Diversity and taxonomy of the genus Amanita (Amanitaceae, Agaricales) in the Yanshan Mountains, Northern China

Globally, the species of Amanita are key components of ectomycorrhizal ecosystems. Some of them are widely known as poisonous or edible fungi. Although many new Amanita species from China have been described, the species diversity of Yanshan Mountains remains unknown. We here describe three new species, namely, A. borealis sp. nov. (Sect. Amanita), A. brunneola sp. nov. (Sect. Caesareae), and A. yanshanensis sp. nov. (Sect. Validae), based on morphological observations and molecular phylogenetic analyses. In addition, nine known species, namely, A. caesareoides (Sect. Caesareae), A. chiui (Sect. Vaginatae), A. muscaria (Sect. Amanita), A. oberwinklerana (Sect. Roanokenses), A. ovalispora (Sect. Vaginatae), A. subglobosa (Sect. Amanita), A. subjunquillea (Sect. phalloideae), A. vaginata var. vaginata (Sect. Vaginatae), and A. virosa (Sect. phalloideae), were reported from Yanshan Mountains for the first time. Our results emphasize that China has a high diversity of Amanita species and that additional studies are required to understand the exact species number. These findings play a crucial role in Amanita toxin research and ecological conservation. This study investigated the areas where Amanita species-related research is lacking. The study also attempted to better understand Amanita distribution and thus contribute to related research. This study enriches the species diversity of Amanita in Yanshan Mountains and offers additional data supporting the macrofungal systematics, toxin research, and diversity and ecological studies of Amanita in future studies.

Based on the traditional morphological and anatomical characteristics, and the molecular phylogeny evidence, the classification of Amanita has also undergone many changes.Corner and Bas (1962) and Bas (1969) considered observing the natural characteristics of Amanita species in the wild important.They applied microscopic characteristics to taxonomy and split the species into two subgenera and six sections.Many mycologists have accepted this taxonomic method as a great historical advance in the Amanita classification (Jenkins, 1977;Hongo, 1982;Jenkins, 1986;Mao, 1990;Pegler and Shah-Smith, 1997).However, the classification of its subgenera remains disputed (Moser, 1967;Garcin, 1984;Singer, 1986).Subsequently, the systematic research on Amanita is gradually deepening with the development and advancement of molecular systematics.A recent comprehensive phylogenetic study introduced the latest classification system for Amanita.According to this system, Amanita was divided into 3 subgenera and 11 sections (Cui et al., 2018).This system is followed by other mycologists (Kumar et al., 2021;Suwannarach et al., 2022;Huang et al., 2023).
The Yanshan Mountains (115°-119°47′E, 39°40′-41°20′N) is located in northern China and has a high plant diversity (Figure 1).The main forest types on these mountains are deciduous broadleaved forests and mixed coniferous and broad-leaved forests.The original dominant ectomycorrhizal plants included Quercus mongolica Fisch.ex Ledeb., Betula platyphylla Suk., Abies nephrolepis (Trautv.)Maxim., Populus tomentosa Carrière, and Pinus tabuliformis Carr.(Wang et al., 2021).The Yanshan Mountains region has a warm-temperate continental monsoon climate with an annual precipitation of 350-700 mm.The peak of precipitation occurs in June-August.The altitude of these mountains ranges from 200 to 2,200 m (Zhou et al., 2022a;Zhou et al., 2022b;Zhou et al., 2022c).Some past records of Amanita species in this area are available (Yang, 2004;Chen et al., 2006; Geographical location and topography of the study area.Zhou et al. 10.3389/fpls.2023.1226794Frontiers in Plant Science frontiersin.orgZhang et al., 2017;Cui et al., 2018;Wu et al., 2020).However, information available on Amanita species on these mountains is incomplete.Taxonomic research has never been considered a popular study, but it can be considered the basis for understanding the world and research in related professional fields and can only be studied and applied if we figure out what the species really is.Especially think of Amanita, which are a very attractive taxa of macrofungi.It plays an important role in toxin research and ecological conservation.In the present study, 36 fresh Amanita specimens were collected from Yanshan Mountains.There were 20 herbarium specimens were loaned from the Herbarium Mycologicum Academiae Sinicae (HMAS, Institute of Microbiology, Chinese Academy of Sciences) for further research.On the basis of morphological examination and inference of phylogeny, three new species and nine known species were reported herein.The study aimed to determine the taxonomic status and phylogenetic position of Amanita species represented by these specimens so as to establish a comprehensive database of macrofungal diversity in Yanshan Mountains, especially Amanita species, and to use this database as a basis for future studies on macrofungal systematics, fungal toxin, diversity, and ecology in this region.
