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A new section and species of Agaricus subgenus Pseudochitonia from Thailand
expand article infoMao-Qiang He§|, Boontiya Chuankid§, Kevin D. Hyde§, Ratchadawan Cheewangkoon, Rui-Lin Zhao|
‡ Chiang Mai University, Chiang Mai, Thailand
§ Mae Fah Luang University, Chiang Rai, Thailand
| Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
¶ University of Chinese Academy of Sciences, Beijing, China
Open Access

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.

Keywords

New taxa, Agaricaceae, Phylogeny, Taxonomy

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, 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.

Materials and methods

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 Qm = 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.

Sequence alignment, phylogenetic analyses and divergence time estimation

A total of 119 specimens representing 87 species were incorporated in phylogenetic analyses. Three new sequences representing A. angusticystidiatus were generated from this study. They are one ITS sequence from specimen BC088 and two LSU sequences from ZRL2085 and ZRL2043 separately. Details of all sequences are listed in Table 1. Sequences were checked in BioEdit V.7.0.4 first (Hall 2007). Alignments were made by Muscle (Edgar 2004) for each region separately, then adjusted by hand and ambiguous regions removed. Alignments were submitted to TreeBase (Submission ID: 22231). Two data matrices were made for different analyses. The first one is an ITS sequence dataset which contains 84 specimens, all belonging to subgenus Pseudochitonia and an outgroup A. campestris. This dataset was used for Bayesian and Maximum Likelihood analyses. Phylogenetic trees generated by Bayesian Inference (BI) analysis were performed in MrBayes 3.1.2. (Ronquist and Huelsenbeck 2003). Best model is GTR + I + G which was indicated by MrModeltest 2.2 (Nylander 2004). Ten million generations were run for six Markov chains and sampled every 100th generation resulting in 100,000 trees. Burn-in was determined in Tracer v1.6 with effective sample sizes (ESS) higher than 200 (http://tree.bio.ed.ac.uk/software/tracer). Remaining trees were used to calculate Bayesian posterior probabilities (PP). Maximum Likelihood (ML) analysis and bootstrap values calculation were performed in raxmlGUI 1.5b1 using GTRGAMMA model with 1000 replicates (Silvestro and Michalak 2012). The second dataset included 63 ITS, 61 LSU and 59 tef1-α gene sequences from specimens representing the six subgenera of Agaricus. The second multi-gene dataset was used for divergence time estimation. Model selections were performed in jModel Test v. 2 (Darriba et al. 2012) for each gene separately. An XML file was generated in BEAUTI v. 1.8. Priors were set according to the previous fossil-calibrated analysis of 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.

Taxa information used in the phylogenetic analyses, new taxa are in bold, “T” refers to type.

