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Mycokeys , 06 Jun , DOI: Two new species of Amanita sect. Phalloideae are described from tropical Africa incl. Madagascar based on both morphological and molecular DNA sequence data. Amanita bweyeyensis sp. It is consumed by local people and chemical analyses showed the absence of amatoxins and phallotoxins in the basidiomata.

Surprisingly, molecular analysis performed on the same specimens nevertheless demonstrated the presence of the gene sequence encoding for the phallotoxin phallacidin PHA gene, member of the MSDIN family. The second species, Amanita harkoneniana sp. It is also characterised by a complete PHA gene sequence and is suspected to be deadly poisonous. Both species clustered together in a well-supported terminal clade in multilocus phylogenetic inferences including nuclear ribosomal partial LSU and ITS This, along with the occurrence of other species in sub-Saharan Africa and their phylogenetic relationships, are briefly discussed.

Macro- and microscopic descriptions, as well as pictures and line drawings, are presented for both species. An identification key to the African and Madagascan species of Amanita sect.

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Phalloideae is provided. The differences between the two new species and the closest Phalloideae species are discussed. Most representatives of Amanita sect. Phalloideae Fr. Currently, the section Phalloideae comprises nearly 60 described species, a number of which were described only recently, mainly from Asia Li et al. Moreover, based on a multigene analysis and morphological data, Cai et al. The phylogenetic analyses made by those authors also resulted in the transfer of several species from sect.

Phalloideae to sect. Most of African mycodiversity remains under-explored with only ca. Very few species belonging to sect. The three poorly known Amanita alliiodora Pat. The latter species is the only Phalloideae known from Central Africa, together with some doubtful mentions of the imported A. Mentions of the species in other regions of the world correspond to either introductions or misidentifications. The exact identity of A. Gilbert syn. Wong, A. African auct. It was described from Australia, growing in association mainly with various species of Eucalyptus e.

Eicker et al. Blake, as well as under pure Casuarina equisetifolia L. Miller et al. Amatoxins and phallotoxins are responsible for the high toxicity of Amanita sect. Nevertheless, apart from Amanita alliiodora , considered toxic by the Madagascan people, and the deadly poisonous A.

In the framework of taxonomic and phylogenetic studies of Amanita sect. Phalloideae , specimens originating from tropical Africa were critically studied. Morphological and multigenic phylogenetic studies proved to be concordant and established the existence of two distinct species that could not be identified as any known taxa. Amanita bweyeyensis from the western province of Rwanda and A. Their phylogenetic affinities with other Amanita species reported from Africa are discussed and a key to African species of Amanita sect. African Amanita phalloides -related specimens held in BR were studied in depth Degreef from Burundi; Degreef and , both from Rwanda.

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Finally, P. Pirot sent us two unnumbered specimens he collected in Madagascar in and We also examined for comparison the type specimen of Amanita marmorata subsp. Weatherhead s.

Cleland s. We unsuccessfully tried to obtain the type specimen of Amanita reidii on loan. Braam Vanwyk informed us that the holotype preserved in PRU had unfortunately been destroyed and no longer exists.

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Although Miller et al. Bailey , K, correct name: Amanita neomurina Tulloss. Macroscopic characters were deduced from herbarium specimens, as well as from specimen labels, field notes and pictures, when available. Measurements were made using a camera lucida and a calibrated scale.

Sequencing was performed by Macrogen Inc. The sequences were assembled in Geneious Pro v. The quality of the sequences was taken into account in selecting the sequences for the phylogenetic analyses. References to sequences retrieved from GenBank: Cai et al. Amanita cf. The alignment was further optimised and manually adjusted as necessary by direct examination with the software Se-Al v. The assignment of codon positions in the protein-coding sequences was confirmed by translating nucleotide sequences into predicted amino acid sequences using MacClade 4.

