Species Diversity of Amanita Section Vaginatae in Eastern China, with a Description of Four New Species

Species of Amanita sect. Vaginatae (Fr.) Quél. are challenging to delimitate due to the morphological similarity or morphostasis among different taxa. In this study, a multi-locus (nuc rDNA region encompassing the internal transcribed spacers 1 and 2 with the 5.8S rDNA, the D1–D3 domains of nuc 28S rDNA, partial sequences of translation elongation factor 1-a, and the second largest subunit of RNA polymerase II) phylogeny was employed to investigate the species diversity of the section in eastern China. Sixteen species were recognized, including four new species; namely, A. circulata, A. multicingulata, A. orientalis, and A. sinofulva. They were documented with illustrated descriptions, ecological evidence, and comparisons with similar species. A key to the species of the section from eastern China is provided.

In China, 33 taxa of A. sect.Vaginatae have been reported [1,2,21,35].Twenty-seven of them can be found in southwestern China.Southern and eastern China are the second and third most species-rich areas, with six and five species.Three species have been reported in central and northeastern China, respectively.The majority of them are distributed in more than one area.There are nine endemic species in southwestern China.Only one species is restricted in the southern, central, and northeastern parts, respectively.The five taxa in eastern China-namely A. cingulata J.W. Liu and Zhu L. Yang, A. hamadae Nagas.and Hongo, A. olivaceofusca Y.Y.Cui et al., A. ovalispora Boedijn, and A. pallidozonata Y.Y.Cui et al.-also occur in central, southern, and southwestern China [2].
During our investigations of macrofungi in eastern China, numerous specimens of A. sect.Vaginatae were collected.In this study, we applied integrative taxonomy, including morphological characters, multi-locus phylogenetic evidence, and ecological data, to elucidate the species diversity of A. sect.Vaginatae in eastern China.A key to all of the species found in the area is provided.

Taxon Sampling
Sixty-one specimens of A. sect.Vaginatae were examined.Among them, 50 were collected from eastern China, and the rest were from southwestern China.For each collection, a part of the basidioma was dried with silica gel for DNA extraction.The remaining materials were then dried at 45-50 • C with an electronic food dehydrator.At the same time, the specimen information, host trees, altitudes, locations, collectors, and dates were recorded, and photos of the fruiting bodies were taken.The location information and ecological habits of the specimens mentioned above are stated in the results section.All specimens examined in this work were deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS), and the Edible-medicinal Fungal Herbarium of Anhui Agriculture University (EFHAAU).

Morphological Observation
The macroscopic descriptions are based on detailed field notes and photographs of fresh basidiomata.Color codes indicated in the descriptions are from Kornerup and Wanscher [36].Microscopic features were studied with light microscopy using dried material rehydrated in 5% KOH and, when necessary, dyed with Congo Red.Melzer's reagent was used to check the amyloidity of basidiospores.In the description of the basidiospores, the abbreviation (n/m/p) represents n basidiospores measured from m basidiomata of p collections.Dimensions for the basidiospores are given using a range notation of the form (a-) b-c (-d).The range b-c contains a minimum of 90% of the measured values.Extreme values, a or d, are given in parentheses.Q represents the 'length/width ratio' of a basidiospore in the side view.Qm means the average Q of all basidiospores measured ± sample standard deviation.

