Study in Agaricus subgenus Minores and allied clades reveals a new American subgenus and contrasting phylogenetic patterns in Europe and Greater Mekong Subregion.
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Abstract
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Study in Agaricus subgenus Minores and allied clades reveals a new American subgenus and contrasting phylogenetic patterns in Europe and Greater Mekong Subregion
Abstract
Within Agaricus subg. Minores, A. sect. Minores remains a little-studied section due generally to its delicate sporocarps often lacking taxonomically relevant morphological characters. To reconstruct the section, using the recent taxonomic system based on divergence times, and to evaluate the species diversity of A. sect. Minores in the Greater Mekong Subregion, 165 specimens were incorporated in phylogenetic analyses. A dated tree based on nuclear ITS, LSU and tef1-α sequence data allowed us to better circumscribe A. subg. Minores and to propose a new subgenus, A. subg. Minoriopsis, which is only known from tropical and subtropical regions of the Americas. A larger tree based on ITS sequences indicated that, with 81 phylogenetic species, the reconstructed section Minores is now one of the largest sections in the genus. Within A. subg. Minores, a new section, A. sect. Leucocarpi, and eleven new species are described from the Greater Mekong Subregion. Thirty-eight species of A. sect. Minores from this region of Asia were distributed in multiple clades that successively diverged over the past 24 million years. In contrast, species reported from Europe mostly grouped in a single non-tropical clade, suggesting a major species diversification following the middle Miocene climatic optimum.
INTRODUCTION
Agaricus (Agaricaceae, Basidiomycota) is a large genus comprising more than 400 species worldwide that are common in forests or grasslands (Zhao et al. 2011, Karunarathna et al. 2016). In the field, Agaricus species are easily distinguishable by having a fleshy pileus with free lamellae which produce brown spores, and an annulate stipe. The taxonomy of the genus has been well developed during the last two decades by using molecular phylogenetic tools (Challen et al. 2003, Kerrigan et al. 2005, 2008, Zhao et al. 2011, Parra 2013, Thongklang et al. 2014, Chen et al. 2015, Gui et al. 2015), which essentially reshaped our understanding of some of the morphologically recognized sections. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA has been largely used for establishing the phylogenetic relationships among the members of the genus, for example, in the phylogenetic study of Zhao et al. (2011), an eight-section taxonomic system was well supported, including seven additional strongly supported clades (TRI to TRVII). The fact that these clades were exclusively from subtropical or tropical regions suggested that geographical and climatic factors had played a major role in the evolutionary history of the genus. Nevertheless, the ITS region alone is generally insufficient to delimit taxa or to resolve their relationships especially for those of higher rank than species (Matheny et al. 2007, Zhao et al. 2016). Zhao et al. (2016) proposed standardization of the taxonomic ranks based on divergence times. And using multi-gene phylogenetic analyses and molecular clock methods, a revised taxonomic system was proposed in which the genus Agaricus was divided into five subgenera and 20 sections (Zhao et al. 2016).
Among the five subgenera of Agaricus considered in Zhao et al. (2016), the present study focuses on A. subg. Minores, which accommodates species with positive reaction to KOH, usually positive, seldom negative Schäffer’s cross-reactions (aniline × nitric acid) at the pileus surface or stipe base, yellowish staining when rubbed or cut and an anise-like or almond odour (Parra 2008, 2013, Zhao et al. 2016). The above traits are also shared by species of A. subg. Flavoagaricus, but taxa of A. subg. Minores could be further recognized by simple annulus (vs bilayered) and microscopically, generally by simple cheilocystidia (vs catenulate) and absence of inflated elements at the lower surface of the annulus.
For the delimitation of the subgenus, we followed the revised system of Zhao et al. (2016) in which clades that diverged 30–33 or 18–26 million years ago (Ma) were ranked as subgenera or sections, respectively. In this recent study A. subg. Minores consisted of three sections: A. sect. Laeticolores, A. sect. Minores, and one unnamed section (Zhao et al. 2016). Agaricus sect. Laeticolores was represented by a single species identified as A. rufoaurantiacus, while A. sect. Minores included not only A. sect. Minores as reported in Zhao et al. (2011), but also the closely related tropical clades TRV to TRVII reported in the same study, because they diverged too recently to be ranked as sections (Zhao et al. 2016). Although they belong in A. sect. Minores and this has been phylogenetically established in the new taxonomic system, a comprehensive morphological study of certain specimens is still needed to better circumscribe the section and its species. Species of A. sect. Minores are commonly distributed in temperate and tropical regions (Zhao et al. 2011, Parra 2013), and can also be found in harsh environments especially the sequestrate (secotioid) species adapted to xeric conditions (Thiers 1984, Lebel 2013).
Agaricus sect. Minores is well documented in Europe with 21 recognized species (Parra 2013). However, the species diversity in other areas is poorly explored. It must be noted that before the application of molecular techniques, few species were described in this group (Peterson et al. 2000). Some species seem to be widespread and have been recorded in several continents, for example, A. brunneolus, A. comtulus, and A. purpurellus (Heinemann 1961, 1962, 1980, 1990, Pegler 1977). Since species of A. sect. Minores usually produce small sporocarps, and lack morphological characters useful for species recognition, the question arises as to whether these species are widely distributed or whether there are any cryptic species, that are morphologically indistinguishable, but genetically distant (Bickford et al. 2007).