As the Iss. To circumvent this problem, DAMBE was used to randomly sample subsets of 4, 8, 16 and 32 OTUs multiple times and to perform the test for each subset to see if substitution saturation exists for these subsets of sequences. Phylogenetic analyses were performed separately for each individual and concatenated loci using Bayesian Inference BI as implemented in MrBayes v3.

All parameters were unlinked across partitions. Bayesian analyses were implemented with two independent runs, each with four simultaneous independent chains for ten million generations, starting from random trees and keeping one tree every th generation. A probability of 0. We provided an additional alignment partition file to force RAxML software to search for a separate evolution model for each dataset.

Note: Iss: index of substitution saturation. P: probability that Iss is significantly different from the critical value Iss. To detect topological conflicts amongst data partitions, the nodes between the majority-rule consensus trees obtained in the ML analysis from the individual datasets were compared with the software compat. A conflict was assumed to be significant if two different relationships for the same set of taxa one being monophyletic and the other not were observed in rival trees.

In these pro-proteins, the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences, with an invariant Pro residue immediately upstream of the toxin regions and as the last amino acid in the toxin regions. Two groups of dried mushrooms, i. For each specimen, mg of dry tissues were ground and homogenised in 3 ml extraction medium methanol:water After 1 hour of incubation, all extracts were centrifuged at rpm for 5 min, the supernatant was filtered using a 0. The solvents used in this study were all HPLC grade.

Chromatography conditions for the procedure followed in this study were reported by Kaya et al.

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The content of the mobile phase was 0. The detection limits were set at 0. 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. We therefore tested the combined dataset for substitution saturation by using A. In this case, no sign of saturation was evidenced, which supports the consistency of the phylogenetic signal in the main Phalloideae clade. We therefore decided to include the introns partitions in the phylogenetic analyses in order to increase the resolution at species level.

The new taxa are highlighted in the shaded box. NT indicates not tested. The topologies obtained by analysing the combined dataset and the ITS-LSU dataset were highly congruent with published trees Zhang et al.

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Several collections from tropical Africa clustered together in a well-supported clade. So far, this clade remains isolated but is notably distantly related to all other Amanita species, as yet reported from Africa Zhang et al. Amanita alliiodora clustered together with the two unnamed species from tropical Africa in all phylogenetic inferences considered individually or concatenated i. For selected nodes, parsimony bootstrap support value and Bayesian posterior probabilities are, respectively, indicated to the left and right of slashes. Morphological examination showed combinations of morphological features unique to and characteristic of each, thereby defining two morphotypes.

The critical morphological features that differentiate them are the following. The first species grows under Eucalyptus. Its bulb at stipe base is sub- globose, neither pointed nor rooting. The ring is striated and the smell sweetish and conspicuous. The second species is not bound with Eucalyptus and has been collected in Miombo woodland and in a garden. The bulb at the stipe base is turnip-shaped to rooting. The ring is smooth or vaguely plicate and the smell weak, resembling raw potato. By using a combination of the degenerated primers cited above, we obtained a complete mer sequence of phallacidin precursor for the three specimens of A.

Surprisingly, this is the first time that a complete PHA sequence has been found in a species of Amanita sect. Phalloideae that does not produce this toxin. This finding is in contrast with the study of Hallen et al. Figs 3 , 4.

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Basidiomata of Amanita bweyeyensis. Western Prov. Degreef BR! Primordium subglobose, smooth, whitish or with a weak olive tint. Pileus 40—73— mm diam. Lamellae free, white, becoming slightly yellowish when old and ochraceous, pinkish-beige to pale pinkish-brown on the exsiccates with a narrow white and fluffy edge; mixed with an equal number of lamellulae which are very variable in length and are usually truncated; sub-distant, 8—9 lamellae and lamellulae per cm at 1 cm from the edge of the pileus, about — lamellae and lamellulae in total counts on 5 basidiomata , 3—14 mm broad, serrate when seen with a magnifying glass.