Species Name Collection Number LSU ITS tef1-α Origin
Agaricus abruptibulbus ZRL2012005 KT951460 KT951356 KT951626 Yunnan, China
A. albosquamosus T LD2012192 KT951520 KT951394 KT951636 Thailand
A. amoenus T ZRL2010072 KT951524 KT951348 KT951638 Yunnan, China
A. angusticystidiatus BC088 MG888054 Thailand
A. angusticystidiatus ZRL2085 MG835413 KT951434 Thailand
A. angusticystidiatus T ZRL2043 MG835412 JF691553 Thailand
A. atrodiscus LD2012185 KT951473 KT284912 KT951653 Thailand
A. benesii LAPAG283 JF797179 Burgos, Spain
A. bernardiformis CA433 KT951467 KT951321 KT951577
A. biannulatus LAPAG611 JF896229 Sardinia, Italy
A. biberi LAPAG687 KR006614 KM657919 KR006642 Hungary
A. bingensis ADK1992 KJ540954 Atakora, Benin
A. bisporiticus LD2012111 KT951507 KJ575611 KT951650 Thailand
A. bisporiticus MCR25 KJ575608 Pakistan
A. bisporus LAPAG446 KR006611 KM657920 KR006640 Burgos, Spain
A. bitorquis CA427 KT951491 KT951320 KT951646
A. bitorquis WZR2012827 KT951492 KM657916 KT951647 Xingjiang, China
A. bohusii LAPAG562 KR006613 KM657928 KR006641 Madrid, Spain
A. boisseletii CA123 DQ182531
A. brunneopictus ADK2564 JF514518 Plateau Atlantique, Bénin
A. brunneopileatus T ZRL2012115 KT951489 KT951404 KT951587 Yunnan, China
A. brunneosquamulosus LD2012105 KJ540968 Thailand
A. brunneosquamulosus ZRL4017 JF691549 Thailand
A. caballeroi AH44503 KJ575605 Spain
A. campestris LAPAG370 KR006607 KM657927 KR006636 Madrid, Spain
A. campestroides LAPAF2 JF727842 Plateaux, Togo
A. candidolutescens T LD2012129 KT951525 KT951335 KT951616 Thailand
A. cf. bernardi CA383 KT951469 KT951319 KT951576
A. cf. goossensiae ADK2171 JF514517 Borgou, Benin
A. chiangmaiensis NTS113 JF514531 Thailand
A. comtulus LAPAG724 KT951448 KT951332 KT951593 Burgos, Spain
A. crassisquamosus T ZRL2012607 KT951510 KT951376 KT951645 Tibet, China
A. cupressicola LAPAG889 KT951465 KT951334 KT951649 Roma, Italy
A. desjardinii WZR2012907 KT951474 KM657901 KT951644 Xinjiang, China
A. dilutibrunneus T ZRL2012010 KT951512 KT951358 KT951569 Yunnan, China
A. dolichopus ZRL2012715 KT951502 KT951382 KT951573 Tibet, China
A. dolichopus ZRL2014120 KT951433
A. duplocingulatus ZRL3064 KJ540966 Thailand
A. erectosquamosus T LD2012165 KT951509 KT951338 KT951565 Thailand
A. erythrosarx MURU6080 JF495068
A. freirei CA186 DQ185553
A. fuscofibrillosus WC913 AY484684
A. fuscopunctatus LD2012115 KJ575612 Thailand
A. fuscovelatus RWK2100 KJ577973
A. gennadii CA339 KT951318 KT951575
A. grandiomyces T ZRL2012611 KR006624 KM657879 KR006652 Tibet, China
A. gratolens ZRL3093 KT951488 JF691548 Thailand
A. haematinus ZRL2109 KT951435 Thailand
A. haematinus ZRL2136 JF691552 Thailand
A. hondensis RWK1938 DQ182513 USA
A. huijsmanii LAPAG639 KT951444 KF447889 KT951571 Navarra, Spain
A. kunmingensis ZRL2012015 KT951506 KT951361 KT951642 Yunnan, China
A. kunmingensis ZRL2012007 KT951427 Yunnan, China
A. lamellidistans T ZRL3099 JF691556 Thailand
A. laskibarii LAPAG115 AY943975 Landes, France
A. leucocarpus T LD2012159 KX083981 KU975101 KX198048 Thailand
A. leucolepidotus T LD201214 KT951519 KT951336 KT951635 Thailand
A. linzhiensis T ZRL2012618 KT951503 KT951378 KT951582 Tibet, China
A. litoralis LAPAG420 KT951483 KT951327 KT951572 Burgos, Spain
A. litoraloides ZRL2011249 KT951523 KT951353 KT951580 Yunnan, China
A. magnivelaris F2389 JF727851
A. martinicensis F2815 KX084032 JF727855 KX198038 MartiniqueFrance
