A large species diversity has recently been discovered in the genus Agaricus. Six subgenera and 23 sections are now recognised. In this study, three specimens collected from Thailand, formed a monophyletic clade in subgenus Pseudochitonia, based on analyses of ITS sequence data. Further analyses, based on multi-gene sequence data (ITS, LSU, tef1-α), using BEAST, revealed that this clade originated 26.7 Ma. According to their distinct morphological characteristics, phylogenetic position and relatively old divergence time, a new section Cymbiformes is proposed and this section is represented by a new species A. angusticystidiatus. This new section is characterised by the strong iodoform odour of basidiomes and cymbiform basidiospores. Descriptions, colour photographs and illustrations are presented.

New taxa, Agaricaceae , Phylogeny, Taxonomy

Agaricus L. 1753 (Agaricaceae, Agaricales) is a well-known genus. Many species in this genus are commercially cultivated and served as food. One of the popular edible mushrooms is A. bisporus (J.E. Lange) Imbach, which is the most extensively cultivated mushroom in the world, accounting for 38% of world production (ISMS Edible mushrooms 2017, http://www.isms.biz/edible-mushrooms/). Another popular edible mushroom, A. subrufescens Peck, is also a medicinal mushroom and contains abundant bioactive compounds, for example, some compounds extracted from the basidiomes can be used as antioxidant (De Silva et al. 2012, 2013a, b, Llarena-Hernández et al. 2017). In the field, Agaricus is easily recognised by its white or brown caps with fibrillose scales on the surface, free lamellae, brown spore print and annulate stipe. Under the microscope, it is characterised by brown basidiospores, single or multiseptate cheilocystidia and often lacks pleurocystidia. Habitats of Agaricus are various, the most common being forests and grasslands, such as A. campestris L. of section Agaricus, which can be found gregariously in small groups or in fairy rings in grasslands. Agaricus also exists in arid habitats, for example, A. colpeteorum T. Lebel and A. lamelliperditus T. Lebel & M.D. Barrett of section Minores, which were discovered in arid zones of Australia (Lebel 2013).

The taxonomic, systematic and species delimitation of Agaricus inferred by morphology are variable (Cappelli 1984, Singer 1986). In the 1990s, the application of molecular techniques brought new perspectives to fungal taxonomic research including the genus Agaricus (White et al. 1990). Using phylogenetic analyses, the taxonomy of Agaricus is becoming more and more stable. Zhao et al. (2011) used ITS sequence data from Agaricus specimens from temperate and tropical areas to build a phylogenetic topology for the genus, which revealed eleven new clades and indicated phylogenetic relationships between temperate and tropical species. Zhao et al. (2016) carried out multi-gene phylogenetic and evolutionary molecular clock analyses. In that study, Agaricus was segregated into five subgenera and 20 sections, according to the phylogenetic position and divergence time of each clade. With the recent discovery of an American subgenus and a new clade found in the Caribbean area, Agaricus now contains six subgenera and 23 sections (Zhao et al. 2016, Chen et al. 2017, Parra et al. 2018).

In this study three interesting specimens found near Chiang Mai, Thailand were analysed morphologically and molecularly. We provide a full description and analyses are presented to support the distinction of this material as a new species and section in subgenus Pseudochitonia.

The Bayesian tree from ITS sequences is shown in Figure 1. A total of 84 sequences are represented from 12 sections of subg. Pseudochitonia and A. campestris was used as outgroup. All sections are well supported both by posterior probabilities (PP) and bootstrap (BS). Phylogenetic trees generated from Bayesian and ML analyses showed identical topologies and are also almost identical with those of Zhao et al. (2016) with the exception of A. dilutibrunneus R.L. Zhao, which clustered with two unknown specimens (A. sp./CA486 and A. cf. goossensiae/ADK2171) and formed a monophyletic clade in our analyses, isolated from all other species in the previous study (Zhao et al. 2016). Our three specimens (ZRL2043, ZRL2085 and BC088) formed a monophyletic clade in subg. Pseudochitonia which is fully supported both in PP and BS values and located at an isolated position (Fig. 1).

Figure 1. 

Phylogenetic tree of Agaricus subgenus Pseudochitonia generated from Bayesian analysis of ITS sequences, rooted with A. campestris. Bayesian posterior probability (PP) values ≥ 0.9 or Bootstrap support (BS) values ≥ 50% are indicated at the internodes (PP/BS). The branches in bold mean the related PP > 0.95, “T” refers to sequences from type specimen.

