Two New Amanita Species in Section Amanita from Thailand

Abstract

Based on a survey of macro-fungi in northern and northeastern Thailand, nine samples collected in 2020 are identified as Amanita and introduced here as two new species, Amanita kalasinensis and A. ravicrocina. Typical macro- and microscopical characteristics indicate that both of these two species belong to Amanita section Amanita, but differ from other currently known species. Amanita kalasinensis is characterized by having a greyish yellow pileus covering with a conical to granuliform, yellowish white volval remnant; the presence of clamps; and a broadly ellipsoid to ellipsoid basidiospore. Amanita ravicrocina is characterized by having a brown to greyish orange pileus covering with a patchy, white volval remnant; a collar-like volval remnant on the stipe; and a subglobose to broadly ellipsoid basidiospore. Multi-gene phylogenetic analysis of partial nuclear rDNA internal transcribed spacer region (ITS), partial nuclear rDNA large subunit region (nrLSU), RNA polymerase II second largest subunit (RPB2), partial translation elongation factor 1-alpha (TEF1-α), and beta-tubulin gene (TUB) also revealed that positions of A. kalasinensis and A. ravicrocina are well-supported within A. section Amanita, but form distinct lineages and do not show any close relationship with any species. The detailed morphological features, line-drawing illustration, and comparison with morphological similar taxa are provided.

1. Introduction

Amanita Pers. is a widespread basidiomycetous genus comprising more than 600 species all over the world [1,2,3,4,5]. According to recent studies [3,4,5], this genus was proposed to be divided into three subgenera and eleven sections [subgenus Amanita Pers., containing: section Amanita Pers., section Amarrendiae (Bougher & T. Lebel) Zhu L. Yang, Y.Y. Cui, Q. Cai & L.P. Tang, section Caesareae Singer ex Singer and section Vaginatae (Fr.) Quél; subgenus Amanitina (E. J. Gilbert) E. J. Gilbert, containing: section Amidella (J. E. Gilbert) Konrad & Maubl., section Arenariae Zhu L. Yang, Y.Y. Cui & Q. Cai, section Phalloideae (Fr.) Quél., section Roanokenses Singer ex Singer, section Strobiliformes Singer ex Q. Cai, Zhu L. Yang & Y.Y. Cui and section Validae (Fr.) Quél., and subgenus Lepidella Beauseigneur, containing section Lepidella Corner & Bas only]. Species in Amanita section Amanita are characterized by having agaricoid basidioma, persistent volval remnants on the pileus, striate pileal margins, truncate lamellulae, basal bulb, and inamyloid basidiospores [1,2,3,4]. To date, there are thirteen species from Amanita sect. Amanita, namely, A. aff. mira Corner & Bas, A. altipes Zhu L. Yang, M. Weiß & Oberw., A. concentrica T. Oda, C. Tanaka & Tsuda, A. digitosa Boonprat. & Parnmen, A. melleialba Zhu L. Yang, Qing Cai & Yang Y. Cui, A. obsita Corner & Bas, A. orientigemmata Zhu L. Yang & Yoshim. Doi, A. rubrovolvata S. Imai, A. siamensis Sanmee, Zhu L. Yang, P. Lumyong & Lumyong, A. sinensis Zhu L. Yang, A. subglobosa Zhu L. Yang, A. submelleialba Yuan S. Liu & S. Lumyong, and A. sychnopyramis f. subannulata Hongo, reported in Thailand [4,6,7,8,9,10,11,12].

During the period of macrofungal investigation in the rainy season of 2020, nine interesting specimens were collected from deciduous forests dominated by Dipterocarpus and Shorea species in northern and northeastern Thailand (Chiang Rai, Kalasin, and Sakon Nakhon Provinces, Thailand). Morphological examination and molecular analyses indicated that these collections herein reported represent two species new to science.

2. Materials and Methods

2.1. Morphological Study

The following information was recorded at the collecting sites: geographic coordinates, forest type, substrate type, and field photographs. Small pieces of tissue from the cap and/or stipe were taken and dried by silica gel to prepare for the molecular material [13], and the remaining specimens were dried at 35–45 °C for at least twelve hours to prepare for the morphological material and later were deposited at the Herbarium of Biology Department (CMUB) and the Herbarium of Sustainable Development of Biological Resources (SDBR), Faculty of Science, Chiang Mai University, Thailand.