2 Materials and methods

Sample collection and morphological analyses
The specimen collection area is shown in Figure 1 (Figure and data provided by the Chinese Academy of Environmental Sciences).The specimens were collected during 2019-2022 from Yanshan Mountains and photographed in the field.Macroscopic features of fresh specimens such as colors and odors were noted.Color codes and designations were assigned after referring to the website ColorHexa (https://www.colourhexa.com).The specimens were dried using a Dorrex dryer at 50°C for approximately 12 h and deposited in the Herbarium of the College of Life Science, Capital Normal University, Beijing, China (BJTC).Other 20 herbarium specimens from the research area were obtained from the HMAS.
To observe microscopic characters, thin sections of the dried material were mounted in 3% KOH or sterilized water.Then, the materials were stained with 1% Congo red to increase the visibility of the structures.Microscopic features (e.g., basidiospores, pileipellis, and volval remnants) were observed and measured under a light microscope (Olympus DP71, Tokyo, Japan).Basidiospore measurements of new species are presented as (a)b − c(d).Among them, b − c, a, and d represent a minimum of 90% of the measured values, minimum extreme values, and maximum extreme values, respectively.Q represents the length/width ratio of the basidiospores, and Q m is the average Q values of all basidiospores measured (Bas, 1969).Q m values ± sample standard deviations are provided.The descriptive terms are in accordance with Cui et al. (2018).

Molecular data analyses and species delimitation
The nrITS-nrLSU-rpb2-tef1-a multi-locus dataset included 216 ingroup samples.These samples were used to infer the phylogenetic status of our Amanita specimens at the phylloclade level.The nrLSU dataset included 204 ingroup samples.These samples were used to analyze the subsection in which the species were located.Furthermore, nrITS was used to infer phylogenetic relationships between new and known Amanita species, as GenBank contains a large amount of nrITS sequence data for this genus.These nrITS sequences of new species were divided into different datasets, given that these sequences are too variable to obtain reliable genus-wide comparisons.Based on previous study findings and the GenBank database of the National Center for Biotechnology Information, reference sequences of all Amanita species in the dataset were selected for phylogenetic analysis (Cui et al., 2018;Su et al., 2022 Cui et al. (2018).
The BI analysis was performed using a Markov chain Monte Carlo (MCMC) algorithm (Rannala and Yang, 1996) and MrBayes 3.1.2(Ronquist and Huelsenbeck, 2003) based on the best substitution model determined by MrModeltest 2.3 (Nylander, 2004), GTR + I + G for nrITS, nrLSU, rpb2, and tef-1a.Two MCMC chains were run from random trees for 10,000,000 generations, stopping when the average standard deviations of split frequencies were less than 0.01.Trees were stored for each 1,000 generations.The first 25% of the trees were excluded as the burn-in stage for each analysis.Branches with significant Bayesian posterior probability (BPP) values were then estimated in the resulting trees (Posada and Crandall, 1998).The ML analysis was performed using a GTR + GAMMA + I locus replacement model (Guindon et al., 2010).The branch support was obtained using the bootstrapping (BS) method of 1,000 replications (Hillis and Bull, 1993).Branches with a bootstrap (BS) support of ≥50% and BPP of ≥0.95 were considered significant (Hillis and Bull, 1993).