The Greater Mekong Subregion (GMS) is a region around the Mekong River basin in Southeast Asia, which includes Cambodia, Laos, Myanmar, Thailand, Vietnam, and Yunnan Province of China. It is also located within the so-called Indo-Burma hotspot, one of the 34 global biodiversity hotspots identified by Conservation International (Fisher & Christopher 2007). A project entitled ‘Inventory and taxonomy of Agaricus species in Thailand, Laos, Malaysia and Yunnan (China); domestication and evaluation of species of nutritional or medicinal interest’ has been carried out since 2010 and has already revealed a high species diversity in this region (Zhao et al. 2011, 2012, 2013, 2016, Chen et al. 2012, 2015, Karunarathna et al. 2016, Thongklang et al. 2014). Most of our samples have been collected in the framework of this project. In other respects, we have also contributed to the study of A. sect. Minores in the monograph of the genus Agaricus in Europe (Parra 2013), and therefore, the European diversity is also widely represented in our phylogenetic analyses. The present study aims to:
phylogenetically reconstruct A. subg. Minores following the new taxonomic system proposed by Zhao et al. (2016) and taking into account allied clades; and
compare the species diversity in GMS and Europe.
MATERIALS AND METHODS
Materials examined and morphological observations
Fresh samples were mostly collected from 2006 to 2015 from Thailand and Yunnan Province, Southwest China, and seven were from Africa, Brazil, Dominican Republic and Malaysia. Specimens are deposited in MFLU (Mae Fah Luang University Herbarium) with duplicates at HMAS (Herbarium Mycologium, Chinese Academy of Sciences, Beijing, China), the African specimens are deposited in BR (the herbarium of the Botanic Garden Meise in Belgium). The Brazilian specimen is deposited in the Eliseo Battistin private herbarium, the Dominican specimens are deposited in JBSD (National Herbarium of Santo Domingo, Dr Rafael M. Moscoso National Botanical Garden), and the Malaysia specimens are deposited in KLU (Herbarium of Kuala Lumpur). In addition, two specimens of A. laeticolor (holotype Goossens5272 and paratype Goossens5371) from Africa were loaned from BR herbarium and three specimens from Martinique and Guadeloupe are from LIP herbarium (Université de Lille, France). Facesoffungi numbers (Jayasiri et al. 2015) are provided for new taxa.
Samples were photographed in situ or in laboratory, and odour and colour change (when rubbed or cut) were recorded in the field. The macroscopic characters were recorded according to the methodology described by Largent (1986). KOH and Schäffer’s reactions were performed as described by Chen et al. (2015). Micromorphological features were examined from dried specimens following the protocols of Largent et al. (1977) including anatomy of lamellae, pileipellis and partial veil, and features of basidiospores, basidia and cystidia. Measurements of anatomical features (basidiospores, basidia and cheilocystidia) were presented based on at least 20 measurements, and include x = the mean of length by width ± SD; Q = the quotient of basidiospore length to width, and Qm = the mean of Q-values ± SD.
DNA extraction, PCR and sequencing
At the Institut National de la Recherche Agronomique (INRA), DNA was isolated from dried specimens following a CTAB protocol as described by Zhao et al. (2011). At the Southwest Forestry University, a commercial DNA extraction kit (E.Z.N.A. Forensic Kit, D3591-01, Omega Bio-Tek) was used for DNA extraction. DNA sequences were obtained from three loci: the internal transcribed spacer (ITS), nuclear large ribosomal subunit (nrLSU) and translation elongation factor 1-alpha (tef-1α). Protocols for amplification of ITS and nrLSU regions followed those of White et al. (1990) with some modifications (Zhao et al. 2010), by using primers ITS4 and ITS5, LR0R and LR5, respectively. Amplification of the tef-1α region using primers EF1-983F and EF1-1567R (Morehouse et al. 2003) followed the procedure described as below:
initial denaturation at 94 °C for 3 min;
denaturation at 94 °C for 30 s;
annealing at 56 °C for 40 s;
extension at 72 °C for 50 s;
repeat for 40 cycles starting at step 2;
leave at 72 °C for 10 min.
Sequencing was performed on ABI Prism Genetic analyzer (Applied Biosystems) at Beckman Coulter Genomics, England or on ABI 3730 XL DNA analyzer (Applied Biosystems) at Shanghai Majorbio Bio-Pharm Technology Co., Ltd, China. Consensus sequences were assembled by using SeqMan package of Lasergene software v. 7.1 (DNAStar, Madison, WI, USA). All sequences have been deposited in GenBank and their accession numbers are given in Table 1.
