4 or overall, changing orange yellow to rusty yellow when its pileus injured. For phylogenetic analysis, the specimens from Huanggang mountain, grouped together with Retiboletus fuscus, but they have some morphological differences, Retiboletus fuscus has slightly narrower basidia, and pileipellis much narrower. Descriptions and hand drawings of the new species and comparisons with similar species are shown below.

Retiboletus is characterized by the following characters: basidiomata small to medium-sized, hymenophore pallid, grayish white or pall yellow, unchanging or yellowish brown or orange-brown when injured; stipe reticulate; context white, pall yellow or bright yellow, unchanging or changing light yellow to orange-brown; clamp connections absent in all tissues; spore greenish brown to yellow-brown in KOH, basidiospores smooth, ellipsoid to subsubfusoid to subfusoid (Binder and  Recently, we investigated the diversity of Macrofungi in Wuyishan Nature Reserve, Jiangxi Province, and collected several specimens of Boletaceae. Through morphological and phylogenetic analysis, we found a new species of Retiboletus.

Specimens collection
The specimens were collected on the ground of coniferous and broad-leaved mixed forest in Wuyishan Nature Reserve, on July 31 and August 23, 2021.
Firstly, clean up the sundries around the specimen, put a scale and label on the edge of the specimen, take photos, and nally dig out the specimen with a shovel and put it in the sampling box.

DNA extraction, ampli cation, and sequencing
The whole genome DNA was extracted from dried specimens (HFJAU10002, HFJAU10003, HFJAU10004) by optimized CTAB method (Doyle 1987;Huang et al. 2000). The primer pairs LR0R-LR5 and TEF1-983F-TEF1-1567R were used to amplify the large ribosomal subunit sequence region (Vilgalys and Hester 1990;James et al. 2006) and the translation elongation factor 1-α region (Wu et al. 2014), respectively. The RNA polymerase II second largest subunit gene (RPB2) was ampli ed following the method described by Réblová et al. (2011). PCR products were detected in 1% agarose gels, and then sent to TSINGKE Biological Technology for sequencing.

Macroscopic and microscopic studies
Macroscopic features were mainly made from the eld records and photographs of the basidiomata, color codes followed Kornerup & Wanscher (1981). Micromorphological descriptions are based on dried materials rehydrated in 5% KOH and stained in ammoniacal Congo red. Freehand sections were done on Nikon SMZ1270 following the standard method described in previous studies (Li et  The number of measured basidiospores is given as n/m/p, which means that the measurements were made on n basidiospores from m basidiomata of p collections. The sizes of basidiospores are given using the notation (a)b-c(d), where the range b-c represents a minimum of 90% of the measured values, and extreme values (a and d) are given in parentheses. Q represents the ratio of length/width of the spores. Q m refers to the average Q of basidiospores ± sample standard deviation.

Phylogenetic analysis
Visualization and editing of the sequences were done with BioEdit v7.0.9 (Hall 1999), and submitted to NCBI online website for Nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) to determine which genus the species belongs to. Based on BLASTn results, all available sequences of this genus used in recent studies and closely related to Retiboletus were downloaded from GenBank, shown in Table 1. For ML analyses, based on the evaluation of nrLSU dataset, using GTRGAMMAI as evolutive model (Stamatakis 2006), choosing the rapid bootstrap analysis with the supports were calculated, using nonparametric bootstrapping with 1000 replicates (Joseph 1985).
For BI analyses, substitution models of partition in the datasets were determined using the Akaike Information Criterion (AIC) implemented in PartitionFinder 2 (Lanfear et al. 2016). For the combined dataset GTR+I+G, SYM+G and SYM+G were chosen as best-t likelihood models for LSU, TEF1-α and RPB2 partitions, respectively. Two MCMC runs with four chains each were run for 4,000,000 generations and sampled every 1000 generations. At the end of the runs, the average deviation of split frequencies was 0.005509; Other parameters were kept at their default settings; Trees were summarized and posterior probabilities (PPs) were calculated after discarding the rst 25% generations as burn-in.