Cyclopeptide Toxins Of Lethal Amanitas: Compositions, Distribution And Phylogenetic Implication

Lethal amanitas(Amanita sect. Phalloideae) are responsible for 90%of all fatal mushroom poisonings. More than ten of new lethal Amanita species were discovered continuously and some of them had caused severe mushroom poisoning in China since 2000. However, the contents and distribution of cyclopeptides in these lethal mushrooms remain poorly known. In this study, the diversity of main cyclopeptide toxins in seven Amanita species from Eastern Asia and three species from Europe and North America were systematically analysed and their phylogenetic relationships based on the cyclopeptides were evaluated. The results are as follow:1、Amatoxins and phallotoxins concentration and distribution in different lethal Amanita species: All lethal Amanita species belonged to the section Phalloideae contained amatoxins and phallotoxins, but A.oberwinklerana of the section Lepidella contained no amatoxins and phallotoxins. there were diversities of the cyclopeptides among the lethal Amanita species, The amounts of total toxins ranged from 3.27-14.19 mg.g-1 dry weight. Among them, the analysis of cyclopeptides in A. rimosa and A. fuligineoides were reported for the first time. The analysis of distribution of amatoxins and phallotoxins in various Amanita speciesdemonstrated that the content of phallotoxins was higher than that of amatoxins in A. phalloides and A. virosa. In contrast, the content of phallotoxins was significantly lower than that of amatoxins in all East Asian lethal Amanita species.2、Concentration and distribution of amatoxins and phallotoxins in different tissues: In this study, four species(A. exitialis, A. fuliginea, A.pallidorosea and A. rimosa) with complete developed carpophores were divided into pileus, stipe and volva, and the contents of amatoxins and phallotoxins in different tissues were determined. The results indicated that the highest amount of total toxins and each amatoxins and phallotoxins were found in the pielus, then in the stipe, and the volva had the lowest amount of toxins. The results also revealed the distribution of amatoxins and phallotoxins varied in different tissues. The total amount of amatoxins was higher than the total amount of phallotoxins in pileus,but the ratio of phallotoxins to amatoxins in stipe and volva was higher than that in pileus, especially in the volva, the amount of phallotoxins was higher than that of amatoxins.3 、 Purification and Mass spectrometry analysis of the main compounds: Nineteen compounds with clearly distinguishable peaks were collected and purified repeatedly by HPLC, then eight affirmative cyclopeptides and three unknown compounds with their accurate masses and formulae were identified by UPLC-QTOF MS/MS method.4、Phylogenetic relationships based on the cyclopeptides of lethal Amanita species: The dendrogram generated by UPGMA method based on the cyclopeptides chromatographic profiles was the first time to be established, the results of the analysis strongly supported two major monophyletic clades, which corresponded respectively to a lethal amanitas group containing cyclopeptides in section Phalloideae and to members containing no cyclopeptides of section Lepidella. Within clade Phalloideae, seven lineages can be divided. The results also demonstrated highly similarity to the phylogeny established previously based on the morphological and the muti-locus DNA sequences.