Table 1
GenBank accession numbers and samples used in the molecular analyses
| Subgenus/section | Species | Taxa no. | Collection | Public database accession number | Geographic origin | ||
|---|---|---|---|---|---|---|---|
| ITS | LSU | TEF | |||||
| Outgroup | Heinemannomyces sp. | ZRL185 | KT951346 | KT951527 | KT951657 | Thailand | |
| Agaricus | A. campestris T | LAPAG370 | JQ903618 | KP739803 | KR006636 | Spain | |
| A. sp. | CA637 | KT951322 | KT951468 | KT951633 | France | ||
| A. sp. | ZRL2012006 | KT951357 | KT951466 | KT951634 | Yunnan, China | ||
| Flavoagaricus/Arvenses | A. arvensis T | LAPAG450 | KF114474 | KP739801 | KX198047 | Spain | |
| A. fissuratus | WC777 | AY484683 | – | – | Denmark | ||
| A. flocculosipes | ZRL2012105 | KT951365 | KT951463 | KT951618 | Yunnan, China | ||
| A. inapertus | ECVel2339 | AF482834 | AF482878 | – | USA | ||
| A. sp. | ZRL2012630 | KT951379 | KT951495 | KT951621 | Tibet, China | ||
| A. subrufescens | ZRL2012722 | KT951383 | KT951451 | KT951632 | Yunnan, China | ||
| Minores/Leucocarpi | A. leucocarpus | LD201226 | KU975102 | KX083982 | KX198049 | Thailand | |
| A. leucocarpus | SCK089 | KU975090 | – | – | Thailand | ||
| A. leucocarpus T | LD201215 | KU975101 | KX083981 | KX198048 | Thailand | ||
| Minores/Minores | A. sp. | 1 | ZRL2012012 | KT951359 | KT951494 | KT951597 | Yunnan, China |
| A. columellatus | 2 | MIN 938394 | KJ912899 | – | – | USA | |
| A. colpetei T | 3 | TL2424 | JX984565 | – | – | Australia | |
| A. aridicola | 4 | CA101 | JF797195 | AF261478 | – | France | |
| A. aridicola | 4 | LAPAG589 | KT951331 | KX084027 | KX198081 | Spain | |
| A. sp. | 5 | CA848 | JF727864 | KT951445 | KT951605 | Thailand | |
| A. sp. | 6 | PS036 | KU975087 | KX084035 | KX198036 | Thailand | |
| A. laeticulus T | 7 | Goossens5272 | KX671705 | – | – | DR Congo | |
| A. laeticulus | 7 | Goossens5371 | KX671704 | – | – | DR Congo | |
| A. sp. | 8 | NTS73 | KU975099 | – | – | Thailand | |
| A. sp. | 9 | NTT33 | JF514535 | – | – | Thailand | |
| A. sp. | 10 | ZRL2011156 | KT951352 | KT951480 | KT951603 | Yunnan, China | |
| A. flavopileatus T | 11 | MS596 | KU975121 | KX084022 | KX198078 | Yunnan, China | |
| A. flavopileatus | 11 | MS603 | KU975122 | KX084023 | KX198045 | Yunnan, China | |
| A. sp. | 12 | ZRLLD013 | KT951384 | KT951516 | KT951604 | Thailand | |
| A. luteopallidus | 13 | SCK121 | KU975092 | – | – | Thailand | |
| A. luteopallidus | 13 | LD2012113 | KU975124 | KX084026 | KX198080 | Thailand | |
| A. luteopallidus | 13 | SCK099 | KU975095 | – | – | Thailand | |
| A. luteopallidus | 13 | LD2012120 | KU975123 | KX084024 | KX198079 | Thailand | |
| A. luteopallidus | 13 | NTF26 | JF514526 | – | – | Thailand | |
| A. luteopallidus | 13 | SCK120 | KU975093 | – | – | Thailand | |
| A. luteopallidus | 13 | SCK138 | KU975094 | – | – | Thailand | |
| A. luteopallidus T | 13 | ZRL3088 | JF691543 | KX084025 | – | Thailand | |
| A. luteopallidus | 13 | NTSCR1 | KU975100 | – | – | Thailand | |
| A. callacii T | 14 | AH42929 | KF447899 | KX083984 | KX198051 | Canary Islands (Spain) | |
| A. chartaceus T | 15 | H6271 | JF495048 | – | – | Australia | |
| A. lamelliperditus T | 16 | MDBF61/96 | JX984559 | – | – | Australia | |
| A. cf. wariatodes | 17 | MEL2058664 | JF495050 | – | – | Australia | |
| A. wariatodes | 18 | TWM1589 | JF495052 | JF495030 | – | Australia | |
| A. parvibicolor T | 19 | LD2012116 | KP715162 | KX084016 | KX198075 | Thailand | |
| A. parvibicolor | 19 | ZRL3091 | JF691546 | KX084015 | – | Thailand | |
| A. purpureofibrillosus | 20 | NTF63 | KU975098 | – | – | Thailand | |
| A. purpureofibrillosus T | 20 | ZRL3080 | JF691542 | KX084021 | – | Thailand | |
| A. sp. | 21 | CA843 | JF727866 | KX084029 | KX198040 | Thailand | |
| A. sp. | 22 | ZRL2012004 | KT951355 | KT951457 | KT951608 | Yunnan, China | |
| A. sp. | 23 | ZRL2012714 | KT951381 | KT951476 | KT951607 | Tibet, China | |