Phylogenetic analyses
The nrLSU dataset contained 209 sequences, including 42 newly obtained sequences.The length of the aligned dataset was 790 bp long.The nrLSU phylogenetic analysis results revealed that our specimens belonged to six sections under Amanita, namely, sections Amanita Pers., Caesareae Singer, Roanokenses Singer, Phalloideae Queĺ., Validae Queĺ, and Vaginatae Queĺ (Figure S1), and were divided into 12 clades.Subsequently, the nrITS-nrLSU-rpb2-tef1-a multi-locus phylogenetic analysis was performed to infer the phylogenetic status of our Amanita specimens at the phylloclade level (Figure 2).The combined nrITS-nrLSU-rpb2-tef1a dataset had 726 sequences, including 143 newly obtained sequences in this study.The aligned dataset was 2,150 bp long including alignment gaps (245 bp for nrITS, 790 bp for nrLSU, 660 bp for rpb2, and 365 bp for tef1-a).Results of the nrITS-nrLSU-rpb2-tef1-a and nrLSU phylogenetic analyses revealed that the subgenera and sections proposed by Cui et al. (2018) were strongly supported with significant BPP values and ML bootstrap (MLB).
The nrITS-nrLSU-rpb2-tef1-a multi-locus phylogenetic analysis showed that our specimens were clustered into 14 clades.Moreover, they formed three distinct and strongly supported new branches, which were nested in sections Amanita, Caesareae, and Validae, respectively.These three new lineages were as follows: two specimens (BJTC Z110 and BJTC L169) formed one clade (BPP = 1.00,MLB = 100%) and were closely related to A. griseopantherina Yang-Yang Cui, Qing Cai & Zhu L. Yang, A. pantherina (DC.)Krombh., and A. subglobosa Zhu L. Yang on Figure 2. The two specimens (BJTC Z087 and BJTC C650) clustered into a branch with a high support (BPP = 1.00,MLB = 100%), which further clustered into a clade containing A. longistriata S. Imai., with moderate support.Eight specimens (BJTC Z049, BJTC Z820, BJTC Z083, BJTC Z824, BJTC Z760, BJTC Z819, BJTC C182, and BJTC Z815) were clustered together, forming a completely supported clade (BPP = 0.99, MLB = 99%).The new branches clustered with A. spissacea S. Imai and formed a sister clade in the phylogenetic tree.The nrLSU phylogenetic analysis revealed topologies similar to those of the multi-locus phylogenetic tree, and the specimens also formed three new lineages.Therefore, based on the results of phylogenetic and morphological analyses, these new clades were identified as three new species herein.
In addition, through morphological and phylogenetic analyses, we identified nine known Amanita species collected from Yanshan The nrITS-nrLSU-rpb2-tef1-a multi-locus phylogenetic tree obtained from the Bayesian analysis.Although the specimen BJTC S233 was clustered with A. cf.angustilamellata (HKAS 89451 and HKAS 83453) in the nrITS-nrLSU-rpb2-tef1-a and nrLSU phylogenetic analyses, additional specimens are required to elucidate its phylogenetic position and morphological characters.The specimens BJTC C654 and HMAS 26491 probably represented undescribed species.However, they could not be described in the present study because of the poor condition of their basidiomata, inadequate number of specimens, and uncertain phylogenetic position in the nrITS-nrLSU-rpb2-tef1a phylogenetic analysis.Therefore, we only temporarily termed them as Amanita sp.
The numbers above the branches represent strong support (BPP ≥0.95 and/or MLB ≥50%).Red font represents t the location of the newly acquired sequences.Table 1 presents the accession numbers of sequence information used.
Distribution: This species is known to be found in northwestern and southwestern China (Cui et al., 2018).
Habitat and distribution: It is present individually or scattered in the broad-leaved forests of Castanea mollissima Blume, with basidioma occurring in summer and autumn.
Habitat and distribution: It is present individually or is scattered in the broad-leaved forests of B. platyphylla Suk.Basidioma occurs in summer and autumn.
Commentary: Yang and Doi (1999) described A. oberwinkleriana from Japan, and it was subsequently reported from China, India, and Republic of Korea (Yang and Li, 2001;Bhatt et al., 2003;Yang, 2005;Kim et al., 2013a;Yang, 2015;Cui et al., 2018).The morphological description of our specimen is consistent with that provided by Yang and Doi (1999).In our multilocus phylogenetic analysis, 10 specimens clustered together with A. oberwinkleriana (HKAS 77330), forming a completely supported clade (BPP = 1.00,MLB = 96%).Based on these characters, our specimens were described as A. oberwinkleriana.Of note, the samples from the herbarium specimens (HMAS) exhibited poor sequence quality because of a long time.The multi-locus and nrLSU phylogenetic trees revealed that the branches of these herbarium specimens were longer (Figures 2, S1).