The multi-gene MCC tree is shown in Figure 2. It was conducted based on the dataset of multi-gene sequences. A total of 63 specimens were included, comprising 43 specimens used in ITS analysis, 19 specimens from five subgenera and an outgroup Heinemannomyces sp. All subgenera and sections are well-supported statistically. Agaricus diverged at the stem age 66 Ma (million years ago), all subgenera diverged between 29.2–33.9 Ma and sections diverged between 20–26.9 Ma. Our three specimens formed a new monophyletic clade in subg. Pseudochitonia with strong PP support and this clade diverged at 26.7 Ma.

Figure 2. 

Maximium Clade Credibility tree of genus Agaricus based on ITS, LSU and tef1-α gene sequences with the outgroup Heinemannomyces sp. Posterior probability values equal or above 0.9 are annotated at the internodes. The 95% highest posterior density of divergence time estimation are marked by horizontal bars.

Taxonomy

Agaricus (Pseudochitonia) section Cymbiformes M.Q. He & R.L. Zhao, sect. nov.

MycoBank No: MB824147

Faceoffungi Number: FoF04104

Type species

Agaricus angusticystidiatus M.Q. He, Desjardin., K.D. Hyde & R.L. Zhao

Etymology

In reference to the cymbiform basidiospores.

Original description

KOH reaction negative, Schäffer’s reaction negative on dry specimens. No discolouration on touching, but discolouration reddish-brown on cutting. Annulus membranous. Smell strong iodoform. Basidiospores cymbiform and cheilocystidia narrow with variable shapes.

Agaricus angusticystidiatus M.Q. He, Desjardin, K.D. Hyde & R.L. Zhao, sp. nov.

MycoBank No: MB825177

Faceoffungi Number: FoF04105

Figure 3

Etymology

refers to the narrow clavate cheilocystidia.

Type

Thailand, Chiang Mai Province, Mae Taeng, Baan Mae Sae village, on Hwy 1095 near 50 km marker, 19°14.599'N, 98°39.456'E, alt. 960 m. In rain forest dominated by Castanopsis armata, Castanopsis sp., Pinus sp., Lithocarpus sp., 26 June 2005, collected by Jennifer Kerekes. Holotype: ZRL2043 (HMAS279593); Isotype: BBH19428 and SFSUZRL2043,

Original description

Pileus 40–80 mm diam., plano-convex, applanate, broadly umbonate; surface concentric squamulose with small skull-cup at disc, appressed, slightly fissured, light brown (6D8), brown (7E3), greyish-brown (5D5), dark brown (6D6) against the grey (8E3) background. Context 4–5 mm thick at disc, fragile, white to grey (8E3) in age. Lamellae free, crowded, lamellulae with 3–4 lengths, 3–4 mm broad, normal to slightly ventricose, brown (7E5) to dark brown (7F7-8), edge colour similar to the gill itself. Stipe 55–100 × 5–8 (base 8–15) mm, cylindrical bulbous, with rhizomorphs in most cases, hollow, surface glabrous to silky, white to dark brown (6D6). Annulus pendent or percurrent; single; upper side membranous, white; lower side surface powdery, light yellow (4B2) grain-like dots in circulate; superior, persistent, edge entire, up to 5 mm broad. Smell of iodoform. No colour change on touching; light dull red, greyish brown (7D4) on cutting.

KOH reaction: negative. Schäffer’s reaction: negative on dry specimens.

Basidiospores 5–6.5 × 3–4 (–4.5) µm [X = 5.6 ± 0.5 × 3.8 ± 0.4, Q = 1.1–2.2, Q_m = 1.52 ± 0.7, n = 20], cymbiform, some endosporium, no germ pore, brown. Basidia 10–15 × 5.5–7 µm, clavate, hyaline, smooth, 4-spored. Pleurocystidia absent. Cheilocystidia 20–30 (–45) × 5–8 µm, occasionally one septum, narrowly clavate to clavate, some with elongated top, rarely subcapitate, hyaline, smooth. Pileipellis cutis consisting of 3–5 µm diam. hyphae, hyaline, smooth, non-constricted at septa. Annulus hyphae same as pileipellis.

Figure 3. 

Morphology of Agaricus angusticystidiatus A, B basidiomes C, D basidiospores E basidia and cheilocystidia.

Habit

Gregarious on soil in rain forest which is mainly dominated by Castanopsis armata, Castanopsis sp., Pinus sp., Lithocarpus sp.

Distribution

Thailand, Chiang Mai Province (type distribution).