Sequence Alignments and Phylogenetic Analyses
According to the most recent comprehensive multi-locus phylogenetic analyses of A. sect.Vaginatae, the ITS sequences were not included [33].Therefore, two datasets, the multi-locus dataset (nrLSU, tef1-α and rpb2) and the ITS sequences matrix, were compiled to infer the phylogeny of the section, respectively.For the ITS dataset, sequences of the new taxa were initially blasted in GenBank.The most closely related sequences (nucleotide identities >90%) were retrieved to complement the ITS matrix with one to two representa-tives per species.For the combined dataset, all known species of the section with sequences of the gene fragments employed in this study were included [2,4,5,21,22,33].According to recent phylogenetic analyses, A. muscaria (L.) Lam., A. parvipantherina Zhu L. Yang et al., A. caesarea (Scop.)Pers., and A. yuaniana Zhu L. Yang of A. sect.Amanita and sect.Caesareae Singer ex Singer were selected as outgroups [2].Sequences of all gene fragments were separately aligned with MAFFT 7 [42] and manually optimized in BioEdit 7 [43].For the two datasets, the introns of tef1-α and rpb2 were excluded because of the difficulty in alignment.The ambiguously aligned regions of nrLSU and ITS were eliminated using Gblocks 0.91b [44] with the "less stringent selection" parameter set.The final alignments of both datasets were deposited in TreeBASE (30635).
Single-gene analyses were carried out for the concatenated matrix to detect possible incongruence among individual genes based on the Maximum Likelihood (ML) method.Because no well-supported bootstrap value (BS > 70%) was detected (Figures S1-S3), the resulting alignments of nrLSU, tef1-α, and rpb2 were concatenated using Geneious v9.1.3[45].The best partition schemes and evolutionary models of the two datasets were selected using PartitionFinder V2.1.1 [46].ITS was divided into three blocks: ITS1, ITS2, and 5.8S.The concatenated matrix was partitioned according to gene fragments and codon positions, including nrLSU, tef1-α_codon1, tef1-α_codon2, tef1-α_codon3, rpb2_codon1, rpb2_codon2, and rpb2_codon3.Thus, three and seven blocks were predefined for the ITS and combined datasets, respectively.The two datasets were then analyzed using RAxML v8.2.4 [47] and MrBayes v3.2.6 [48] for ML and Bayesian Inference (BI) analyses, respectively.In the ML analyses, the statistical supports were obtained using rapid bootstrapping with 1000 replicates, and the other parameters used the default settings.Some of the selected models could not be implemented in RAxML; thus, the GTR + I + G model, which included all of the parameters of the selected model, was used for all partitioned data.For BI analyses, four Markov Chain Monte Carlo (MCMC) chains were run simultaneously for 5 million generations under the best partition schemes and evolutionary models selected using PartitionFinder, and trees were sampled every 1000 generations.Runs were automatically terminated when the average standard deviation of split frequencies fell below 0.01 and the ESS values exceeded 200 [48].Tracer v1.5 (http://tree.bio.ed.ac.uk/software/tracer/, accessed on 20 January 2023) was used to confirm the chain convergence.Subsequently, the sampled trees were summarized, and posterior probabilities were obtained by discarding the first 25% of generations as burn-in.Quotation marks are added to indicate the uncertain taxonomic positions, -represents missing corresponding sequences.

Phylogenetic Analyses
Overall, 163 sequences-including 56 for nrLSU, 42 for tef1-α, 37 for rpb2, and 28 for ITS-were newly generated in this study, and they were aligned with the sequences downloaded from GenBank.The sequences retrieved from GenBank and obtained in this study are listed in Tables 1 and S1.The concatenated dataset (nrLSU, tef1-α, and rpb2) included 429 sequences from 201 samples representing 112 taxa (Table 1).The raw concatenated dataset comprised 2146 positions, and the final matrix retained 1846 positions, with 646 parsimony-informative sites, after excluding introns and poorly aligned regions.In the ITS dataset, 128 sequences from 64 taxa were included (Table S1).The dataset comprised 903 positions, with 384 parsimony-informative sites, and 570 positions of the ambiguously aligned regions were excluded.Six and two subsets were selected for the combined and ITS datasets, respectively.The best partition schemes and corresponding best-fits models are summarized in Table 2.The phylogenetic trees inferred from the ML and BI analyses were similar in topology.Therefore, only the trees obtained from the ML analyses were presented (Figures 1 and S1-S4).In the phylogenetic tree based on the combined matrix, the collections from eastern China were clustered into 19 lineages, including 12 known species and seven undescribed taxa (Figure 1).Four of them were described as new species, namely A. circulata, A. multicingulata, A. orientalis, and A. sinofulva (Figures 1 and S1-S4).Amanita circulata formed a monophyletic clade with A. flavidocerea Thongbai et al. from Thailand, A. pekeoides G.S. Ridl.from New Zealand, A. verrucosivolva Zhu L. Yang from China, and three undescribed taxa (A.aff.fulva HKAS29518 China, Amanita sp.RET 732-8, and 374-3 USA, A. cf.circulata China) (Figures 1  and S4).Amanita multicingulata was sister to A. liquii Zhu L. Yang et al. from China.Amanita orientalis formed a monophyletic group with A. griseofolia Zhu L. Yang and another two species from South Korea, erroneously identified as A. ceciliae (Berk.& Broome) Bas.In the phylogenetic tree inferred from the ITS dataset, the taxon was more closely related to the sample (JL2) from China, which was labeled as A. griseofolia, and two collections (SUA441 and SUA510) from Pakistan, with moderate support (Figure S4).Amanita sinofulva was clustered in the clade formed by A. orientifulva Zhu L. Yang et al., A. suborientifulva Raspé et al., and another five collections from China, Japan, and South Korea, which were labeled as A. cf.suborientifulva (EFHAAU4437 and EFHAAU5291), or erroneously identified as A. orientifulva (STDS-2-10 and KA12-0642) and A. fulva Fr. (ASIS26398), respectively (Figures 1 and S4).
The remaining lineages represented three putatively new taxa.As only one or two collections were included in every species, they will be described in the future with adequate samples.