| A. sp. | 24 | ZRL2011039 | KT951351 | KT951449 | KT951606 | Yunnan, China | |
| A. sp. | 25 | LD201252 | KU975103 | KX083983 | KX198050 | Thailand | |
| A. sp. | 26 | ADK2751 | JF514519 | – | – | Bénin | |
| A. sp. (A. semotus) | 27 | PDD68575 | AF059224 | AF059224 | – | New Zealand | |
| A. campbellensis T | 28 | GAL9420 | DQ232644 | DQ232657 | – | New Zealand | |
| A. sp. | 29 | GAL5812 | EF460364 | EF460389 | – | USA | |
| A. sp. | 30 | ZRL3056 | JF691541 | KX084020 | – | Thailand | |
| A. megalosporus | 31 | LD2012142 | KU975120 | KX084019 | KX198077 | Thailand | |
| A. megalosporus | 31 | ZRL2012199 | KT951367 | KT951470 | KT951595 | Yunnan, China | |
| A. megalosporus T | 31 | LD030 | JF514521 | – | – | Thailand | |
| A. sp. | 32 | CA846 | JF727865 | KT951452 | KT951601 | Thailand | |
| A. fimbrimarginatus T | 33 | LD201250 | KU975119 | KX084017 | KX198076 | Thailand | |
| A. sp. | 34 | ZRL2044 | JF691540 | KX084018 | – | Thailand | |
| A. robustulus | 35 | ADK2905 | JF514520 | – | – | Bénin | |
| A. robustulus T | 35 | CA847 | KU975086 | KX084034 | KX198039 | Thailand | |
| A. robustulus | 35 | AK075 | KU975088 | – | – | Malaysia | |
| A. robustulus | 35 | MAR145 | KU975089 | – | – | Malaysia | |
| A. robustulus | 35 | ZRL2012357 | KT951369 | KT951496 | KT951610 | Yunnan, China | |
| A. robustulus | 35 | NT055 | JF727846 | – | – | Thailand | |
| A. purpurellus | 36 | LAPAG682 | KF447903 | KX083993 | KX198059 | Italy | |
| A. purpurellus | 36 | LAPAG944 | KU975076 | KX083994 | KX198060 | Czech Republic | |
| A. jacobi | 37 | LAPAG942 | KU975081 | KX083995 | – | Spain | |
| A. jacobi T | 37 | AH44505 | KF447895 | KX083996 | KX198061 | Spain | |
| A. marisae | 38 | LAPAG138 | KU975083 | KX083998 | KX198065 | Spain | |
| A. marisae T | 38 | LAPAG111 | JF797182 | – | – | Spain | |
| A. edmondoi T | 39 | LAPAG80 | KF447902 | – | – | Spain | |
| A. edmondoi | 39 | LAPAG412 | KT951326 | KT951481 | KT951590 | Spain | |
| A. kerriganii | 40 | LAPAG808 | KT951306 | KT951442 | KT951589 | Spain | |
| A. kerriganii T | 40 | AH44509 | KF447893 | KX083999 | KX198066 | Spain | |
| A. cf. kerriganii (A. diminutivus) | 41 | WC912 | AY484681 | – | – | USA | |
| A. dulcidulus | 42 | PRM909627 | KF447894 | – | KX198064 | Czech Republic | |
| A. iesu-et-marthae | 43 | LAPAG41 | KF447904 | – | – | Spain | |
| A. brunneolus | 44 | LAPAG654 | KU975077 | – | KX198063 | Czech Republic | |
| A. brunneolus | 44 | LAPAG938 | KU975082 | KX083997 | KX198062 | Spain | |
| A. sp. | 45 | GAL3083 | EF460374 | EF460399 | – | USA | |
| A. friesianus T | 46 | F156208 | KF447907 | – | – | Sweden | |
| A. friesianus | 46 | LAPAG592 | KT951316 | KX083992 | KT951594 | France | |
| A. matrum | 47 | LAPAG916 | KU975080 | KX083990 | KX198057 | Spain | |
| A. matrum T | 47 | AH44506 | KF447896 | KX083991 | KX198058 | Spain | |
| A. heinemannianus | 48 | LAPAG302 | KF447906 | – | KX198056 | Spain | |
| A. heinemannianus T | 48 | AH19381 | KF447905 | – | – | Spain | |
| A. pallens | 49 | LAPAG441 | KF447898 | – | KX198067 | Spain | |
| A. pallens | 49 | LAPAG580 | KF447897 | – | – | Spain | |
| A. arrillagarum | 50 | LAPAG810 | KF447900 | KX083985 | KT951592 | Spain | |
| A. arrillagarum T | 50 | AH44508 | KF447908 | – | – | France | |
| A. gemlii T | 51 | AH44510 | KF447891 | KX083989 | – | Spain | |
| A. gemlii | 51 | LAPAG286 | KU975079 | KX083988 | KX198055 | Spain | |
| A. comtulus | 52 | LAPAG724 | KT951332 | KT951448 | KT951593 | Spain | |
| A. comtulus | 52 | LAPAG303 | KU975078 | KX083986 | KX198052 | Spain | |
| A. luteomaculatus | 53 | CA331 | KF447901 | – | KX198053 | France | |
| A. sp. | 54 | ZD1528 | KU975104 | KX083987 | KX198054 | Yunnan, China | |
| A. gemloides T | 55 | ZRL2014084 | KT633271 | – | – | Yunnan, China | |
| A. gemloides | 55 | ZRL2014009 | KT633272 | – | – | Yunnan, China | |
| A. sp. | 56 | ZRLWXH3067 | KT951387 | KT951497 | KT951611 | Jiangxi, China | |
| A. sp. | 57 | ZRL3102 | JF691545 | KX084028 | – | Thailand | |
