Population Genetics Of Trogia Venenata, Molecular Phylogeny And Biogeography Of Cantharellus Cibarius Species Comples (CCSC)

The mushroom Trogia venenata is mainly distributed in Yunnan, southwestern China. Recently, epidemiological and toxicological analyses suggested this mushroom as the leading culprit for SUDs. To find out the true pathogenic mechanism of Trogia venenata, researchers analyzed several possible factors, such as two non-protein amino acids, barium concentration and so on. In this study, we analyzed the genotypic diversity, population structure, reproductive mode in nature populations of Trogia venenata. Cantharellus cibarius species complex (CCSC) is of significant economic and ecological importance. Yunnan is one of the centers of modern species diversity, however, the molecular systematics and biogeography researches of CCSC are insufficient in this area, and the existing systematics researches are mainly confined to samples in the Europe, United States and Africa. Here, phylogenetic and biogeographical analyses were conducted using multiple sequences of four loci for CCSC. The main results are as follows:1. Population genetic analyses of T. venenataBased on the distributions of SUDs from earlier epidemiological surveys, we chose seven sites for this study. These sites cover the known geographic distribution of SUDs and are located in five counties:Tengchong, Dayao, Xiangyun, Binchuan and Heqing in Yunnan Province. All seven sites are located in difficult-to-access and remote mountainous regions. The fruiting bodies of our samples were obtained from the surface of tree stumps, fallen tree trunks, and rotten wood. A total of 232 fruiting bodies were collected during the rainy season (June-August) in 2010 and 2011. Four nuclear molecular markers (ITS, β-tub、rpb2 and tef1-α) were used to conduct population genetic analyses for 232 samples of T. venenata. The sequences were analyzed to identify the sequence variation at four DNA fragments and the patterns of genetic variation within and among populations. Our ITS sequence analyses confirmed that all the isolates belonged to the same species. Within individual geographic populations, we found significant deviations of genotype frequencies from Hardy-Weinberg expectations, with the overall observed heterozygosity lower than that expected under random mating, consistent with prevalent inbreeding within local populations. The geographic populations were overall genetically differentiated. Interestingly, while a positive correlation was found between population genetic distance and geographic distance, there was little correlation between genetic distance and barium concentration difference for the geographic populations. Our results suggest frequent inbreeding, geographic structuring, and limited gene flow among geographic populations of T. venenata from southwestern China.2. Molecular phylogeny and biogeography of Cantharellus cibarius species complex (CCSC)414 samples of Cantharellus cibarius species complex (CCSC) fruitbody collected from Yunnan and Europe were studied. Firstly, three single copy genes (tef1-a, rpbl and chsl) and one mitochondrial gene (ATP6) were used to reconstruct phylogenetic relationship and identify phylogenetic species.309 tefl-a sequences were successfully amplified and sequenced from the samples. Through comparisons with our sequences, there were 6 closely related species retrieved from GenBank.47 unique sequence types or genotypes were identified from the tefl-a data set by GenAlEx from 309 tefl-a sequences aligned by MAFFT and selected conserved blocks by Gblocks. Secondly, the composed set of 47 tefl-a genotype sequences and 47 reference sequences was used reconstruct phylogeny.7 branches were identified from the phylogenetic tree-4 branches (A, E, F and G) only included samples from Yunnan and 3 branches (B, C and D) only included Europe samples. Genealogical Concordance Phylogenetic Species Recognition (GCPSR) identified 4 known species (C. tenuithrix、C. amethysteus、C. ferruginascens and C. lewisii), one new phylogenetic species, clade A (Provincially adopted as C. tuberculosporus) and C (Provincially adopted as C. cibarius) include more cryptic species. Evident endemism was detected in CCSC and Yunnan was identified as the center of species diversity. Furthermore, molecular dating suggested that CCSC was divergent from the estimated 209±32 Mya (121-335Mya,95% HPD) during the evolutionary processes and chanterelles may be more primitive than gilled fungi. Historical distribution reconstructions based on Maximum Likelihood method and Bayesian Binary MCMC analysis strongly supported that Southern Africa and Southern USA were the original regions for CCSC, then spread to northern Africa, Eurasia. However, the endemism and disjunctions of CCSC between different continents would be from the allopatric speciation raised by multiple dispersal and isolation events during the Ice Age, or due to recent human-mediated introductions.