Amanita sect. Phalloideae: two interesting non-lethal species from West Africa

The members of Amanita sect. Phalloideae (Fr.) Quél. are responsible for many fatalities worldwide. However, some species in this section have previously been reported as non-lethal and lacking deadly toxins. Sequences of five genes (ITS, nrLSU, RPB2, TEF1-α, TUB2) of species belonging to the section from tropical Africa, America, Asia, Australia, and Europe were included in this study to elucidate the phylogenetic relationships among the species. The results indicated that the lethal species are in one clade (subclade I) while the non-lethal species are divided into two clades (subclades II and III) within the section. Moreover, two non-lethal species from tropical Africa, namely A. ballerinoides and A. bulbulosa are newly described based on both morphology and molecular approaches. Phylogenetically, they cluster in the same subclade III with other known non-lethal amanitas, including A. ballerina, A. chuformis, A. franzii, A. levistriata, and A. pseudogemmata. Neither amatoxins nor phallotoxins were detected in A. ballerinoides and A. bulbulosa by LC-HRMS, which agrees with their placement in the non-lethal subclade III within A. sect. Phalloideae. Finally, a key to the West African species of Amanita sect. Phalloideae is provided.


Introduction
Amanita Pers. (Agaricales, Basidiomycota) is a cosmopolitan genus comprising about 1000 species names in the literature, with about 700 species presently accepted worldwide (Bas 2000;Yang 2005;Yang et al. 2018;Cui et al. 2018). It contains both choice edible and deadly poisonous species (Cai et al. 2014;Li et al. 2015;Cui et al. 2018;Codjia et al. 2020). Most of the species form ectomycorrhizal associations with vascular plants, which play important roles in the maintenance of the ecosystem (Reid 1980;Pegler and Shah-Smith 1997;Wood 1997;Yang 1997Yang , 2005Tulloss 2005;Wolfe et al. 2012;Corrales et al. 2018;Davison et al. 2021). The modern classification, that is based on morphological characters and molecular approaches, split the genus into three subgenera comprising eleven sections .
Recent studies have reported that some species in A. sect. Phalloideae, A. ballerina Raspé, Thongbai (Cai et al. 2014;Thongbai et al. 2017;Fraiture et al. 2019;Cui et al. 2018). Among those species, A. bweyeyensis represents a particular case. The species belongs to the lethal group of A. sect. Phalloideae and does indeed contain the genes responsible for producing the toxins. However, these genes are not expressed, and the species does not produce the toxins (Fraiture et al. 2019). The lack of toxins in that species has also been confirmed by the fact that local people in Rwanda know the species and eat it without any problem (Fraiture et al. 2019). In this study, we re-examined the phylogenetic positions of taxa of A. sect. Phalloideae by adding specimens from tropical Africa. Their macro-and micromorphology characteristics, phylogenetic relationships with other Amanita species are discussed along with a key to the West African species of Amanita sect. Phalloideae. The screening of the new species for the known toxins occurring in Amanita is also reported.

Collections and preservation
Collections were made in five (05) West African countries (Benin, Guinea, Ivory Coast, Mali, Togo) during the rainy seasons from June to September (2018−2020) (Fig. 1). Basidiomata were collected from forests dominated by Fabaceae/Leguminosae (Isoberlinia Craib & Stapf ex Holland, Anthonotha P. Beauv., Berlinia Sol. ex Hook. f.), Phyllanthaceae (Uapaca Baill.) and Dipterocarpaceae (Monotes A. DC.). A digital camera type Canon EOS 60D was used to photograph the specimens in situ. The description of macro-morphological characteristics on fresh basidiomata follows Tulloss and Yang (2011). Color codes from fresh basidiomata are reported according to Kornerup and Wanscher (1981). The fresh basidiomata were air-dried using an electric dryer Stöckli Dörrex at 45°C during 24 h thereafter stored as exsiccates with their label in sealable plastic bags type minigrip. Holotypes and isotypes of the newly described species are deposited in the Mycological Herbarium of the University of Parakou (UNIPAR). Duplicate specimens are conserved at the Herbarium of Cryptogams at the Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS). For molecular investigations, small pieces of fresh basidiomata were also stored in CTAB lysis buffer (2% cetyltrimethylammonium bromide, 100 mM Tris-HCl, 20 mM EDTA, 1.4 M NaCl) and dried with silica gel. The nomenclature aspects and authorities for scientific names were double-checked against Index Fungorum (www.mycology. net) and on the Studies in the Amanitaceae website (Tulloss and Yang 2021).