Macroscopic characters were described based on field notes and field images. Color codes and names were recorded according to Kornerup and Wanscher [14]. Marginal striations on the pileus were expressed as a proportion of the ratio of striation length to the radius of the pileus (nR). Microscopic features were observed from dried specimens mounted in distilled water, 5% aqueous KOH (w/v), 1% Congo red (w/v), and Melzer’s reagent under a Leica DM500 microscope to depict all tissues [2,3]. Sections of the pileipellis were cut radial-perpendicularly, and halfway between the center and margin of the pileus, and sections of the stipitipellis were taken from the center of the stipe, along the middle part along the longitudinal axis. For the description of basidiospores, the term [n/m/p] represents that n basidiospores were measured from m basidiomata of p collections. Dimensions for basidiospores are given as (a–) b–c (–d), in which ‘b–c’ represents a minimum of 90% of the measured and extreme values ‘a’ and ‘d’ are given in parentheses whenever necessary. Q denotes the ratio of length divided by width of the basidiospore in the side view, Qm denotes the average Q of n measured basidiospores, and SD is their standard deviation. The results are presented as Q = Qm ± SD. Basidiomata size and spores shape are defined according to Bas [15].

2.2. DNA Extraction, PCR Amplification, and Sequencing

Methods of DNA extraction, PCR amplification, and sequencing protocols were conducted based on previous studies [4,12,16]. Five primer pairs ITS1F/ITS4 [17,18], LR0R/LR5 [19], EF1-983F/EF1-1567R [20], Am-6 F/Am-7 R, and Am-β-tubulin F/Am-β-tubulin R [21] were used to amplify ITS, nrLSU, TEF1-α, RPB2, and TUB, respectively. Sequences generated in this study were subjected to BLASTn (http://www.ncbi.nlm.nih.gov (accessed on 15 December 2021)) analysis and submitted to GenBank.

2.3. Phylogenetic Analyses

Detailed information about the sequences retrieved from GenBank and the sequences newly generated in this study was analyzed (

Table 1. Species names, voucher numbers, countries, and their respective GenBank accession numbers of the taxa used in this study.