| A. coccyginus T | 58 | ZRL2012485 | KU245979 | – | – | Tibet, China | |
| A. coccyginus | 58 | ZRL2012576 | KT951372 | KT951499 | KT951596 | Tibet, China | |
| A. coccyginus | 58 | ZRL2014430 | KU245980 | – | – | Yunnan, China | |
| A. huijsmanii | 59 | LAPAG639 | KF447889 | KT951444 | KT951571 | Spain | |
| A. sp. | 60 | PYP014 | KU975091 | – | – | Thailand | |
| A. sp. | 61 | ADK3580 | KU975097 | – | – | Bénin | |
| A. sp. | 62 | NT62 | JF727845 | – | – | Thailand | |
| A. patris | 63 | ZRL3101 | JF691544 | KX084013 | – | Thailand | |
| A. patris T | 63 | LD201224 | KU975118 | KX084012 | KX198073 | Thailand | |
| A. sodalis T | 64 | LD2012159 | KP715161 | KX084014 | KX198074 | Thailand | |
| A. sodalis | 64 | LD2011029 | KP715160 | – | – | Thailand | |
| A. pseudolutosus T | 65 | AH11488 | KF447890 | – | – | Spain | |
| A. pseudolutosus | 65 | LAPAG454 | KT951329 | KT951453 | KT951602 | Spain | |
| A. sp. | 66 | MATA774 | JF727871 | – | – | Mexico | |
| A. sp. | 67 | ZRLWXH3076 | KT951388 | KT951458 | KT951612 | Fujian, China | |
| A. sp. | 68 | ZRLWXH3150 | KT951390 | KT951447 | KT951609 | Guangdong, China | |
| A. sp. (A. diminutivus) | 69 | Vellinga2360 | AF482831 | AF482877 | – | USA | |
| A. viridopurpurascens T | 70 | Horak68/79 | JF514525 | – | – | New Zealand | |
| A. sp. | 71 | TL2154 | JF495059 | – | – | Australia | |
| A. sp. | 72 | TL2307 | JF495058 | – | – | Australia | |
| A. brunneolutosus | 73 | MS541 | KU975112 | KX084007 | – | Yunnan, China | |
| A. brunneolutosus T | 73 | MS514 | KU975111 | KX084006 | – | Yunnan, China | |
| A. sp. | 74 | MS386 | KU975113 | KX084008 | KX198044 | Yunnan, China | |
| A. sp. | 75 | CA935 | KU975085 | KX084036 | KX198034 | Thailand | |
| A. badioniveus T | 76 | LD2012131 | KU975117 | – | KX198072 | Thailand | |
| A. flammicolor | 77 | ZRL2012270 | KU975116 | KX084011 | KX198071 | Yunnan, China | |
| A. flammicolor | 77 | LD201225 | KU975115 | KX084010 | KX198070 | Thailand | |
| A. flammicolor T | 77 | LD201502 | KU975114 | KX084009 | KX198042 | Thailand | |
| A. sp. | 78 | CA845 | KU975084 | KX084033 | KX198035 | Thailand | |
| A. sp. | 79 | NTT72 | JF514539 | – | – | Thailand | |
| A. fulvoaurantiacus | 80 | MS316 | KU975106 | KX084001 | KX198043 | Yunnan, China | |
| A. fulvoaurantiacus T | 80 | LD201404 | KU975107 | KX084002 | KX198069 | Yunnan, China | |
| A. fulvoaurantiacus | 80 | MS549 | KU975105 | KX084000 | KX198068 | Yunnan, China | |
| A. luteofibrillosus | 81 | ZRL2014136 | KU245974 | – | – | Yunnan, China | |
| A. luteofibrillosus | 81 | LD201501 | KU975108 | KX084003 | KX198041 | Thailand | |
| A. luteofibrillosus T | 81 | ZRL2013484 | KU245972 | – | – | Yunnan, China | |
| A. luteofibrillosus | 81 | ZRL2110 | KU975109 | KX084004 | – | Thailand | |
| A. luteofibrillosus | 81 | ZRL3039 | KU975110 | KX084005 | – | Thailand | |
| A. luteofibrillosus | 81 | NTT37 | JF514537 | – | – | Thailand | |
| Minores/sect. 1 | A. candidolutescens T | LD2012129 | KT951335 | KT951525 | KT951616 | Thailand | |
| A. sp. | LAPAM14 | KT951312 | – | KT951613 | Dominican Republic | ||
| A. sp. | LAPAM45 | KX671701 | – | – | Dominican Republic | ||
| A. sp. | ZRLWXH3161 | KT951391 | KT951526 | KT951615 | Guangdong, China | ||
| Minoriopsis | A. martinicensis | F2815 | JF727855 | KX084032 | KX198038 | Martinique (France) | |
| A. martinicensis | LAPAM16 | KX671699 | KX671709 | KX671706 | Dominican Republic | ||
| A. rufoaurantiacus | LAPAM15 | KT951313 | KX671708 | KT951641 | Dominican Republic | ||
| A. aff. rufoaurantiacus | CL/GNAD05090 | JF727857 | KX084031 | – | Guadeloupe (France) | ||
| A. sp. | LAPAM28 | KX671700 | KX671710 | KX671707 | Dominican Republic | ||
| A. sp. | LAPAM34 | KX671703 | – | – | Dominican Republic | ||
| A. sp. | LAPAM66 | KX671702 | – | – | Brazil | ||
| A. sp. (A. comtulus) | HAI0386 | AJ884624 | – | – | USA | ||
| A. sp. (A. johnstonii) | F1779 | JF727853 | KX084030 | KX198037 | Martinique (France) | ||