Taxonomy
Amanita circulata Y.Y.Cui, Q. Cai and Zhu L. Yang, sp.nov., Figures 1-3.Fungal Names: FN 571585.Etymology: circulata from circular, referring to its circular zone on the pileus.Diagnosis: Similar to A. pallidozonata, but differs in its more filamentous hyphae in the volval remnants on the stipe base.
Habitat: Solitary to scattered on soil in subtropical mixed forests with Fagaceae and Pinaceae.
Distribution: known from eastern and southwestern China.Additional specimens examined: CHINA.ANHUI PROVINCE: Huangshan, in a broad-leaved forest with trees of Fagaceae, altitude 620 m, 13  Notes: Amanita circulata is somewhat related to A. flavidocerea (Figure 1).However, the latter can be easily distinguished from the former species by its non-umbonate pileus, which is yellow and lacks a ring-like zone at the proximal end of marginal striations [4].Amanita pallidozonata and A. zonata Y.Y.Cui et al. might be confused with A. circulata due to the pronounced ring-like zones at the proximal end of the marginal striations.However, A. pallidozonata differs from A. circulata by its more inflated cells in the inner part of volval remnants on the stipe base [2].Amanita zonata has relatively smaller basidiospores (9-10.5 × 8.5-10 µm) [2].
Diagnosis: Close to A. liquii, but differs in its longer striations on pileal margin, white to dirty white lamellae without obvious color change when dried, smaller basidiospores and distributions in subtropical forests dominated with Fagaceae, sometimes mixed with Pinus.
Habitat: Solitary to scattered on soil in subtropical broad-leaved forests dominated with Fagaceae, sometimes in mixed forests with fagaceous and Pinus plants.
Amanita orientalis Q. Cai, Y.Y.Cui and Zhu L. Yang, sp.nov., Figures 1, 2 and 5. Fungal Names: FN 571587 Etymology: orientalis means eastern, namely after its type locality from East Asia.Diagnosis: Close to A. griseofolia but differs in its more brownish pileus and mostly subglobose to broadly ellipsoid basidiospores.
Lamellar trama bilateral.Habitat: Solitary to scattered on soil in subtropical forests dominated with Fagaceae, sometimes mixed with Pinus.
Distribution: Known from eastern, central, and southwestern China.Based on the phylogenetic tree inferred from the ITS dataset, it also occurs in Tibet autonomous region and Hunan province (Figure S4).
Additional Notes: Amanita orientifulva and A. suborientifulva can be confused with A. sinofulva.According to our multi-gene phylogenetic analysis (Figure 1), the first two species also share close relationships with A. sinofulva.However, A. orientifulva has slightly narrower basidiospores (10.0-14.0× 9.5-13.0µm, Q = 1.0-1.12,Qm = 1.06 ± 0.04) and is found in subalpine forests dominated by trees of Abies and Picea [2,54].The non-umbonate pileus and globose to subglobose or broadly ellipsoid basidiospores of A. suborientifulva set it apart from A. sinofulva [4].The European A. fulva is also similar to A. sinofulva, but differs in the globose to subglobose basidiospores and in the saccate volva, with inflated cells dominant in its outer part [1,2].