Species Name	Voucher	Country	GenBank Accession Number
			ITS	nrLSU	RPB2	TEF1-α	TUB
‘Amanita austrowellsii’	RET 302-1	USA	MN963578	MN963578	—	—	—
‘A. austrowellsii’	RET 576-2	USA	MN963579	MN963579	—	—	—
A. cruziiT	BARONI 8998 (CORT)	Dominican Republic	KC855222	KC855222	—	MH508750	MH485478
A. cruzii	BARONI 9791 (CORT)	Dominican Republic	KC855223	KC855223	—	MH508751	—
A. elata	HKAS 83449	China	MH508334	MH486486	MH485965	MH508763	MH485488
A. frostiana	RET 547-2	USA	KP313581	—	—	—	—
A. frostiana	RET 588-6	USA	KP313583	—	—	—	—
A. kalasinensis	SDBR-STO-2020-231	Thailand	OM040561	OM040552	—	—	—
A. kalasinensisT	CMUB-39966	Thailand	OM040562	OM040553	OM066913	OM066919	OM066925
A. kalasinensis	SDBR-STO-2020-253	Thailand	OM040563	OM040554	OM066914	OM066920
A. kalasinensis	SDBR-STO-2020-303	Thailand	OM040564	OM040555	—	—	—
A. kalasinensis	SDBR-STO-2020-398	Thailand	OM040565	OM040556	—	—	—
A. mira	HKAS 91953	China	MH508437	MH486646	MH486097	—	—
A. pakistanica	RET 317-6	Pakistan	KX365198	KX365199	—	—	—
A. pakistanica	RET 411-7	India	MG991745	MG991814	—	—	—
A. pantherina	HKAS 56702	Czech Republic	MH508487	KR824782	KR824789	KR824825	MH485670
A. pantherina	MB-102863	Germany	MH508488	MH486743	MH486167	MH508976	MH485671
A. parcivolvata	RET 504-5	USA	KP313586	—	—	—	—
A. parcivolvata	RET 511-10	USA	KP313584	—	—	—	—
A. parcivolvata	RET 614-4	USA	MN963585	MN963584	—	—	—
A. parvipantherina	HKAS 54723	China	MH508495	KR824780	KR824802	KR824807	MH485676
A. parvipantherinaa	HKAS 67907	China	MH508498	KR824781	KR824803	KR824808	MH485679
A. pseudopantherina T	HKAS 80007	China	MH508514	MH486777	MH486191	MH509004	MH485698
A. pseudopantherina	HKAS 57611	China	MH508511	MH486774	MH486188	—	MH485695
A. pseudosychnopyramis	HKAS 82293	China	MH508529	MH486790	MH486204	—	MH485712
A. pseudosychnopyramis T	HKAS 87999	China	MH508530	KR824778	KR824790	KR824824	MH485713
A. rubrovolvata	BZ2015-68	Thailand	KY747465	KY747477	KY656882	—	KY656863
A. rubrovolvata	HKAS 54491	China	JN943178	JN941153	JQ031116	KR824823	—
A. rufoferruginea	HKAS 101395	China	MH508578	MH486839	MH486249	—	MH485753
A. rufoferruginea	HKAS 79616	China	MH508579	MH486842	MH486252	—	MH485756
A. siamensis	HKAS 67855	China	MH508592	MH486864	MH486271	—	MH485773
A. siamensis	HKAS 83681	China	MH508593	MH486866	MH486273	—	MH485774
A. sinensis var. sinensis T	HKAS 25761	China	AB096059	AF024474	—	—	AB095864
A. sinensis var. sinensis	HKAS 100492	China	MH508594	MH486867	MH486274	—	MH485775
A. sinensis var. sinensis	HKAS 100493	China	MH508595	MH486868	MH486275	—	MH485776
A. subglobosa	HKAS 54787	China	MH508618	MH486900	MH486301	MH509121	MH485801
A. subglobosa	HKAS 56893	China	JN943176	JN941157	JQ031120	KR824826	MH485802
A. submelleialba T	CMUB-S1	Thailand	MZ045688	MZ045693	MZ048619	MZ048624	MZ048629
A. submelleialba	HKAS 112958	Thailand	MZ045685	MZ045690	MZ048616	MZ048621	MZ048626
A. submelleialba	HKAS 112959	Thailand	MZ045686	MZ045691	MZ048617	MZ048622	MZ048627
A. subparvipantherina	HKAS 56817	China	JN943171	JN941160	JQ031114	KR824815	MH485818
A. subparvipantherina	HKAS 58891	China	MH508628	MH486918	MH486316	MH509136	MH485819
A. sychnopyramis f. subannulata	HKAS 101427	China	MH508631	MH486922	—	MH509139	MH485824
A. sychnopyramis f. subannulata	HKAS 101437	China	MH508632	MH486923	MH486319	MH509140	MH485825
A. sychnopyramis f. subannulata	HKAS 101442	China	MH508633	MH486925	MH486321	MH509142	MH485826
A. sychnopyramis f. subannulata	HKAS 75485	China	MH508634	MH486926	—	MH509143	MH485827
A. wellsii	RET 387-5	Canada	KU248115	OK285332	—	—	—
A. wellsii	RET 654-2	USA	OK299151	OK299151	—	—	—
A. wellsii	RET 726-6	USA	OK299169	OK299169	—	—	—
A. ravicrocina	SDBR-STO-2020-229	Thailand	OM040566	OM040557	OM066915	OM066921	OM066926
A. ravicrocina	SDBR-STO-2020-235	Thailand	OM040567	OM040558	OM066916	OM066922	OM066927
A. ravicrocinaT	CMUB-39967	Thailand	OM040568	OM040559	OM066917	OM066923	—
A. ravicrocina	SDBR-STO-2020-283	Thailand	OM040569	OM040560	OM066918	OM066924	—
Outgroup
A. caesareoides	HKAS 92009	China	MH508285	MH486421	MH485901	MH508708	—
A. caesareoides	HKAS 92017	China	MH508286	MH486422	MH485902	MH508709	—
A. yuaniana	HKAS 58807	China	MH508653	MH486954	MH486347	MH509174	MH485852
A. yuaniana	HKAS 68662	China	MH508654	MH486957	MH486350	MH509177	MH485854

Newly generated sequences in this study are in black bold, while holotypes are marked with “T”.