| Pseudochitonia | A. bisporus | LAPAG446 | KM657920 | KR006611 | KR006640 | Spain | |
| A. bitorquis | WZR2012827 | KM657916 | KT951492 | KT951647 | Xinjiang, China | ||
| A. sinodeliciosus | WZR2012822 | KM657907 | KT951518 | KT951648 | Xinjiang, China | ||
| Spissicaules | A. albosquamosus T | LD2012192 | KT951394 | KT951520 | KT951636 | Thailand | |
| A. gratolens T | ZRL3093 | JF691548 | KT951488 | – | Thailand | ||
| A. leucolepidotus T | LD201214 | KT951336 | KT951519 | KT951635 | Thailand | ||
| A. litoralis | LAPAG475 | KT951393 | KX083980 | KX198046 | Spain | ||
| A. litoraloides | ZRL2011249 | KT951353 | KT951523 | KT951580 | Yunnan, China | ||
| A. sp. | AW145 | KT951308 | – | KT951637 | Canada | ||
Note New taxa are in bold. ‘T’ refers to type specimen. Species numbering in A. sect. Minores follows the order observed in the ITS ML tree of Fig. 3. Species name of the sequences deposited in GenBank is included in parenthetical citation after the identification adopted in this work whether when both differ due to a clear misidentification (A. comtulus), the same identification is indicated in GenBank for divergent sequences of obviously unrelated samples (A. diminutivus), or identification is based on a dubious and confused name (A. semotus) or based on material not matching the original description (A. johnstonii).
Sequence alignment, divergence time estimation and phylogenetic analyses
A total of 165 specimens were incorporated in phylogenetic analyses. In addition to the sequences generated from this study, 109 ITS sequences, 43 nrLSU sequences and 39 tef-1α sequences were retrieved from GenBank (Geml et al. 2008, Zhao et al. 2011, 2016, Lebel & Syme 2012, Lebel 2013, He & Zhao 2015, Liu et al. 2015, Bates et al. 2016, Li et al. 2016) and their accession numbers are given in Table 1. Sequences were aligned, for each region independently using MAFFT (Katoh & Standley 2013), then manually adjusted in BioEdit v. 7.0.4 (Hall 2007). The ITS alignment was treated with Gblocks 0.91b (Castresana 2000), eliminating poorly or ambiguously aligned positions or DNA segments. Alignments have been submitted to TreeBase (submission ID 19813).
Divergence times were estimated using BEAST v. 1.8 (Drummond et al. 2012) based on 111 sequences. We first constructed an XML file with BEAUTI v. 1.8. Per-gene alignments were imported as separate partitions. Clock and substitution models were set to be unlinked (independently estimated for each gene partition), while the tree prior parameters were set to be linked across partitions (concatenation). Substitution models were chosen based on jModelTest v. 2 (Darriba et al. 2012). In this case, we used normal distribution prior on the treeModel.rootHeight parameter, which has an initial value of 66 Myr for the genus Agaricus and a standard deviation of 1 Myr. The initial value is according to the previous fossil-calibrated analysis of Zhao et al. (2016). We ran an independent Monte Carlo Markov Chains of 50 million generations, logging states every 5 000 generations. The log file was opened in Tracer v. 1.6 (Rambaut et al. 2014) to evaluate convergence and mixing, and to ensure that Effective Sample Sizes were at least 200. An ultrametric maximum-clade-credibility (MCC) tree was summarized using TreeAnnotator 1.8, discarding 10 % of states as burn-in and annotating clades with ≥ 0.8 posterior probability.
Maximum Likelihood analysis was performed using RAxML-HPC2 v. 8.2.4 (Stamatakis 2014) as implemented on the Cipres portal (Miller et al. 2010), under a GTRGAMMA model with one thousand rapid bootstrap (BS) replicates for each gene. A reciprocal 70 % bootstrap support approach was used to compare the tree topologies from individual genes. There was no significant incongruence between the datasets, so the ITS, nrLSU, and tef-1α sequences were concatenated in BioEdit v. 7.0.4 (Hall 2007) for subsequent phylogenetic analyses.