Habitat and distribution: It is present individually or scattered in the coniferous forests and mixed coniferous and broad-leaved forests of Pinus tabuliformis Carr.and Juglans mandshurica Maxim., with basidioma occurring in summer and autumn.Zhou et al. 10.3389/fpls.2023.1226794Frontiers in Plant Science frontiersin.organd described their basidiospores.According to Cui et al. (2018), no sequence of A. ovalispora is available from its type locality to delimit this species accurately.The morphological description of our specimens is consistent with that provided by Boedijn (1951).In our multi-locus phylogenetic analysis, two specimens clustered together with A. oberwinklerana (HKAS 79625 and HKAS 101406), forming a completely supported clade (BPP = 1.00,MLB = 96%) (Figure 2).The nrLSU phylogenetic analysis exhibited topologies similar to those of the multi-locus phylogenetic tree (Figure S1).According to these characters, our specimens were described as A. ovalispora.
Habitat and distribution: It is present solitary or is scattered in the pine, broad-leaved, or mixed forests of Fagaceae and Pinaceae trees.Basidioma occurs in summer and autumn (Cui et al., 2018).
The nrLSU phylogenetic analysis revealed topologies similar to those of the multi-locus phylogenetic tree (Figure S1).Based on these characters, we described our specimen as A. subglobosa.In addition, DNA sequences from another herbarium specimens (HMAS 34658) could not be generated.However, we also examined the morphology of these specimens, and the results proved that these specimens were A. subglobosa.
Habitat and distribution: It is present individually or is scattered in coniferous forests and mixed coniferous and broadleaved forests of Pinus tabuliformis Carr., J. mandshurica Maxim, and P. davidiana Dode., with basidioma occurring in summer and autumn.
This species belongs to section Validae and is closely related to A. spissacea on the multi-locus phylogenetic tree (Figure 2).In the single loci phylogenetic trees of both nrITS and nrLSU, A. yanshanensis clustered into an independent clade (Figures S1, S4).Moreover, basidiomata of A. yanshanensis with a gray-brown to gray pileus are also comparable with those of A. spissacea and A. fritillaria (Sacc.)Sacc.However, A. fritillaria has a basal bulb covered with conical, blackish, dark-gray to gray-brown volval remnants and a dirty white to gray annulus (Corner and Bas, 1962;Kumar et al., 1990;Yang, 2005;Yang, 2015;Cui et al., 2018).

Discussion
In our study, the molecular phylogenetic analyses further supported the delineation of Amanita into two subgenera, namely, subgen.Amanita Pers.and Amanitana (E.-J.Gilbert) E.-J.Gilbert, as suggested by Cui et al. (2018) (Figures 2, S1).Based on the macroscopic morphology and the preliminary comparison of the original sequence we obtained, the sections where the specimens were located were initially known.Therefore, the sequence information of subgen.Cui et al. (2018) mentioned that, for a better understanding of the range of variation in characters, new species should be described based on several specimens, but the technology of molecular systematics used in the present study improved the accuracy of our description of species.As specified in the results, the quality of sequences of these old herbarium specimens (HMAS 283800, HMAS 253802, HMAS 263406, and HMAS 253796) identified as A. oberwinkleriana was not good, but its systematic position and combined morphology could still be somewhat helpful in specimen identification.In addition, we could only depend on morphological observations for identifying these specimens as their sequences could not be obtained because of the poor condition of their basidiomata.
Studies have found and recorded 169 Amanita species in China, with most of them being concentrated in the southwest, northwest, and south of China, including Yunnan, Guangdong, Heilongjiang, Liaoning, and Hunan provinces (Yang, 1994;Yang, 1997;Yang et al., 2004;Yang, 2005;Deng et al., 2014;Li and Cai, 2014;Ariyawansa et al., 2015;Li et al., 2015;Yang, 2015;Cai et al., 2016;Deng et al., 2016;Liu et al., 2017;Cui et al., 2018;Su et al., 2022).Records of Amanita species in Yanshan Mountains, northern China, are few.In this study, 12 Amanita species from Yanshan Mountains were recognized.Of them, 10 species or approximately 83% of the species were new or recorded for the first time in this area.Therefore, accelerating the discovery and description of Amanita species by using both morphological and molecular approaches in this area is necessary.