). Sequences of five gene regions were aligned with MAFFT v.7 [22] using the G-INS-i iterative refinement algorithm, and then checked visually and manually optimized using BioEdit v.7.0.9 [23]. Gblocks v. 0.91b [24] was used to check and exclude the ambiguously aligned regions for ITS, based on two options “Allow smaller final blocks” and “Allow gap positions within the final blocks”. Maximum likelihood analyses were carried out for each single gene dataset using the same settings used for concatenated analysis to test potential conflicts among the five genes. Sequence Matrix v.100.0 was applied to combine the five gene fragments for further phylogenetic analysis and the concatenated dataset was deposited in TreeBASE under the number of 29155. Phylogenetic tree inference was performed using both Bayesian inference (BI) and maximum likelihood (ML), as detailed in Dissanayake et al. [25]. The best-fit model of nucleotide substitution was determined for each single gene dataset using MrModeltest v. 2.3 [26] following the default parameters.

The ML analysis was performed at the CIPRES web portal [27] using RAxML v.8.2.12 as part of the “RAxML-HPC BlackBox” tool [28] with default settings, except the “Estimate proportion of invariable sites (GTRGAMMA+I)” was set to be “yes” for both single-gene and combined gene analyses. Phylogenetic inference was first performed on each single-gene alignment, and as there was no evident conflict (with ML bootstrap support ≥75%), then multiple-gene alignments and trees were built. The Bayesian analysis was performed using MrBayes v.3.1.2 [29]. Posterior probabilities [30] were determined by Markov chain Monte Carlo sampling (MCMC) [31] in MrBayes v.3.1.2. Six simultaneous Markov chains were run from random trees for 1 million generations and trees were sampled every 100th generation (the critical value for the topological convergence diagnostic is 0.01). The first 25% of trees were discarded and the remaining trees were used for calculating posterior probabilities in the majority rule consensus tree. The phylogenetic tree was visualized with FigTree v.1.4.4 [32].

3. Results

3.1. Phylogenetic Analyses

The best-fit models for the five genes were as follows: general time reversible + proportion of invariable sites + gamma distribution (GTR + I + G) for ITS, nrLSU, and TEF1-α; Hasegawa-Kishino-Yano (HKY) + I + G for RPB2; and Kimura 2-parameter (K80) + G for TUB. The concatenated dataset was partitioned into five parts by sequence region. The model HKY + I + G and K80 + G could not be implemented in RAxML, thus the GTR + I + G model, which included all parameters of the selected model, was used instead.

The multi-gene dataset comprised 212 sequences, including 33 newly generated and 179 retrieved from GenBank. Amanita yuaniana (HKAS58807), A. yuaniana (HKAS68662), A. caesareoides (HKAS92009), and A. caesareoides (HKAS92017) from Amanita section Caesarea were set as the outgroup taxa. The concatenated dataset comprised 2769 positions (ITS: 1–445; nrLSU: 446–1343; RPB2: 1344–2014; TEF1-α: 2015–2576; and TUB: 2577–2803) after alignment, including the gaps.

Bayesian and RAxML analysis of the combined dataset resulted in phylogenetic reconstructions with largely similar topologies, thus the result of maximum likelihood (RAxML) tree is shown in Figure 1. In our phylogenetic results, five collections representing Amanita kalasinensis and four collections representing A. ravicrocina, respectively, formed a monophyletic lineage from other extant species with credible support values, which could be recognized as two new species.

3.2. Taxonomy

Amanita kalasinensis Yuan S. Liu & S. Lumyong, sp. nov. (Figure 2 and Figure 3).

Mycobank number: 842342.

Holotype: Thailand, Kalasin Province: Kham Muang District, Na Bon, 16°53′05″ N, 103°41′49″ E, alt. 239 m, 12 August 2020, Yuan S. Liu, STO-2020-233 (CMUB-39966).

Etymology: The specific epithet ‘kalasinensis’ refers to the Kalasin province of Thailand, where the holotype was collected.

Description: Basidioma small to medium-sized. Pileus 3.0–5.7 cm diam., convex, plano-convex to applanate, slightly depressed at center, white (1A1) to greyish yellow (4B3–5), often darker at center and becoming paler towards margin; volval remnants on pileus conical, pyramidal to granuliform, 1–2 mm dia., white (1A1) to yellowish white (4A2), densely arranged on the disk; margin striate (ca. 0.3–0.5), non-appendiculate; context white, unchanging. Lamellae free, crowded, white (1A1); lamellulae truncate, plentiful. Stipe 3.5–8.0 cm long × 0.6–0.9 cm diam., subcylindrical and slightly tapering upward, with apex slightly expanded, white (1A1) to yellowish white (4A2), sometimes with greyish yellow (4B3–4) tinge, covered with white (1A1) fibrils, often becoming floccose near basal bulb; context white (1A1), stuffed; basal bulb globose to subglobose, 0.9–1.4 cm diam., white (1A1); volval remnants on stipe base granuliform to floccos, white (1A1) to light yellow (4B4–5). Annulus absent. Odor not recorded.