The combined dataset was partitioned into ITS1, 5.8S, ITS2, nrLSU, tef-1α intron and tef-1α coding sites. The best substitution model for each partition was inferred with the program MrModeltest 2.2 (Nylander 2004): GTR+I+G for ITS1, 5.8S, ITS2, nrLSU, and tef-1α intron sites, and SYM+I+G for tef-1α coding sites. Bayesian Inference (BI) analysis was performed with MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003). Six Markov chains were run for five million generations and sampled every 100th generation. Burn-in was determined by checking the likelihood trace plots in Tracer v. 1.6 (Rambaut et al. 2014) and subsequently discarded. Maximum parsimony (MP) analysis was performed in PAUP* 4.0b10 (Swofford 2004), by heuristic searches with unordered characters, random addition of sequences, gaps treated as missing data, and the tree bisection reconnection (TBR) branch swapping. Bootstrap values (BS) were obtained from 1 000 replicates. A node is considered to be strongly supported if at least two of the three analyses show a bootstrap support value (BS) ≥ 70 % and/or a posterior probability (PP) ≥ 0.95.
Phylogenetic species recognition criterion
Because the taxa belonging to A. sect. Minores often lack distinctive morphological characters, we applied the multilocus genealogical phylogenetic species recognition approach (Taylor et al. 2000, Dettman et al. 2003) to delimit the species boundaries. A phylogenetic species is recognized when it matches either of the two criteria:
a genealogical concordant group that is present in the majority of the single-locus genealogies; or
a clade that is strongly supported by at least one single-locus genealogy and is not contradicted by any other locus (Ge et al. 2014).
Automatic Barcode Gap Discovery (ABGD) method was used for primary species delimitation (Puillandre et al. 2012). In certain cases, species circumscription was improved by examining polymorphisms in ITS alignment, taking into account insertions/deletions and heteromorphisms which are relatively frequent in species of Agaricus and reflect allelic polymorphisms and heterozygosity.
RESULTS
Phylogenetic analyses
We generated 166 new sequences for this study, including 56 of ITS, 60 of nrLSU, and 50 of tef-1α. For dating analysis, a maximum clade credibility (MCC) tree is represented in Fig. 1. For this analysis 111 samples were used. Representatives of each five subgenera recognized by Zhao et al. (2016) were included and a specimen from the genus Heinemannomyces was used as an outgroup taxon. Eighty-six of these samples belong to A. subg. Minores.
Maximum Clade Credibility tree of Agaricus resulting from analysis of ITS, LSU, tef-1α sequence data with the outgroup Heinemannomyces sp. Posterior probabilities which are equal and above 80 % are indicated. The 95 % highest posterior density of divergence time estimation are marked by horizontal bars. Species sampled from GMS and Europe are in red and blue, respectively; new taxa are in bold.
In the multi-gene analyses as in the ITS analysis below, some redundant sequences were deleted, only subgenera related to A. subg. Minores were represented and A. campestris was used as outgroup. For multi-gene analyses, the final alignment contained 99 samples and was 2004 characters in length. The likelihood value of the final ML tree was -16287.144026. The topologies of the trees generated by the Bayesian and the maximum parsimony analyses were very similar to the ML tree (Fig. 2) except for few ungrouped samples such as A. aridicola/LAPAG589, A. callacii/AH42929, A. sp./CA843, A. sp./ZRL3080, A. sp./ZRLWXH3150, A. sp./ZRL2012004, and A. sp./ZRL2012012.
Maximum likelihood phylogram of Agaricus sect. Minores resulting from analysis of ITS, LSU, tef-1α sequence data. The best scoring RAxML multi-gene tree is rooted with A. campestris. The bootstrap support values greater than 50 % are indicated above or below the nodes (ML/MP), and branches with Bayesian posterior probabilities greater than 0.95 are in bold. The eleven subclades of Agaricus sect. Minores are indicated. Species sampled from GMS and Europe are in red and blue, respectively; new species are in bold. T = Type specimen.
For the ITS ML analysis 45 specimens belonging to 20 species were added. The aligned ITS dataset consisted of 150 sequences and was 751 nucleotides in length. The final alignment contained 676 characters after excluding ambiguous regions. Maximum likelihood analysis resulted in one ML tree with optimization likelihood value -8012.130834. To facilitate comparison between the trees and more specifically in A. sect. Minores, the major clades and the species of this section were numbered following the order in which they appeared in the multi-gene tree (from I to XI in Fig. 2) and in the ITS tree (from 1 to 81 in Fig. 3), respectively. Despite the different number of sequences used in multi-gene and ITS trees the same 11 major clades of A. sect. Minores were represented in both trees.
Maximum likelihood phylogram of Agaricus sect. Minores resulting from analysis of ITS sequence data. The best scoring RAxML ITS tree is rooted with A. campestris. The bootstrap support values greater than 50 % are indicated. The eleven subclades of Agaricus sect. Minores are indicated. Species sampled from GMS and Europe are in red and blue, respectively; new species are in bold. T = Type specimen. * an ungrouped sample in the multi-gene trees of Fig. 1 and and2,2, and which therefore, probably, does not belong to the clade A-XI. Circle symbol indicates secotioid species.
New subgenus and sections based on divergence times
We used the taxonomic system of classification introduced by Zhao et al. (2016) with the following criteria to recognize subgenera and sections in the genus Agaricus:
they must be monophyletic and statistically well-supported in the multi-gene analyses;
estimated stem ages for subgenera and sections are c. 30 Ma and c. 20 Ma, respectively; and
they should be identifiable phenotypically, whenever possible.
