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Squamanitaceae and three new species of Squamanita parasitic on Amanita basidiomes

The systematic position of the enigmatically mycoparasitic genus Squamanita (Agaricales, Basidiomycota) together with Cystoderma, Phaeolepiota, Floccularia, and Leucopholiota is largely unknown. Recently they were recognized as Squamanitaceae, but previous studies used few DNA markers from a restricted sample of taxa from the family and lacked a formal taxonomic treatment. In this study, with newly generated sequences of the type of the genus Squamanita, S. schreieri, and several additional species of the family, the phylogeny is reinvestigated with a concatenated (18S-5.8S-nrLSU-RPB2-TEF1-α) dataset. This study reveals that Cystoderma, Phaeolepiota, Squamanita, Floccularia, and Leucopholiota are a monophyletic clade with strong statistical support in Bayesian analysis and form Squamanitaceae. Phaeolepiota nested within Cystoderma; Squamanita, Leucopholiota, and Floccularia clustered together as two monophyletic subclades; and Squamanita was present as a monophyletic clade with strong statistical support in both Maximum Likelihood and Bayesian analyses. The family name Squamanitaceae is formally emended and a detailed taxonomic treatment is presented to accommodate the five genera. Meanwhile, another concatenated (18S-ITS-nrLSU-RPB2-TEF1-α) dataset is used to investigate phylogenetic relationships and species delimitation in Squamanita. Our data indicates that “S. umbonata” from the Northern hemisphere forms two species complexes, one complex includes six specimens from North America, Europe, and East Asia, the other includes two specimens from Central America and North America respectively. Futhermore, species of Squamanita can parasitize species of Amanita, besides other fungal species. Squamanita mira parasitizes A. kitamagotake (A. sect. Caesareae), while S. orientalis and S. sororcula are parasites of species belonging to the A. sepiacea complex (A. sect. Validae). “Squamanita umbonata” from Italy occurs on A. excelsa (A. sect. Validae). Three new species of Squamanita from East Asia, viz. S. mira, S. orientalis and S. sororcula are documented with morphological, multi-gene phylogenetic, and ecological data, along with line drawings and photographs, and compared with similar species. A key for identification of the global Squamanita species is provided.


INTRODUCTION
Squamanita is one of the most enigmatic genera of the Agaricales (Halama 2016;Mondiet et al. 2007;Redhead et al. 1994), and the members of this genus are extremely rare and sporadic all over the world (Griffith et al. 2019;Holden 2005;Matheny and Griffith 2010). Squamanita was originally described from riverine forest in Switzerland. After examining the type material, Horak (1968) presented a full re-description of the microscopic characters including features not reported in the protologue. Almost all the species of Squamanita are biotrophic parasites on other agaric species (Halama 2016;Harmaja 1987;Henrici 2013;Matheny and Griffith 2010;Nagasawa et al. 1990;Redhead et al. 1994;Reid 1983). The basidiomes of Squamanita grow from other agaric species and deform the host basidiomes so that they become incorporated into an enlarged base of the stipe of the Squamanita. Eventually, the host is completely deformed and more or less unrecognizable (Halama 2016;Redhead et al. 1994). Parasitized host tissue has been labelled as "sclerotial bodies", "protocarpic tubers" (Bas 1965;Singer 1986), "galls" (Redhead et al. 1994), "cecidiocarp" (Bas and Thoen 1998) or "mycocecidium" (Griffith et al. 2019;Vizzini and Girlanda 1997), and sometimes multiple basidiomes come out from a "mycocecidium" (Bas 1965;Mondiet et al. 2007).