Lamellar trama bilateral. Mediostratum 15–20 μm wide, composed of abundant clavate to cylindrical inflated cells (54–145 × 13–30 μm); filamentous hyphae abundant, 4–12 μm wide; vascular hyphae scarce. Lateral stratum composed of abundant ellipsoid to clavate inflated cells (32–66 × 13–37 μm), diverging at an angle of ca. 30° to 45° to mediostratum; filamentous hyphae abundant to very abundant, 3–10 μm wide. Subhymenium 20–30 μm thick, with 2–3 layers of globose, ellipsoid, or irregular inflated cells, 6–24 × 6–18 μm. Basidia (Figure 3b) 37–52 × 12–16 μm, clavate, four-spored; sterigmata 4–6 μm long; basal septa clamped. Basidiospores (Figure 3a) [100/3/3] (8.5–) 9.0–11.5 (–13.0) × (6.0–) 7.0–8.5 (–10.0) μm, avl X avw = 10.3 × 7.4 μm, Q = (1.19–) 1.25–1.53 (–1.67) μm, Qm = 1.40 ± 0.09, broadly ellipsoid to ellipsoid, inamyloid, colorless, thin-walled, smooth; apiculus short but wide, width up to 1.5 μm. Lamellar edge appearing as a sterile strip, composed of very abundant to nearly dominant globose, subglobose, ellipsoid, or irregular inflated cells (11–26 × 8–25 μm), single and terminal or in chains of 2–3, thin-walled, colorless; filamentous hyphae scattered, 3–6 μm wide, irregularly arranged or parallel to lamellar edge. Pileipellis 130–205 μm thick, composed of radial, thin-walled, colorless, filamentous hyphae 3–17 μm wide; vascular hyphae scarce. Volval remnants on pileus (Figure 3c) composed of vertically to subvertically arranged elements: filamentous hyphae fairly abundant to abundant, 2–8 μm wide, colorless, thin-walled, branching, anastomosing; inflated cells abundant, globose, subglobose, to ellipsoid, sometimes irregular, 23–65 × 15–55 μm, colorless, thin-walled, terminal or in chains of 2–3; vascular hyphae scarce. Volval remnants on stipe base is semblable with the structure of volval remnants on pileus, composed of irregularly arranged elements: filamentous hyphae very abundant to nearly dominant, 2–11 (–15) μm wide, colorless, thin-walled, branching, anastomosing; inflated cells fairly abundant, globose, subglobose to ellipsoid, sometimes irregular, 12–88 × 10–55 μm, colorless, thin-walled; vascular hyphae scarce. Stipe trama composed of longitudinally arranged, clavate terminal cells, 90–265 × 20–48 μm; filamentous hyphae abundant, 2–16 μm wide; vascular hyphae scarce. Clamps present in all parts of basidioma.

Habitat: Solitary to scattered on soil in tropical deciduous forests dominant by Dipterocarpus and Shorea. Basidioma occurs in the rainy season during May to October.

Distribution: Currently known from northern and northeastern Thailand.

Additional collections examined: Thailand, Kalasin Province: Kham Muang District, Na Bon, 16°53′05″ N, 103°41′49″ E, alt. 239 m, 12 August 2020, Yuan S. Liu, STO-2020-231 (SDBR-STO-2020-231); Kalasin Province: Somdet District, Mahachai, 16°48′38″ N, 103°46′13″ E, alt. 197 m, 14 August 2020, Yuan S. Liu, STO-2020-253 (SDBR-STO-2020-253); Sakon Nakhon Province: Kut Bak District, Na Mong, 17°06′04″ N, 103°54′32″ E, alt. 208 m, 15 August 2020, Yuan S. Liu, STO-2020-303 (SDBR-STO-2020-303); Chiang Mai Province: Mueang Chiang Mai District, Suthep, 18°48′10″ N, 98°57′02″ E, alt. 343 m, 26 August 2020, Yuan S. Liu, STO-2020-398 (SDBR-STO-2020-398).