Species Delimitation and Recognition within Amanita sect. Vaginatae
Our data revealed that several macro-and microscopic characteristics could be useful for the delimitation of species in A. sect.Vaginatae.Five of them are most informative, viz. the color of the basidiomata, the striations on the pileal margin, the presence or absence of the annulus, the volval remnants on the stipe base, and the size of the basidiospores.In this study, A. cingulata is the only species with a white basidioma, while the other species from eastern China have basidiomata ranging from yellow, to gray, to brown.The striations on the pileal margin of A. zonata, A. pallidozonata, and A. circulata form a ring-like zone at the proximal end, while the remaining taxa in eastern China are without this zone [2].Most species in the section are ringless, with only seven taxa with an annulus [12,21,50].The volval remnants on the stipe base of several species are saccate, while some of them are tomentose, arranged in incomplete rings, viz. A. griseofolia, A. multicingulata, and A. orientalis.Species with an annulus, a ring-like zone at the proximal end, or with incomplete rings of volval remnants on the stipe base are clustered in non-monophyletic groups.
Given that it is difficult to delimitate these species based solely on morphological studies, integrative taxonomy is indispensable in recognizing species of the section.This method, which delimits and describes taxa by integrating information from different types of data and methodologies (e.g., phylogeny, comparative morphology, habitat and preference of hosts, and behavior), is proven to be useful for species recognition in plants, animals, and fungi [2,[60][61][62][63][64].In this study, species with similar morphological characteristics are successfully recognized using this method.
For example, A. zonata, A. pallidozonata, and A. circulata are morphologically similar due to the pronounced ring-like zones at the proximal end of the marginal striations.However, they occupy different positions in the phylogenetic tree and are distantly related (Figure 1).Following detailed morphological studies, A. pallidozonata can be distinguished from A. circulata by its more inflated cells in the inner part of the volval remnants on the stipe base [2].Amanita zonata differs from A. circulata by its smaller basidiospores (9-10.5 × 8.5-10 µm) [2].
Amanita sinofulva is phylogenetically close and morphologically similar to A. orientifulva.However, they are clustered into two independent lineages (Figures 1 and S4), and differ in their geographic distributions and host plants.The former species is restricted to the subtropical forests dominated by the trees of Fagaceae and Pinus, while the latter is found in the subalpine forests under Picea spp., Abies spp., and Quercus spp.[2,54].Furthermore, the latter can be distinguished from the former by its narrower basidiospores (10-14 × 9.5-13 µm).
Overall, the combination of morphological characteristics, multi-locus phylogeny, and ecological data can make the result of species delimitation more reliable and objective.According to our phylogenetic analyses, species of the section from the southern parts of China are closely related to those reported from Southeast Asia.For example, of the 13 species reported from Southeast Asia [4,29,65,66], 7 of them also occur in the southern parts of China (Figure 1).Among them, A. angustilamellata (Höhn.)Boedijn, A. brunneoprocera Thongbai et al., A. cinctipes, and A. pallidocarnea (Höhn.)Boedijn are typical tropical elements restricted in the tropical areas of China.The other three species, viz. A. brunneosquamata Thongbai et al., A. suborientifulva, and A. subovalispora, extend their distribution from Southeast Asia to subtropical China.In addition, several taxa found in subtropical or subalpine temperate areas in southern parts of China are phylogenetically close to species from Southeast Asia.For example, A. circulata and A. zonata, reported from the subtropical regions of China, are sister to A. flavidocerea and A. flavidogrisea Thongbai et al. from Southeast Asia, respectively (Figure 1).Amanita pallidozonata from the subtropical areas and A. orientifulva from the subalpine forests of the southern parts of China are closely related to A. pallidocarnea from Southeast Asia and tropical China (Figure 1).Therefore, the species in the southern parts of China may have historical affinities in common with those of tropical Asia [67].This was also consistent with the results of Codjia [33], in which part of the taxa in East Asia were indicated to have migrated from Southeast Asia.
Previously, only five species of A. sect.Vaginate were reported from eastern China [2].In this study, 16 species were delimitated, with four new species and 12 newly recorded species.For the convenience of recognition, a key to them is provided.

Figure 1 .
Figure 1.Phylogenetic tree of Amanita sect.Vaginatae inferred from maximum likelihood analyses based on the combined dataset (nrLSU, tef1-α and rpb2).Bootstrap values over 50% and Bayesian posterior probabilities over 0.90 are shown along the branches.Sequences from type collections are indicated with (T), and new species are in boldface.

4. 2 .
Phylogenetic Relationships of Amanita sect.Vaginatae Species in Southeast Asia and Southern Parts of China In this study, 40 taxa of A. sect.Vaginatae were delimitated in China, including 33 known taxa [1,2,21], four species new to sciences (A.circulata, A. multicingulata, A. orientalis and A. sinofulva), and three species new to China (A. brunneoumbonata Thongbai et al., A. suborientifulva and A. subovalispora Thongbai et al.).Thirty-nine of them are reported from the southern parts of China-namely, southwestern, central, eastern, and southern China.

Table 1 .
Information on specimens used in multi-locus phylogenetic analyses and their GenBank accession numbers.Sequences newly generated in this study are indicated in bold.

Table 2 .
The best partition schemes and models selected by PartitionFinder.