The genus Squamanita was assigned on the basis of morphology to different families in the past, including Squamanitaceae and Cystodermataceae. Based on phylogenetic analysis of combined nuclear ribosomal RNA genes, Matheny and Griffith (2010) suggested that Squamanita, Cystoderma, and Phaeolepiota represent a monophyletic clade. In the subsequent molecular works by Matheny et al. (2015), Griffith et al. (2019) and Vizzini et al. (2019), Squamanita and allied genera were referred as Squamanitaceae. Recently, Squamanita, Cystoderma, Phaeolepiota, Floccularia, and Leucopholiota were classified into Squamanitaceae (http://www.agaric.us) (Kalichman et al. 2020), but without a formal taxonomical treatment. In addition, the host species of Squamanita have been identified mainly based on morphological data and ecological evidence (Bas 1965; Mondiet et al. 2007), except for a few studies (Griffith et al. 2019;Matheny and Griffith 2010;Mondiet et al. 2007), which used molecular phylogenetic techniques to identify the hosts.
In the survey of macrofungi in China, we collected three species of Squamanita and two collections of Amanita sect. Caesareae and one collection of A. sect. Validae (Cui et al. 2018) with similar "mycocecidia" of two Squamanita species in the nearby localities respectively. To validate the taxonomical, phylogenetic and ecological traits, detailed morphological and anatomical studies and molecular phylogenetic analyses are carried out. To understand the species recorded in China, additional specimens collected in other parts of the world are examined and included in the present report.

MATERIAL AND METHODS
Morphology, sampling, DNA extraction, PCR amplification and sequencing Specimens studied are listed in Tables 1 and 2. For morphological study, we follow Cui et al. (2018) and the references therein. To verify the mycoparasitic features of the target species, routine samples (HKAS100826) for DNA extraction were separately taken both from the basidiome (five samples for basidiome labeled from C1 to C5) and the mycocecidium (six samples labeled from B1 to B6 as illustrated in Fig. 6). In addition, samples of other specimens were taken from different locations from their basidiomes and mycocecidia respectively, and then mixed for improving the success probability of DNA extraction in case of poor sample quality. Particularly, the volval remnant-like structure on the cap of the Squamanita specimen (HKAS74862A) was sampled. All Chinese collections are deposited in the Herbarium of Cryptogams of Kunming Institute of Botany, Chinese Academy of Sciences, China (HKAS).
PCR products which failed in direct sequencing were firstly purified with the Cycle-pure-kit (Omega, USA) or Gel Extraction and PCR Purification Combo Kit Tubariomyces sp.   (Spin-column) (Bioteke, China), and then cloned using pClone007 simple vector kit (Tsingke, Beijing). For the recently collected specimen (HKAS100826) and the volval remnants like structure on the cap of a Squamanita specimen (HKAS74862A), 10 clones of each ITS and nrLSU PCR products of each sampling point were randomly selected from a 90 mm petri dish for sequencing with primer pair M13-47/M13-48 to investigate the mycelium distribution of hosts and parasitising fungi. The cloning, PCR amplification and sequencing followed the protocols described by Cai et al. (2016) and Cui et al. (2018).

Results of sequencing
For specimen of HKAS100826, the ITS and nrLSU sequences were successfully amplified from all eleven sampling points (C1-C5, B1-B6). Among them, there are two bands occurring in gel electrophoresis diagram of each of the PCR products of ITS from six sampling points of mycocecidium (B1, B2, B3, B4, B5, B6), see Fig. 1. By cloning and sequencing all of the purified PCR products of ITS and nrLSU, a total of 50 ITS and 50 nrLSU sequences were generated from all points (C1-C5). After alignment and comparison, all of them belong to the same species, namely the mycoparasitic species itself. For the mycocecidium, each band of PCR productions with two bands were excised from gel respectively, and then purified and sequenced, generating a total of 120 ITS and 60 nrLSU sequences from sampling points B1-B6. After analysis, two types of mushroom sequences were detected for each DNA locus. Statistically, 50% ITS, 90% nrLSU matched to the potential mycoparasitic species and 50% ITS, 10% nrLSU belong to the potential host species. For the volval remnants on the cap of the Squamanita specimen (HKAS74862A), 60% ITS, 90% nrLSU were the potential mycoparasitic species and 20% ITS, 0% nrLSU were assigned to the potential host species, others are Trichoderma hirsutum or vector sequences.