Notes: Amanita kalasinensis is similar to several species that have a light yellow tinge pileus covered by pyramidal to granuliform, white volva remnants, such as A. parvipantherina Zhu L. Yang, M. Weiß & Oberw., A. sychnophyramis f. subannulata Hongo, and A. sychnophyramis Corner & Bas f. sychnopyramis. However, both A. parvipantherina [33,34] and A. sychnophyramis f. subannulata [1,2,3,35] differ from A. kalasinensis by having a white or brownish annulus on its stipe, as well as a darker brownish pileus. Amanita sychnophyramis f. sychnophyramis, occurring in Singapore, China, and Malaysia [3,36,37], is discerned from A. kalasinensis by having a larger and darker brownish pileus. Furthermore, compared with the former species, which is short of clamp and has a globose to subglobose basidiospore (6.5–8.5 × 6–8 μm, Q = 1.01–1.11, Qm = 1.06 ± 0.03), A. kalasinensis has obvious clamps and a broadly ellipsoid to ellipsoid basidiospore (9.0–11.5 × 7.0–8.5 μm, Q = 1.25–1.53 μm, Qm = 1.40 ± 0.09).

Amanita ravicrocina Yuan S. Liu & S. Lumyong sp. nov. (Figure 4 and Figure 5).

Mycobank number: 842343.

Holotype: Thailand. Sakon Nakhon Province: Phu Phan District, Khok Phu, 17°00′09″ N, 103°57′34″ E, alt. 291 m, 15 August 2020, Yuan S. Liu, STO-2020-282 (CMUB-39967).

Etymology: ravicrocina, from ravus = greyish, and crocinus = orange, refers to its greyish orange pileus.

Description: Basidioma small to medium-sized. Pileus 3.0–8.5 cm diam., convex to plano-convex, often slightly depressed at center, brown (5E5), greyish orange (5B4–5) to orange white (5A2), often darker at center and becoming paler towards margin; volval remnants on pileus often persistent as large, thick, white (1A1) patches slightly attached on pileus; margin striate (ca. 0.3–0.5), non-appendiculate; context white, unchanging. Lamellae free, crowded, white (1A1); lamellulae truncate. Stipe 5.6–13.1 cm long × 0.8–1.4 cm diam., slender, subcylindrical and slightly tapering upward, with apex slightly expanded, white (1A1), covered with white (1A1) fibrils, becoming floccose near basal bulb; context white (1A1), fistulose; basal bulb globose to subglobose, 1.8–2.9 cm diam., white (1A1); volval remnants on stipe base formed a collar-like or shortly limbate volva on limit between stipe and basal bulb, white (1A1). Annulus absent. Odor not recorded.

Lamellar trama bilateral. Mediostratum 15–30 μm wide, composed of abundant clavate to cylindrical inflated cells (25–160 × 12–35 μm); filamentous hyphae abundant, 2–7 μm wide; vascular hyphae scarce. Lateral stratum composed of abundant ellipsoid to clavate inflated cells (50–155 × 7–18 μm), diverging at an angle of ca. 30° to 45° to mediostratum; filamentous hyphae abundant, 2–8 μm wide. Subhymenium 20–30 μm thick, with 2–3 layers of subglobose, pyriform, or irregular cells, 8–19 × 5–10 μm. Basidia (Figure 5b) 38–52 × 10–14 μm, clavate, four-spored; sterigmata 3–6 μm long; basal septa lacking clamps. Basidiospores (Figure 5a) [100/5/4] (7.0–) 8.0–9.5 (–10.5) × (6.0–) 7.0–8.5 (–9.0) μm, avl X avw = 8.7 × 7.5 μm, Q = (1.00–) 1.06–1.29 (–1.36) μm, Qm = 1.16 ± 0.08, subglobose to broadly ellipsoid, inamyloid, colorless, thin-walled, smooth; apiculus small. Lamellar edge appearing as a sterile strip, composed of abundant subglobose to ellipsoid inflated cells (8–25 × 6–12 μm), single and terminal or in chains of 2–3, thin-walled, colorless; filamentous hyphae fairly abundant, 2–8 μm wide, irregularly arranged or parallel to lamellar edge. Pileipellis 60–100 μm thick, composed of radial, thin-walled, colorless, filamentous hyphae 2–10 μm wide; vascular hyphae scarce. Volval remnants on pileus (Figure 5c) composed of irregularly arranged elements: filamentous hyphae abundant to very abundant, 2–10 μm wide, colorless, thin-walled, branching, anastomosing; inflated cells abundant, globose, subglobose, fusiform to ellipsoid, sometimes irregular, 20–90 × 14–46 μm, colorless, thin-walled, terminal or in chains of 2–4; vascular hyphae scarce. Volval remnants on stipe base is semblable with the structure of volval remnants on pileus, composed of irregularly arranged elements: filamentous hyphae very abundant to nearly dominant, 2–9 (–12) μm wide, colorless, thin-walled, branching, anastomosing; inflated cells fairly abundant to abundant, globose, subglobose, ellipsoid to clavate, sometimes irregular, 18–90 × 11–35 μm, colorless, thin-walled; vascular hyphae scarce. Stipe trama composed of longitudinally arranged, clavate terminal cells, 75–235 × 18–36 μm; filamentous hyphae abundant, 3–11 μm wide; vascular hyphae scarce. Clamps absent in all parts of basidioma.

