Taxonomic Novelties and New Records of Amanita Subgenus Amanitina from Thailand

The Amanita subgenus Amanitina contains six sections, and the species diversity of this subgenus has still not been explored in Thailand. Twenty samples collected in 2019 and 2020, which had the morphological characteristics of the Amanita subgen. Amanitina, were observed in this study. Both the microscopical characteristics and multi-gene phylogenetic analyses of the ITS, nrLSU, RPB2, TEF1-α, and TUB gene regions revealed that the 20 samples represented nine species and dispersed into four sections. Remarkably, three taxa were different from any other currently known species. Here, we describe them as new to science, namely A. albifragilis, A. claristriata, and A. fulvisquamea. Moreover, we also recognized six interesting taxa, including four records that were new to Thailand, viz. A. cacaina, A. citrinoannulata, A. griseofarinosa, and A. neoovoidea, as well as two previously recorded species, A. caojizong and A. oberwinkleriana. Moreover, we provide the first RPB2 and TEF1-α gene sequences for A. cacaina. Detailed descriptions, illustrations as line drawings, and comparisons with related taxa are provided.


Introduction
Amanita Pers. is an important basidiomycetous genus comprising about 700 species [1][2][3][4]. It contains both well-known edible and deadly poisonous species. In addition, Amanita species are regarded as key organisms involved in nutrient and carbon cycling in forest ecosystems on account of their ability to form ectomycorrhizal relationships with more than 10 families of vascular plants, e.g., Dipterocarpaceae, Fagaceae, Myrtaceae, and Pinaceae [2,[5][6][7].
Since the genus Amanita was formally established in 1797 [8], many mycologists have continued to contribute to and improve the taxonomic knowledge of this genus [9][10][11][12][13][14][15][16][17][18][19][20][21][22]. Corner and Bas [15] and Bas [16] proposed splitting the genus Amanita into two subgenera and six sections, which had important significance for the taxonomy of Amanita. Yang [17] revised the classification of this genus and split it into two subgenera and seven sections. Although the above classifications have been widely adopted, the delimitation within this genus is still controversial [23,24]. Until 2018, according to multi-gene phylogenetic analysis, morphological examinations, and ecological studies, Cui et al. [2] proposed the division of the genus Amanita into three subgenera and eleven sections as follows. The Newly generated sequences in this study are in red. Holotypes are marked with " T ".
Sequences of each gene fragment were separately aligned with MAFFT v.7 [39] using the G-INSi iterative refinement algorithm and then manually optimized with AliView v.1.28 [40]. Gblocks v.0.91b [41] was used to exclude the ambiguously aligned regions for ITS with two options: "Allow smaller final blocks" and "Allow gap positions within the final blocks". Sequence Matrix v.100.0 was applied to concatenate the five gene fragments for further phylogenetic analysis. MrModeltest v.2.3 [42] was adopted to determine the best fitting model of nucleotide substitution for each single-gene dataset by applying the default parameters.
Phylogenetic trees were inferred using both maximum likelihood (ML) and Bayesian inference (BI), as detailed in [43]. The ML analysis was performed at the CIPRES web portal [44] using RAxML v.8.2.12 as part of the "RAxML-HPC BlackBox" tool [45] with the default settings, and the option "Estimate proportion of invariable sites (GTRGAMMA+I)" was set to "yes" for both the single-gene and the concatenated gene analyses. The phylogenetic analyses were initially performed on each single-gene alignment, and since there was no evident conflict (with ML bootstrap support of ≥75%), the concatenated dataset was built, and the multi-gene ML analysis was performed. The Bayesian analysis was carried out with MrBayes v.3.1.2 [46]. The posterior probabilities [47] were determined via Markov chain Monte Carlo sampling (MCMC) [48]. Six simultaneous Markov chains were run from random trees for one million generations, and the trees were sampled every 100th generation (the critical value for diagnosing topological convergence was 0.01). The first 25% of the trees were discarded, and the remaining trees were used for calculating the posterior probabilities in the majority-rule consensus tree. The phylogenetic trees were visualized with FigTree v. 1.4.4 [49].