For the other specimens of Squamanita and nearby Amanita, all sequences were amplified then directly sequenced or obtained by cloning from PCR products. One hundred forty-five sequences have been submitted to GenBank and used for phylogenetic analyses (Tables 1 and 2). The sequences of the two potential species of hosts are the same as those of the coexisting Amanita species respectively, and were finally identified to belong to A. kitamagotake (Fig. 4) and the A. sepiacea complex (Fig. 5). The potential mycoparasitic species are clustered into the genus Squamanita (Figs. 2 and 3).

DNA sequence alignment
Sequences used in study are listed in Tables 1 and 2 with their Herbarium ID and accession numbers. Four Table 2 Specimens used to identify the mycocecidia of new species of Squamanita in this study are listed with their Herbarium ID and accession numbers. Newly generated sequences are highlighted in boldface (Continued) datasets, namely 18S-5.8S-nrLSU-RPB2-TEF1-α, 18S-ITS-nrLSU-RPB2-TEF1-α, ITS-nrLSU-TEF1-α, and ITS were used in our study to reinvestigate the phylogeny of Squamanitaceae, identify the phylogenetic position of the basidiomes and mycocecidia of the mycoparasitic species. From the first dataset to the last, a total of 4100, 4743, 1878 and 693 characters were used in the phylogenetic analyses, respectively. Moreover, two phylogenetic trees which only use ITS and nrLSU sequences were used to investigate the phylogeny of Squamanitaceae are provided as additional files (Additional files 1 and 2), respectively. The final alignments have been submitted to TreeBase (https://www.treebase.org /, nos.: 27,493, 27, 494, 27,496, 27,497, 27,498, 27,499). For each dataset, the sequences were aligned using MAFFT v6.8 (Katoh et al. 2005), manually edited with BioEdit v7.0.9 (Hall 1999) and concatenated with Phyutility v2.2.1 (Smith and Dunn 2008). Unsampled gene regions were coded as missing data. In the concatenated datasets, all introns of RPB2 and TEF1-α were excluded because of the difficulty in alignment. Maximum likelihood (ML) analyses were performed using IQ-TREE 1.6 (Trifinopoulos et al. 2016). Bayesian Inference (BI) analyses were used to analyze the datasets with MrBayes v3.1.6 (Ronquist et al. 2012). The optimal substitution models for each dataset were determined by using the Akaike Information Criterion (AIC) implemented in MrModeltest v2.4 (Nylander 2004), with 18S, 5.8S/ITS and nrLSU treated as a single block. In ML analyses, the substitution model options for four datasets were auto evaluated after provided partition file by using IQ-TREE 1.6 (http://iqtree.cibiv.univie.ac.at/), clade support for the ML analyses was assessed using an SH-aLRT test with 1000 replicates (Guindon et al. 2010) and 1000 replicates of the ultrafast bootstrap (UFB) (Hoang et al. 2018). In the ML analyses, nodes with support values of both SH-aLRT ≥80 and UFB ≥ 95 were considered well supported, nodes with one of SH-aLRT ≥80 or UFB ≥ 95 were weakly supported, and nodes with both SH-aLRT < 80 and UFB < 95 were unsupported, and the other parameters use the default settings. For BI analyses, the selected models for four datasets were 18S-5.8S-nrLSU(GTR + I + G)-RPB2(GTR + I + G)-TEF1-α(GTR + I + G), 18S-ITS-nrLSU(GTR + I + G)-RPB2(SYM + I)-TEF1-α(SYM + I + G), ITS(SYM + G)-nrLSU(HKY + I)-TEF1-α(SYM + G), and ITS (GTR + G) respectively. Bayesian analyses used the selected models and four chains were run simultaneously for 2 million generations with trees sampled every 100 generations. The sampling of the posterior distribution was considered to be adequate when the average standard deviation of split frequencies was lower than 0.01. Chain convergence was
Notes: Here we fix the application of the generic name Squamanita by lecto-and epitypfiying the type species of the genus, S. schreieri, and describe the new species discovered in this study.