Habitat: Solitary to scattered on soil in tropical deciduous forests dominant by Dipterocarpus and Shorea. Basidioma occurs in the rainy season during May to October.

Distribution: Currently known from northeastern Thailand.

Additional collections examined: Thailand, Kalasin Province: Kham Muang District, Na Bon, 16°53′05″ N, 103°41′49″ E, alt. 239 m, 12 August 2020, Yuan S. Liu, STO-2020-229 (SDBR-STO-2020-229); Yuan S. Liu, STO-2020-235 (SDBR-STO-2020-235); Sakon Nakhon Province: Phu Phan District, Khok Phu, 17°00′09″ N, 103°57′34″ E, alt. 291 m, 15 August 2020, Yuan S. Liu, STO-2020-283 (SDBR-STO-2020-283).

Notes: Amanita ravicrocina has a collar-like volva remnant on the limit between stipe and inflated basal bulb, which is not a common characteristic in A. section Amanita. Coupled with a brown tone pileus surface, A. ravicrocina could be easily singled out from other species in A. section Amanita, except A. ibotengutake T. Oda, C. Tanaka & Tsuda, A. pseudopantherina Zhu L. Yang ex Yang-Yang Cui, Qing Cai & Zhu L. Yang, and A. subglobosa Zhu L. Yang. Nevertheless, the latter three taxa are distinguished from A. ravicrocina by having the membranous annulus and pyramidal to subconical volva remnants on pileus [1,4,38]. In addition, A. parvipantherina [4,33] also possesses close likeness with A. ravicrocina. Both of them have small to medium-sized basidiomata and a light grey to brown pileus surface. However, A. parvipantherina has a white to brownish annulus, and its volva remnants on pileus are verrucose to pyramidal, while A. ravicrocina has a short annulus and appears as patchy volva remnants on pileus.

4. Discussion

As mentioned above, thirteen species in section Amanita have been described in Thailand. Although most of these species are based on both of morphologic and phylogenetic evidences, four species, namely, A. aff. mira, A. obsita, A. siamensis, and A. subglobosa, have only morphologic data [6], which need to be supplemented with more molecular data to confirm their taxonomic status. Our study, along with other previous studies, allowed to discover the high diversity of Amanita species in Thailand [6,7,8,9,10,11,12], indicating that more taxa remain to be discovered and documented.

In our multi-gene phylogenetic analyses, both Amanita kalasinensis and A. ravicrocina formed a well-supported (BS = 100%, PP = 1.0) monotypic clade. However, the position of A. ravicrocina is not stable. Despite that many potential causes could lead to these unstable topologies, the primary reason might be that A. ravicrocina has significant differences from any other existing Amanita species in each single gene (the highest similarity and its query cover of initial BLAST searches results in GenBank for ITS and nrLSU of A. ravicrocina samples were from following: A. frostiana—RET 588-6 (KP313583) with 89.23% similarity and 99% query cover for ITS, and A. altipes—BZ2013-42 (MH716040) with 97.58% similarity and 100% query cover for nrLSU).

The erratic positions of Amanita ravicrocina and its distinct sequences provide indirect evidence that there are probably some new taxa related to A. ravicrocina that remain to be discovered. Therefore, further exploration of Amanita diversity is critical, which could reveal more members in this section.