Other materials examined

Thailand, Chiang Mai Province, Mae Taeng, Ban Mae Sae Village, on Hwy 1095 near 50 km marker, 19°14.599'N, 98°39.456'E, elev. ca. 960 m, 3 July 2004, collected by Thitiya Boonpratuang, ZRL2085 (HMAS279594, BBH19468 and SFSUZRL2085); Thailand, Chiang Mai Province, Mae Taeng, Mushrooms research center, 30 July 2014, collected by Boontiya Chuankid, BC088 (MFLU 14-0903).

Notes

This new species is morphologically distinguished from other Agaricus species by its strong iodoform smell, context reddish-brown discolouration on cutting, cymbiform basidiospores and narrow cheilocystidia with variable shapes. Phylogenetic analyses confirmed it is a member of the subgenus Pseudochitonia with an isolated phylogenetic position in Agaricus. This new species is similar to A. iodolens Heinem. & Gooss.-Font. of section Xanthodermatei, because both have relatively slender basidiomes and odour of iodine (Naritsada et al. 2014). However, this new species has cymbiform basidiospores and a bulbous stipe, while those of A. iodolens are ellipsoid and an equal stipe (Zoberi 1972). Agaricus lamellidistans R.L. Zhao and A. variicystis L.J. Chen, K. D. Hyde & R. L. Zhao of section Crassispori resemble this new species, because all have greyish-brown pilei and cymbiform basidiospores. These species lack discolouration on cutting, while those of A. angusticystidiatus have dull red discolouration on cutting (Zhao et al. 2016).

Based on phylogenetic and morphological studies, we propose A. angusticystidiatus as a new species in subgenus Pseudochitonia. Furthermore, the dating analysis, based on multi-gene sequences, indicated that A. angusticystidiatus diverged at 26.7 Ma which is slightly older than other sections in Agaricus (18–26 Ma, in Zhao et al. 2016). Therefore, a new section Cymbiformes is proposed, which presently only contains species A. angusticystidiatus. Thus up to now, there are six subgenera and 24 sections in the genus Agaricus (Zhao et al. 2016; Chen et al. 2017; Parra et al. 2018).

Zhao et al. 2016 had conducted a reconstruction of the taxonomic system of Agaricus. In that study, they used the following criteria to recognise subgenera and sections: “(i) they must be monophyletic and statistically well-supported in the multi-gene analyses; (ii) their respective stem ages should be roughly equivalent and subgenera stem ages must be older than section stem ages; and (iii) they should be identifiable phenotypically, whenever possible” (Zhao et al. 2016). That means divergence time has been used as an additional criterion to rank taxa of above species level in Agaricus. Later, the criterion of divergence time, along with phylogenetic, monophyletic and morphological support, has been accepted in other new subgenus and section recognitions in Agaricus, such as a new subgenus Minoriopsis (Chen et al. 2017); and a new section Kerrigania (Parra et al. 2018).

As mentioned before, this proposed new section Cymbiformes has a closely phylogenetic relationship with sections Trisulphurati and Crassispori. In morphology, all of them differed with other sections of Agaricus by the combination of negative Schäffer’s reaction, chemical odours such as phenol, ink or carbolic acid and basidiospores endosporium and often cymbiform. However, section Trisulphurati has woolly squamules on the surfaces of the pileus and stipe and the other two sections only have appressed squamules at the centre of the pileus. Furthermore, this new section Cymbiformes could be separated from section Crassispori by its negative KOH reaction and developed annulus (the latter is positive KOH reaction and with fragile annulus) (Zhao et al. 2016).

So far, section Cymbiformes is only known from a tropical area. The cymbiform basidiospores are rare in Agaricus species. Presently there are three Agaricus species from tropical areas which have this kind of basidiospores. They are A. angusticystidiatus of section Cymbiformes and A. lamellidistans and A. variicystis of section Crassispori (Zhao et al. 2016). In phylogenetic analyses, these two sections also show a close phylogenetic position, which is similar to previous studies (specimens ZRL2043 and ZRL2085 were treated as A. sp. in Zhao et al. 2011; Zhao et al. 2016). The presence of cymbiform basidiospores is a common character in another genus Micropsalliota of Agaricaceae. In phylogenetic analyses, Agaricus is sister to Hymenagaricus, then sister to Chlorophyllum, Heinemannomyces and Micropsalliota (Zhao et al. 2017) and all of them have tropical distribution habitats. Thus we hypothesised that cymbiform basidiospores have formed at least twice in evolutionary events and are associated with tropical environments.

This project was conducted under the financial support of the National Key R&D Program of China (Project No. 2018YFD0400200), the National Natural Science Foundation of China (Project ID:31470152 and 31360014) and Beijing Innovative Consortium of Agriculture Research System (Project ID: BAIC05-2018).