Phylogenetic Analyses
The best fitting model for each gene fragment was as follows: general time reversible + proportion of invariable sites + gamma distribution (GTR + I + G) for nrLSU, RPB2, TEF1-α, and TUB; Hasegawa-Kishino-Yano (HKY) + I + G for ITS. The concatenated dataset was partitioned into five parts according to the sequence region. Because the model HKY + I + G could not be implemented in ML, the GTR + I + G model was used, as it included all the parameters of the selected model.
The multi-gene dataset comprised 466 sequences, of which 71 were newly generated and 395 were retrieved from GenBank. The resulting topologies of the ML and BI analyses were congruent; therefore, an ML tree is shown in Figure 1. In our phylogenetic analyses, all six sections of the subgenus Amanitina showed similar mutual relationships as those in previous studies [2,4], as well as the species in each section. The three novel species formed a clearly monophyletic lineage that was distinct from other extant species with credibly supported values. Notes: Species from section Amidella have a series of remarkable characteristics, such as the color of the basidiomata changing to a brownish or reddish tone when injured, a striate and appendiculate pileal margin, the lamellae changing to a brown tone upon drying, truncate lamellulae, the amyloid basidiospores, and the absence of clamps [2,16,50]. The above combination of characteristics is unique to the section Amidella and is not found in any other section of Amanita.
Habitat: Solitary to scattered on soil in tropical deciduous forests dominated by Dipterocarpus and Shorea species. Basidiomata occurs in the rainy season.
Habitat: Solitary to scattered on soil in tropical deciduous forests dominated by Dipterocarpus and Shorea species. Basidiomata occurs in the rainy season.
Phylogenetically, Amanita albifragilis is closely related to A. griseorosea Q. Cai, Zhu L. Yang and Y.Y. Cui and A. molliuscula Q. Cai, Zhu L. Yang and Y.Y. Cui. Amanita griseorosea can be easily distinguished from A. albifragilis by having a grayish-brown pileus with darkgray fibrils and pinkish lamellae [2,19,38,56]. Compared to the newly described species, A. molliuscula has much more abundantly inflated cells in structures of pileipellis and in the interior of volval remnants on stipe base, as well as the wider and globose to subglobose basidiospores (7.5-9.0 × 7.0-8.0 µm, Qm = 1.07 ± 0.06) [2,38,56] [2,25,26,28]. In this study, four taxa belonging to the section Roanokenses were recognized and are presented below. Habitat: Solitary to scattered on soil in tropical deciduous forests dominated by Dipterocarpus and Shorea species. Basidiomata occurs in the rainy season.
Distribution: This species is currently known in China [2,57], Japan [58], Korea [59], and Thailand [26, [2]. It is worth noting that our Thai collections had a much wider color range on the pileus, i.e., milk white to greyish yellow or greyish brown.
Habitat: Solitary to scattered on soil in tropical deciduous forests dominated by Dipterocarpus and Shorea species. Basidiomata occurs in the rainy season.
Specimens Notes: Amanita caojizong, reported from China, is a common edible mushroom found in Yunnan province. It is morphologically similar to a number of taxa, such as A. pseudoporphyria, A. pseudomanginiana Q. Cai, Y.Y. Cui and Zhu L. Yang, A. griseoturcosa T. Oda, C. Tanaka and Tsuda, A. roseolifolia Y.Y. Cui, Q. Cai and Zhu L. Yang and A. modesta Corner and Bas. Detailed comparisons between A. caojizong and these similar species can be found in Cui et al. [2]. It is worth noting that our Thai collections had a much wider color range on the pileus, i.e., milk white to greyish yellow or greyish brown.
Notes: Amanita griseofarinosa was first reported from Japan [60] and was then found in China and Korea [2,18,19,62]. Our two specimens possess small-to medium-sized basidiomata, pileus and stipe densely covered by floccose to pulverulent, brownish-grey or yellowish-white squamules, appendiculate margin, attenuate lamellulae, and fragile and fugacious annulus. All these features are consistent with the type specimen.
Habitat: Solitary to scattered on soil in tropical deciduous forests dominated by Dipterocarpus and Shorea species. Basidiomata occurs in the rainy season.
Specimens Notes: Amanita oberwinkleriana was firstly reported from Japan [61] and has also been found in other Asian countries, e.g., China, India, Korea, and Thailand [2,[18][19][20]26,67,68]. It is characterized by small-to medium-sized basidiomata, a smooth and white pileus often tinged yellowish in the center, non-appendiculate pileal margin, attenuate lamellulae, fusiform to napiform stipe base surrounded by limbate volval remnants, as well as the membranous annulus. Our Thai materials are consistent with all the above features.
Notes: Our Thai materials possess very large basidiomata, pyramidal to verrucose and dark-brown pileal remnants, non-striate and appendiculate margin, solid stipe densely covered with floccose to pulverulent white to reddish-brown squamules, globose to subglobose stipe base surrounded by verrucose to squarish, reddish-brown warts. All these features are consistent with the original description of Amanita cacaina, which was recently published in China in 2023 [69]. Morphologically, A. cacaina is similar to a number of taxa, e.g., A. pseudosculpta L.P. Tang and T. Huang, A. sculpta, and A. westii (Murrill) Murrill. Detailed comparisons between A. cacaina and the related species above can be found in Huang et al. [69].  Figures 2l and 11.