Literature review revealed that 14 taxa of Amanita were identified from Yanshan Mountains.Three species were identified by Yang (2004) (Cui et al., 2018).The morphology of HMKS 75237 was consistent with that of A. vaginata var.vaginata, but we could not obtain the DNA sequence from the specimen.The specimen HMAS 26491 was collected from Baihuashan Mountains, Beijing, and was originally identified as A. subglobosa Zhu L. Yang.However, the results of morphological and phylogenetic analyses conducted in the present study were inconsistent with the original identification.We tentatively named this specimen as Amanita sp. because of the poor status of the specimen and await the subsequent collection of additional specimens for further study.In addition, some Amanita species have been recorded in the literature even in the absence of any detailed information about the specimen, and so, the accuracy of the information about these species needs to be further verified by collecting specimens and obtaining molecular data.These species with only distribution records available are as follows.A. caesarea, A. inaurata Gillet, and A. yuaniana are distributed in Wulingshan Mountains, Hebei Province (Wang et al., 2005); A. flavoconia Alk., A. phalloides (Vaill.ex Fr.) Link, and A. subjunquillea are distributed in Dahaituo Mountains National Nature Reserve, Hebei Province (Wu et al., 2017); A. orientigemmata is distributed in Songshan Mountains National Nature Reserve, Beijing (Wu et al., 2020); A. verna Bull ex Lam. is distributed in Badaling Forest Park, Beijing (Zhang et al., 2017); and A. panterina (DC.ex Fr.) Schrmm is distributed in Dayangshan National Forest Park, Beijing (Chen et al., 2006).Therefore, specimens of Amanita spp.must be more extensively collected from the Yanshan Mountains region to improve the study of their biodiversity.
Amanita deserves special attention because of its unique research and popular science education value.However, because some Amanita species are similar in morphology and color, distinguishing them in the field is difficult.Moreover, many casualties have been caused through mistakenly consumed poisonous Amanita species in many places in China (Li et al., 2020;Li et al., 2021a;Li et al., 2021b).The China Center for Disease Control and Prevention reported eight incidents of mushroom poisoning in Beijing in 2020, with 23 people poisoned.Seven incidents of mushroom poisoning were reported from Hebei Province around Beijing, with 33 people poisoned.In these incidents, the main species causing poisoning were A. rimosa, A. subjunquillea, and A. oberwinkleriana (Li et al., 2021a;Li et al., 2021b).The present study is the initial report on Amanita's biodiversity in the region of Yanshan Mountains, including northern part of Beijing, and Tianjin and Hebei provinces.Given the large area of North China and its diverse forest types, many more Amanita species may be discovered in this region in future.

Conclusions
In this research, 20 Amanita specimens deposited in Chinese herbaria and 36 newly collected specimens from North China were studied based on the results of morphological and phylogenetic analyses.In total, 12 phylogenetic species were found.Of them, three species were described as new species, namely A. borealis sp.nov., A. brunneola sp.nov., and A. yanshanensis sp.nov.Furthermore, nine known species were identified, namely, A. caesareoides, A. chiui, A. muscaria, A. oberwinklerana, A. ovalispora, A. subglobosa, A. subjunquillea, A. vaginata var. vaginata, and A. virosa.Our results underscore that China has a very high biodiversity of Amanita species and that additional studies are required to completely determine the exact number of species.It plays a crucial role in Amanita toxin research and ecological conservation.This study investigated the areas where Amanita species-related research is lacking.The study attempted to better understand Amanita distribution and thus contribute to related research.This study improves the knowledge regarding the species diversity of Amanita in Yanshan Mountains and provides new data for the macrofungal systematics, toxin research, and diversity and ecological studies of Amanita in subsequent studies.

TABLE 1
Information of sequences used in the nrITS-nrLSU-rpb2-tef1-a phylogenetic analysis in this study.