Study of three interesting Amanita species from Thailand: Morphology, multiple-gene phylogeny and toxin analysis

Abstract

Amanita ballerina and A. brunneitoxicaria spp. nov. are introduced from Thailand. Amanita fuligineoides is also reported for the first time from Thailand, increasing the known distribution of this taxon. Together, those findings support our view that many taxa are yet to be discovered in the region. While both morphological characters and a multiple-gene phylogeny clearly place A. brunneitoxicaria and A. fuligineoides in sect. Phalloideae (Fr.) Quél., the placement of A. ballerina is problematic. On the one hand, the morphology of A. ballerina shows clear affinities with stirps Limbatula of sect. Lepidella. On the other hand, in a multiple-gene phylogeny including taxa of all sections in subg. Lepidella, A. ballerina and two other species, including A. zangii, form a well-supported clade sister to the Phalloideae sensu Bas 1969, which include the lethal “death caps” and “destroying angels”. Together, the A. ballerina-A. zangii clade and the Phalloideae sensu Bas 1969 also form a well-supported clade. We therefore screened for two of the most notorious toxins by HPLC-MS analysis of methanolic extracts from the basidiomata. Interestingly, neither α-amanitin nor phalloidin was found in A. ballerina, whereas Amanita fuligineoides was confirmed to contain both α-amanitin and phalloidin, and A. brunneitoxicaria contained only α-amanitin. Together with unique morphological characteristics, the position in the phylogeny indicates that A. ballerina is either an important link in the evolution of the deadly Amanita sect. Phalloideae species, or a member of a new section also including A. zangii.

Two new species of Amanita sect. Phalloideae from Africa, one of which is devoid of amatoxins and phallotoxins

“…We therefore included an ITS partition, excluding the poorly aligned positions identified by Gblocks, in the combined dataset. Regarding the introns partitions, according to Thongbai et al (2017), A.zangii and the A.ballerina clade (Fig. 1), should be considered to belong in a different section ( Amanitaincertaesedis ), sister to the Phalloideae sensu Bas (1969).…”

“…Sequencing was performed by Macrogen Inc. (Korea and The Netherlands) using the same primer combinations as for PCR, except for Am-β-tub-F, which was replaced by the shorter primer Am-β-tub-F-Seq (5’-CGGAGCRGGTAACAAYTG-3’) following Thongbai et al (2017). The sequences were assembled in Geneious Pro v. 6.0.6 (Biomatters).…”

“…Note: cl_ stands for clone. References to sequences retrieved from GenBank: Cai et al (2012), Cai et al (2014), Cui et al (2018), Davison et al (2017), Henry et al (2015), Houles et al (2018), Li et al (2015), Thongbai et al 2017, Tulloss (continuously updated).…”

“…Amanitacf.spissacea voucher OR1214 and Amanitasubjunquillea voucher HKAS63418 were used as outgroups for the combined and ITS-LSU datasets, respectively (Thongbai et al 2017, Cui et al 2018).…”

“…are famous worldwide for their high, often deadly, toxicity. Currently, the sectionPhalloideae comprises nearly 60 described species, a number of which were described only recently, mainly from Asia (Li et al 2015, Cai et al 2016, Thongbai et al 2017). Moreover, based on a multigene analysis and morphological data, Cai et al (2014) identified 14 phylogenetic clades potentially representing new species.…”