Distribution: Currently known from Jiangxi and Yunnan Province, central and Southwest China.
Notes: In this study, molecular evidence confirms that the hosts of S. mira as well as two collections of Amanita in the nearby area, within 2 km of S. mira, are A. kitamagotake (Figs. 4, 6). Morphologically, S. mira highly resembles the informally published "S. tropica" ("nom. Prov.") (Bas 1965), because both are parasitic on basidiomes of Amanita and form a volva-like structure at the base of the stipe. Furthermore, they share abundant tawny squamules on the pileus surface, serrate-dentate or subundulate lamellae edges, irregular ring analogues on the upper part of the stipe and ellipsoid to subreniform basidiospores. However, S. mira differs from S. tropica in its subconical to convex pileus with a distinct umbo. The material of S. tropica is lost (Bas 1965).
Distribution: Currently known from Yunnan Province, Southwest China.
Notes: Our morphological data and molecular phylogenetic evidences confirm that the host of S. orientalis and the collection of Amanita in the nearby area within two kilometers' range of S. orientalis are A. sepiacea (Figs. 5,8,10). Interestingly, some volval remnants of A. sepiacea are found on the center of the pileal surface of S. orientalis (Fig. 8), and its anatomical features are those of A. sepiacea (Yang 2005) (Fig. 10), and the filamentous hyphae with clamp connection belong to S. orientalis (Fig. 10). Squamanita orientalis is similar to S. schreieri. However, the latter species has no cystidia. Furthermore, the former is a parasite on A. sepiacea, while S. schreieri is possibly associated with A. strobiliformis or A. echinocephala (Bas 1965).
Squamanita orientalis is also similar to S. sororcula and S. umbonata. However, S. orientalis differs from S. sororcula by its irregular fibrillose annular zone on the upper part of the stipe and ciliate squamules on the pileal margin, and larger cystidia (90-105 × 17-27 μm). In addition, there are ca. 50 and ca. 40 base differences in ITS and nrLSU regions between the two species respectively, and even though their hosts are identified as A. sepiacea for both species, there are ca. 25 different bases in the ITS region from host material. Squamanita umbonata differs from S. orientalis by its umbonate pileus, and narrower cystidia (60-95 × 9-20 μm), cylindrical to clavate fusiform mycocecidia.
Distribution: Currently known from Hunan and Yunnan Provinces, central and Southwest China.
Notes: Squamanita sororcula is similar to S. mira, S. orientalis, S. schreieri, S. umbonata, and other collections assigned to the "S. umbonata" complex. The differences between the first two and S. sororcula have been discussed above. Besides, S. sororcula differs from S. schreieri by the presence of cystidia and differs from S. umbonata by its subglobose mycocecidia. Wang and Yang (2004) treated two collections (HKAS38127 and 38149) as "S. umbonata" collected from Hunan province, central China. Unfortunately, the collections have not been traced by us. However, the two collections are without an annular zone, and should be close to S. sororcula rather than S. orientalis. Liu et al. IMA Fungus (2021) (Fig. 2). Oberwinkler (1976) and Singer (1986) supposed that Horakia (now included in Verrucospora) belonged to Thelephorales or Cystodermateae of Agaricales, respectively, which are incorrect placements based on our molecular phylogenetic data. Phylogenetic placements of Ripartitella, and Cystodermella, which was separated from Cystoderma by Harmaja (2002), are unclear at present, although previous research based on nLSU, RPB1 and ITS molecular sequences indicated that Ripartitella and Cystodermella are near Cercopemyces (Baroni et al. 2014). Our study (Fig. 2) is consistent with Baroni et al. (2014), and these three genera are close to Hydnangiaceae in our phylogenetic tree (Fig. 2). Saar et al. (2016) treated Phaeolepiota aurea as Cystoderma aureum because it was nested within Cystoderma. However, P. aurea, with large inamyloid fusoid and asperulate spores, differs from Cystoderma, species of which have amyloid, ellipsoid, oblong or fusiform and smooth spores. In our multigene phylogenetic tree (Fig.  2), and the supplementary trees of Varga et al. (2019), P. aurea nested within Cystoderma, but clustered with Cystoderma superbum (Fig. 2), a unique species commonly reported to be amyloid but in only a small area of the basidiospore surface, which is a morphotaxonomic character that differs from other species of Cystoderma. In the study of Matheny and Griffith (2010), and supplementary trees of that study (Additional files 1 and 2), a close relationship among P. aurea, Cystoderma and C. superbum was not well supported. Therefore, for the moment, we continue to recognize Phaeolepiota for P. aurea. Further studies with more samples and using more DNA makers are necessary to clarify the position of P. aurea and C. superbum in relation to other species of Cystoderma.