Micromorphological studies
Micromorphological data were recorded from dried materials and examined at 1000× magnification by mean of a microscope type Zeiss Axioskop-40. Microscopic characteristics, measurements, and line drawings were obtained from slide preparations mounted in 5% KOH and stained with Congo Red. Melzer's reagent was used to test the amyloidity of basidiospores. A minimum of 20-30 basidiospores from each basidioma were measured in side view. In addition, basidia, hyphal elements of the subhymenium, pileipellis, and stipe trama, volval elements, were measured from each type specimen. In the descriptions of basidiospores, the term (n/m/p) means n basidiospores from m basidiomata of p collections. The dimensions of basidiospores are provided with the notation (a)b−c(d). The range b−c contains a minimum of 90% of the measured values. Extreme values, i.e., a and d, are provided in parentheses. Q is used for the ratio length/width of a spore in side view; Q m is the average Q of all basidiospores ± sample standard deviation. The statistical analysis of measurements for the basidiospores was conducted with Piximetre v5.10 (Henriot and Cheype 2020). The descriptive terms follow Bas (1969), Yang (2005Yang ( , 2015, Cai et al. (2016), Cui et al. (2018Cui et al. ( , 2021, and Codjia et al. (2020).
The generated sequences were assembled using Sequencher 4.1.4 (Gene Codes Corporation, Ann Arbor, Michigan).

Phylogenetic analyses
In this study, all newly generated sequences were submitted to GenBank, and accession numbers are shown in Table 1. Additional sequences were retrieved from previously published papers and GenBank (Table 1). Species within A. sect. Lepidella, including Amanita flavofloccosa Nagas. & Hongo and Amanita manicata (Berk. & Broome) Pegler, were used as outgroup (Table 1). Alignments were performed using MAFFT v7.310 (Katoh and Standley 2013), edited manually when necessary in BioEdit v7.0.9 (Hall 1999). The Amanita chuformis   ambiguously aligned portions and divergent regions were eliminated using Gblocks v0.91b (Castresana 2000;Talavera and Castresana 2007). A concatenated dataset (including ITS, nrLSU, RPB2, TEF1-α, TUB2) comprising 370 sequences were constructed in Geneious v7.0.2 (Kearse et al. 2012) and used for phylogenetic analyses. The concatenated alignment was submitted to TreeBASE (submission ID 29481). The Incongruence Length Difference (ILD) test in PAUP v4.0a169 (Swofford 2002) was performed to determine if there were any incongruence between genes before concatenating them. As no incongruence (P-value = 0.884000) was detected, the maximum likelihood (ML) and Bayesian inference (BI) were used for phylogenetic tree inference. The ML and BI were performed using IQ-TREE v1.6.12 (Nguyen et al. 2015) and MrBayes v3.2 (Ronquist et al. 2012), respectively. Nucleotide substitution models, as well as the  (Zhang et al. 2020). For the ML analysis, the best partitioning schemes, the ultrafast bootstrap replicates at 1000, and the Shimodaira-Hasegawa and approximate likelihood ratio test (SH-aLRT) test with 1000 replicates (Guindon et al. 2010;Hoang et al. 2018) were used. The BI was conducted with the following parameters: 2 runs, each with four simultaneous Metropolis-Coupled Markov chains, and trees were sampled every 1000 generations. The analyses were completed after 20,000,000 generations when the average standard deviation of split frequencies was 0.017963 for the five-gene analysis, and the first 25% generations were discarded as burn-in. The phylogenetic trees inferred from ML and BI analyses were visualized with FigTree v1.4.4 (Rambaut 2018) and then edited in Adobe Illustrator CS6.

Analysis of toxins by LC-HRMS
Dried basidiomata of the target taxa were analyzed for the most notorious toxins, namely α-amanitin, β-amanitin, phalloidin, and phallacidin (standards provided by Sigma Chemical Co, USA). The liquid chromatography-high-resolution mass spectrometry (LC-HRMS) using 1290 Infinity II HPLC systems coupled with 6540 UHD precision mass Q-TOF instruments was applied to screen the compounds. Toxin extraction, as well as LC-HRMS analyses, follow the method of Codjia et al. (2020).