A. megacystidiatus LD2012179 KF305946 Thailand
A. microvolvatulus LD201271 KT951508 KJ575614 KT951651 Thailand
A. murinocephalus ZRL3044 JF691555 Thailand
A. nevoi LAPAG257 KR006606 KM657922 KR006635 Burgos, Spain
A. nevoi LAPAG535 KT951330 KT951574 Teruel, Spain
A. nigrobrunnescens DEH632 JX308267 Hawaii, USA
A. nigrogracilis T ZRL2012014 KR006621 KM657882 KR006647 Yunnan, China
A. niveogranulatus LD201124 KJ540959 Thailand
A. padanus WZR2012903 KR006616 KM657903 KR006644 Xingjiang, China
A. pallidobrunneus T ZRL2012358 KT951471 KT951370 KT951566 Yunnan, China
A. parvitigrinus CA158 AY899267
A. pattersoniae RWK1415 AY943974
A. phaeolepidotus CA217 DQ185552
A. pilosporus LAPAG227 KT951425 Burgos, Spain
A. pseudolangei ZRL3012 JF691551 Thailand
A. rufoaurantiacus LAPAM15 KX671708 KT951313 KT951641 Dominican Republic
A. silvaticus ALG07 213 KT951307 KT951567 Algonquin, ON, Canada
A. sinodeliciosus WZR2012822 KT951518 KM657907 KT951648 Xingjiang, China
A. sordidocarpus LD201237 KJ540946 Thailand
A. subrufescens ZRL2012722 KT951451 KT951383 KT951632 Yunnan, China
A. subsaharianus ADK4732 JF440300 Ouagadougou, Burkina Faso
A. sylvaticus LAPAG382 KR006608 KM657929 KR006637 Burgos, Spain
A. sylvaticus ZRL2012013 KT951500 KT951360 KT951570 Thailand
A. sylvaticus ZRL2012568 KT951501 KT951371 KT951568 Tibet, China
A. tibetensis ZRL2012585 KR006633 KM657895 KR006658 Tibet, China
A. tollocanensis CA235 AY703913
A. toluenolens CA911 KJ540947
A. trisulphuratus complex LAPAF7 KR006605 KM657924 KR006634 Plateaux, Togo
A. trisulphuratus complex Swk079 KT951472 KT951343 KT951561 Lanjak-Entimau, Malaysia
A. trisulphuratus complex ZRL2014023 KT951428 China
A. trisulphuratus complex ZRL2014024 KT951429 China
A. trisulphuratus complex ZRL2014030 KT951432 China
A. trisulphuratus complex ZRL2132 JF691558 Thailand
A. tytthocarpus ZRLWXH3077 KR006618 KM657889 KR006645 Fujian, China
A. variabilicolor ZRL4002 KT951438 Thailand
A. variabilicolor ZRL4007 KT951439 Thailand
A. variabilicolor ZRL4012 KT951440 Thailand
A. variicystis LD201228 KT951426 Thailand
A. variicystis T LD201234 KT951517 KT951339 KT951562 Thailand
A. xanthodermulus CA160 AY899273
A. xanthodermus CA15 AY899271
A. xanthodermus LAPAG387 KR006609 KM657923 KR006638 Soria, Spain
A. xanthosarcus Goossens5415 JF514523
A. sp. CA486 JF797189
A. sp. CA820 JF727861
A. sp. LD2012162 KT951493 KT951337 KT951563 Thailand
A. sp. NT020 JF797197 Thailand
A. sp. Swk014 KT951482 KT951342 KT951654 Lanjak-Entimau, Malaysia
A. sp. ZRL133 KT951505 KT951344 KT951656 Thailand
A. sp. ZRL2010010 KT951511 KT951347 KT951639 Thailand
A. sp. ZRL2010099 KT951479 KT951349 KT951564 Yunnan, China
A. sp. ZRL2012267 KT951504 KT951368 KT951655 Yunnan, China
A. sp. ZRL2012629 KR006627 KM657890 KR006656 Tibet, China
A. sp. ZRLWXH3078 KT951464 KT951464 KT951643 Fujian, China
A. sp. ZRLWXH3161 KT951526 KT951391 KT951615 Guangdong, China
A. sp. ZRLWXH3140 KT951441 Guangdong, China
Heinemannomyces sp. ZRL185 KT951527 KT951346 KT951657 Thailand

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).

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, Qm = 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).

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 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.

Acknowledgements

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).

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