A new section and species of AgaricussubgenusPseudochitonia from Thailand

Abstract 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.


Introduction
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, 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.

Morphological examination
Photographs were taken immediately in situ, in the field in Thailand. Basidiomes were wrapped in aluminium foil or kept in plastic boxes separately. Macro morphological characteristics were recorded when specimens were fresh. Every specimen was completely dried in an electrical food drier at 60 °C, then kept in a plastic ziplock bag and deposited in Herbarium Mycologicum Academiae Sinicae (HMAS), Mae Fah Luang University Herbarium (MFLU), Biotec Bandkok Herbarium (BBH) and the Thiers Herbarium at San Francisco State University (SFSU). Colour terms and notations in parentheses are those of Kornerup and Wanscher (1978). Anatomical and cytological characteristics including basidiospores, basidia, cystidia and pileipellis were observed using an Olympus CX31 microscope. Scanning electron microscope (SEM) photos for basidiospores were captured through a Hitachi SU8010 Field Emission SEM (Tokyo, Japan). Measurements were analysed and recorded as X = the mean of length by width ± SD, Q = the quotient of basidiospore length to width and Q m = the mean of Q values ± SD. All the protocols of morphological studies followed Largent's methodology (Largent 1986).

DNA extraction and PCR
At the Institute of Microbiology Chinese Academy of Science, genomic DNA was extracted from dry specimens by using an E.Z.N.A. Forensic DNA Extraction Kit (D3591-01, Omega Bio-Tek) following the manufacturer's protocol. PCR amplification was performed following He et al. (2017). Primers for the internal transcribed spacer (ITS), large ribosomal subunit (LSU) and translation elongation factor (tef1-α) were ITS4/ITS5, LR5/LROR and 983f/1567r, respectively (White et al. 1990, Moncalvo et al. 2000, 2002, Morehouse et al. 2003. PCR products were sent to a commercial company for sequencing and both directions were sequenced to ensure accuracy. At the Botanic Garden Meise (BR), genomic DNA was extracted from dry specimens using a CTAB isolation procedure adapted from Doyle (1990). Ca. 10 mg of tissue was ground with a Retsch 300 beadmill. ß-mercaptoethanol (0.2%) was added to the CTAB lysis buffer just prior to extraction; samples were lysed for 1 hour at 60 °C; proteins and polysaccharides were removed by two consecutive extractions with chloroform: isoamylalcohol (24:1), after which DNA was precipitated by the addition of 0.8 volume isopropanol to the aqueous phase. The pellet was washed once in 600 μl 70% ethanol, air-dried and suspended in 100 μl TE pH 8.0. RNA was then digested with RNase A. For PCR amplification of the ITS1-5.8S-ITS2 region of rDNA, ITS1-F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) primers were used. Amplifications were performed in 20 μl reactions containing 2 μl 10× polymerase buffer, 0.2 μM of each dNTP, 200 μg μl -1 bovine serum albumin (BSA), 0.25 μM of forward and reverse primers and 0.5 U Taq polymerase (DreamTaq, Thermo Scientific, St. Leon-Rot, Germany). Cycling was carried out using the following programme: 3 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 52 °C, 60 s at 72 °C; 5 min at 72 °C. PCR products were purified by adding 1 U of Exonuclease I and 0.5 U FastAP Alkaline Phosphatase (Thermo Scientific, St. Leon-Rot, Germany) and incubated at 37 °C for 1 h, followed by inactivation at 80 °C for 15 min. Sequencing was performed by Macrogen Inc. (The Netherlands) with PCR primers.    Zhao et al. (2016). An independent Monte Carlo Markov Chain of 50 million generations was run and log states every 5,000 generations by BEAST v1.8 (Drummond et al. 2012). The log file was checked in Tracer v. 1.6 (Rambaut et al. 2014) to ensure ESS (Effective Sample Sizes) value higher than 200. An ultrametric maximum-clade-credibility (MCC) tree was summarised using TreeAnnotator 1.8, discarding 10% of states as burn-in and annotating clades with ≥ 0.8 posterior probability.

Results
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). 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. Etymology. In reference to the cymbiform basidiospores.
Habit. Gregarious on soil in rain forest which is mainly dominated by Castanopsis armata, Castanopsis sp., Pinus sp., Lithocarpus sp.
Distribution  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).

Discussion
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 multigene 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 ; 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 ) 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.