Taxonomy
Habitat. Solitary or in small group on the ground in woodland and gallery forests, associated with Berlinia grandiflora or Isoberlinia doka (Fabaceae/Leguminosae) and Uapaca guineensis (Phyllanthaceae).
Distribution Notes. Amanita ballerinoides presents the following characteristics: small, white to whitish basidiomata, floccose pileus with small adherent squamules, or patchy universal veil that are whitish to gray orange, striate at maturity, and marginate basal bulb. Phylogenetically, the species clustered in the non-lethal subclade III, including A. ballerina, A. chuformis, A. franzii, A. levistriata, A. pseudogemmata, and forms a well-distinct lineage in the phylogenetic tree.
Among these species, A. ballerinoides is nearly identical morphologically to A. ballerina. However, A. ballerina can be separated from A. ballerinoides by its slightly viscid pileus when moist covered with dull white to pale yellowish white thin adherent areolate squamules or patchy volval remnants (Thongbai et al. 2017). Ecologically, A. ballerinoides occurs in woodland or gallery forests, dominated by Berlinia grandiflora or Isoberlinia doka (Fabaceae/Leguminosae) and Uapaca guineensis (Phyllanthaceae), whereas A. ballerina occurs in evergreen Fagaceae hill forest or mixed deciduous Dipterocarpaceae/Fagaceae forest (Thongbai et al. 2017).
Distribution. Currently known from Benin and Guinea. Notes.Amanita bulbulosa is characterized by its small, white basidiomata, floccose pileus with small adherent squamules or patchy universal veil that are white to gray orange, striate at maturity, and marginate basal bulb. The phylogenetic analyses clustered this species in the non-lethal subclade III like A. ballerinoides, A. ballerina, A. chuformis, A. franzii, A. levistriata, A. pseudogemmata, and it forms a well-distinct lineage in the phylogenetic tree.
At first sight, this species has some morphological similarities with A. ballerina, A. ballerinoides, A. chuformis, Amanita franzii, A. levistriata, A. pseudogemmata. These species have a striate pileal margin and a marginate basal bulb like A. bulbulosa. Although A. ballerinoides is very close to A. bulbulosa, it differs from the latter by its medial, broad, skirt-like annulus and smaller basidiospores (8.5-9 × 6.5-7 μm). Amanita ballerina differs from A. bulbulosa by the presence of a membranous annulus, medial, persistent, cottony, skirt-like, having a thickened edge, striate inside and white to dull white, and smaller basidiospores (7.5-8.9 × 6-7.5 μm) (Thongbai et al. 2017). Amanita chuformis differs from A. bulbulosa by its brownish gray, brownish to dirty white pileus, and grayish, brownish to dirty white stipe (Cui et al. 2021). Amanita franzii can be distinguished from A. bulbulosa by its relatively bigger and elongate basidioma, and a dirty white to pale yellow-brown pileus which becomes paler towards margin . Amanita levistriata can be separated from A. bulbulosa by its smaller basidioma with an ocher yellow to pale yellow pileus, a whitish to pale yellow stipe, and smaller basidiospores (7.5-9.5 × 6.5-8.0 μm) (Jenkins 1988;Tulloss and Yang 2021). Amanita pseudogemmata can be distinguished from A. bulbulosa by its dirty yellow to pale yellow-brown pileus with yellow to yellow-brown volval remnants, a yellow stipe with its basal bulb covered with collar-like, white to yellowish volval remnants, and smaller basidiospores (7-9.5 × 6-8.5 μm) (Hongo 1974;Yang and Doi 1999;Yang 2005Yang , 2015Cui et al. 2018).

Analysis of toxins by LC-HRMS
No corresponding monoisotopic masses were identified for αamanitin, β-amanitin, phalloidin, and phallacidin. Thus, neither amatoxins nor phallotoxins were found in A. ballerinoides, A. bulbulosa, and A. sp. 0595. This result confirms the placement of those species within the nonlethal subclade III of A. sect. Phalloideae.