Diversity of the "S. umbonata" species complex Our study indicated that material of "Squamanita umbonata" from the Northern Hemisphere clustered into two species complexes each consisting of several different species (Figs. 2 and 3), including S. orientalis, S. sororcula, and several undefined specimens. Morphological characteristics of collection R. E. Halling 7691 (NY79971) (Fig. 13) from Costa Rica are mostly consistent with the descriptions of the type (NY27684) by Sumstine (1914) and Bas (1965), with an umbonate pileus, cylindrical to clavate fusiform mycocecidia, and thin-walled cystidia. However, considering that the type of S. umbonata was from Pennsylvania, USA, we are reluctant to identify R. E. Halling 7691 as S. umbonata until molecular data from the type are available.
Host preference or specificity of Squamanita species Our study reveals that the basidiomes of S. mira are composed of its own hyphae, while the mycocecidia also include hyphae of the host, which is consistent with the observations on S. paradoxa by Mondiet et al. (2007) and Griffith et al. (2019). Interestingly, host hyphae are found in the volval remnants that are attached to the pileal surface of S. orientalis (Figs. 5,8,10). This character may provide additional help for the host identification of Squamanita. Although sometimes the basidiomes of Squamanita may macromorphologically deform the hosts, most of the time the shapes of infected hosts (mycocecidia) still largely maintain consistent morphological characteristics with nearby uninfected basdiomes of the same species. Our study showed that S. orientalis, S. sororcula and "S. umbonata" (HKAS107325A) from Italy, with subglobose mycocecidia, are parasitic on A. sect. Validae, while S. mira, with the sheathing volva arising from the margin of a bulb, is parasitic on A. kitamagotake. Therefore, the shape and the size of the mycocecidia could be a reliable morphological character at species level.

CONCLUSION
The monophyly of the family Squamanitaceae was confirmed by multi-gene Bayesian phylogenetic analysis, with five genera, namely Cystoderma, Phaeolepiota, Squamanita, Floccularia and Leucopholiota falling in the family. Three new species from China, parasitizing two different species from two sections of Amanita, were uncovered and described based on morphological and molecular evidence. Furthermore, a multi-gene phylogenetic analysis on "Squamanita umbonata" from North America, Central America, Europe, and East Asia showed that it represents two species complexes harboring eight subclades. Further morphological studies are needed to reveal the species diversity and distribution patterns of "Squamanita umbonata".

Supplementary Information
The online version contains supplementary material available at https://doi. org/10.1186/s43008-021-00057-z. Fig. 13 Specimens of "Squamanita umbonata" included in this study. a Fresh basidiomes of R. E. Halling 7691 with cylindrical to clavate fusiform mycocecidium from Costa Rica. b C. Bas 3808 from USA with cylindrical mycocecidium. c H. E. Bigelow 17431 from USA with subglobose mycocecidium, a lump of clay is attached on the center of pileus of the specimen on the right, and the apical part of volval remnants on mycocecidium can be observed between clay and pileus under anatomical lens. d HKAS107325A from Italy with subglobose mycocecidium. Bars a = 25 mm, b-d = 20 mm Liu et al. IMA Fungus (2021) 12:4