Discussion
Phylogeny of Amanita sect. Phalloideae In the present study, the phylogenetic analyses strongly support Cui et al. (2018Cui et al. ( , 2021) that recognize three subclades within A. sect. Phalloideae (ML ultrafast bootstrap = 95%; SH-aLRT = 100%, ML ultrafast bootstrap = 100%, BPP = 1.0). The subclade I contains the lethal species, and the subclades II and III contain the non-lethal species. The non-lethal subclades comprise few species until now. Previously, seven (07) species (including two species in subclade II and five species in the subclade III) were reported mostly from Asia and America (Jenkins 1988;Thongbai et al. 2017;Cai et al. 2014;Cui et al. 2018Cui et al. , 2021. In contrast, no species from those non-lethal subclades have been reported from Africa previously. This study represents the first contribution of that group with two new species from tropical Africa. The African collections in our phylogenetic tree form well-distinct lineages. Amanita ballerinoides and A. bulbulosa, newly described from Africa, are clustered in the same subclade III with other non-lethal species, namely A. ballerina, A. chuformis, A. franzii, A. levistriata, A. pseudogemmata reported previously by Jenkins (1988), Thongbai et al. (2017), and Cui et al. (2018Cui et al. ( , 2021. In general, what makes lethal amanitas easily distinguishable from other groups is the morphological characteristics and the presence of deadly poisonous substances. The screening for the most notorious toxins by LC-HRMS revealed the absence of α-amanitin, β-amanitin, phalloidin, and phallacidin in A. ballerinoides and A. bulbulosa. In previous studies, the screening for both α-amanitin and phalloidin were also performed in A. ballerina, A. franzii, and A. pseudogemmata (Thongbai et al. 2017;Fraiture et al. 2019). The results from those studies showed that toxins were not present in those species. Thus, Thongbai et al. (2017) proposed the separation of non-lethal species of A. sect. Phalloideae into a new section. However, the phylogenetic result was not statistically supported, and the group comprises few species. Moreover, the nonlethal species (A. ballerina, A. ballerinoides, A. bulbulosa, A. franzii, A. pseudogemmata) are reminiscent of some species belonging to A. sect. Lepidella subsect. Limbatulae, such as A. limbatula Bas, A. parva Murrill, A. mutabilis Beardslee, and A. praelongispora (Murrill) Murrill (Beardslee 1919;Murrill 1941Murrill , 1945Bas 1969;Jenkins 1979Jenkins , 1986Tulloss 1984). Phylogenetically, those species within A. sect. Lepidella might probably cluster in the non-lethal subclades within A. sect. Phalloideae. Still, there is not enough molecular evidence to confirm the phylogenetic relationships of those species within A. sect. Lepidella with the non-lethal amanitas of A. sect. Phalloideae.
On the other hand, the second non-lethal subclade II in the multigene phylogenetic tree comprises A. hesleri and A. zangii, which were also reported as non-lethal taxa (Cai et al. 2014;Fraiture et al. 2019). Both species have some similar morphological characteristics with the non-lethal species of subclade III. The elongate to ventricose basal bulb is characteristic for both non-lethal subclades. However, the main morphological characteristic that differentiates the species of subclade II from those of subclade III is their appendiculate pileal margins (Yang et al. 2001;Thongbai et al. 2016;Cui et al. 2018). All those morphological characteristics of the non-lethal species do not fit the circumscription of A. sect. Phalloideae (Bas 1969), which makes their placement uncertain (Thongbai et al. 2017). Thus, based on our results, the non-lethal species could be considered either as early diverging lineages in A. sect. Phalloideae, or as new sections (Thongbai et al. 2017).

Distribution and ecology of new species
The new species reported here are only known from West Africa and were collected during the rainy season. Amanita ballerinoides was found in Benin, Guinea, Ivory Coast, and Mali. It occurs in woodlands and gallery forests, associated with Berlinia grandiflora or Isoberlinia doka (Fabaceae/ Leguminosae) and Uapaca guineensis (Phyllanthaceae). Amanita bulbulosa was found in Benin and Guinea in woodlands and gallery forests where it is associated with Uapaca guineensis or U. togoensis (Phyllanthaceae) and Isoberlinia doka (Fabaceae/Leguminosae). Both woodlands and gallery forests of West Africa are home to a high diversity of mycorrhizal fungi (Bâ et al. 2012).
The genus Amanita is one of the most ecologically and economically important groups within the fungi kingdom, even if some species are deadly poisonous and cause a lot of fatalities around the world (Cai et al. 2014;Li et al. 2015;Cui et al. 2018;Codjia et al. 2020). As ectomycorrhizal fungi, Amanita species represent key and indispensable actors for the good functioning of forest ecosystems (woodland, gallery forests) and strongly influence the diversity and productivity of tropical African forests (Bâ et al. 2011). Albeit, many taxa are not yet fully documented, many representatives within the genus Amanita are consumed and sold by local people in West Africa (Yorou et al. 2014;Boni and Yorou 2015;Kamou et al. 2017;Fadeyi et al. 2017Fadeyi et al. , 2019Soro et al. 2019). Further ethnomycological investigations could help to check whether both newly described taxa are choice edibles or not and thus support their phylogenetic placement within the non-lethal groups of the section Phalloideae. So, increasing the investigations in the genus Amanita is crucial, especially in tropical Africa, so far remaining underexplored.
Key to the West African species of Amanita sect. Phalloideae