In the genus Agaricus, MCC tree revealed that six well-supported clades had a stem age over 30 Ma (Fig. 1). Five of them correspond to subgenera that were previously recognized as A. subg. Agaricus, A. subg. Flavoagaricus, A. subg. Minores, A. subg. Peudochitonia, and A. subg. Spissicaules by Zhao et al. (2016). The remaining clade was well supported in the MCC tree (PP ≥ 0.99; Fig. 1), in the multi-gene ML tree (ML: BS = 98, MP: BS = 90, PP > 0.95; Fig. 2) and in the ITS ML tree (BS = 80; Fig. 3). Its stem age was estimated to 31.02 Ma (Table 2). Therefore, this clade represents a new subgenus and is named below as A. subg. Minoriopsis. It includes five species in the MCC tree distributed in two sister clades which are well supported (PP ≥ 0.99; Fig. 1) and represent two sections since they diverged 22.83 Ma ago (Table 2). Using a broader sampling, the new subgenus includes eight species in the ITS ML tree and the two new sections remain well supported with BS values of 99 and 91, respectively. The new subgenus corresponds to the clade TRII in Zhao et al. (2011) and was represented by a single specimen (LAPAM15) in Zhao et al. (2016), which was included in A. [subg. Minores] sect. Laeticolores. Using more samples, our analysis indicates that this previous placement was incorrect as well as the name of the section. Indeed, we obtained the ITS sequence of a type specimen of A. laeticulus (a nom. nov. for the illegitimate name A. laeticolor, the type of the section) and this sample was placed in A. sect. Minores in the ITS tree (Fig. 3; see also the taxonomic treatment of A. sect. Minores).
Table 2
Mean stem ages of well-supported clades (P ≥ 0. 99) within or related to A. subg. Minores.
| Subgenera | Section/major clades | Mean of stem age (Ma) |
|---|---|---|
| Flavoagaricus | 31.02 | |
| Arvenses | 31.02 | |
| Minores | 33.15 | |
| Leucocarpi | 27.64 | |
| Minores | 30.06 | |
| A-I | 17.56 | |
| A-II | 12.88 | |
| A-III | 17.40 | |
| A-IV | 14.21 | |
| A-V | 14.21 | |
| A-VI | 22.28 | |
| A-VII | 14.30 | |
| A-VIII | 14.26 | |
| A-IX | 12.89 | |
| Minoriopsis | 31.02 |
The clade corresponding to A. subg. Minores was subdivided in three clades A, B, and C, which are well supported in the MCC tree (PP ≥ 0.99; Fig. 1) and in the multi-gene ML tree (ML: 95, 97, and 100; MP: 84, 75, and 100; PP > 0.95; Fig. 2). In the system of classification adopted here, they represent three sections, respectively, since their stem ages are over 20 Ma (Fig. 1, Table 2). The clade A corresponds to A. sect. Minores since it includes the type (A. comtulus) of this section. The clade B was previously reported as clade A2 by Zhao et al. (2016) and now corresponds to an unnamed section, while clade C represents a new section of the subgenus. Clades B and C are sister and thus have the same stem age which was estimated to 27.64 Ma (Table 2). In the ITS ML tree, clades corresponding to the different subgenera and sections were recovered except for A. subg. Minores. Therefore, using only ITS sequence data, it was not possible to circumscribe A. subg. Minores and the phylogenetic relationships between the three sections of this subgenus remained unresolved. In the ITS ML tree (Fig. 3), only clades B and C are well supported (BS of 80 and 100, respectively), while clade A, corresponding to A. sect. Minores is poorly supported (BS < 50). However, the placement of the species in the three sections/clades of the ITS tree does not differ from their placement in the multi-gene MCC or ML trees.
Major clades in A. sect. Minores
In the multi-gene ML tree, 11 subclades were revealed within A. sect. Minores and numbered from A-I to A-XI (Fig. 2). Except clade A-XI, all subclades received moderate to strong support. The clade A-VII is a core clade of A. sect. Minores and includes the type A. comtulus. It contains 20 species: 16 of the 19 species from Europe included in this study, two from China (A. gemloides and a putative sister taxon represented by ZD1528), and two unnamed species represented by samples from North America GAL3083 and WC912, respectively. The latter, originally identified as A. diminutivus is closely related or could belong to A. kerriganii. The tropical clades TRV, TRVI, and TRVII previously revealed in the phylogenetic analysis of Zhao et al. (2011) were distributed in clades A-III, A-IV, and A-X, respectively.
In the MCC tree, clades A-I to A-X were well supported (PP ≥ 0.99; Fig. 1) but the samples of clade XI were distributed in several clades forming a group paraphyletic to clade A-X. Species are distributed in the 11 clades as in the multi-gene and ITS ML trees except one sample [57] ZRL3102 which appears to group with A. huijsmanii. The estimated mean stem and crown ages of A. sect. Minores were 30.06 and 24.19 Ma. In this section the broadest clade TRVII diverged relatively late, since its estimated stem and crown ages were 14.30 and 10.63 Ma, respectively.
In the ITS ML tree (Fig. 3), A. sect. Minores and the same 11 major clades were recovered, but were phylogenetically poorly supported, except the four clades A-III, -IV, -VI, and -X. Agaricus laeticulus clustered in A. sect. Minores, showing close affinities with two undescribed species in clade A-III.
Phylogenetic species recognition
In total, 60 species-level groups were recognized belonging to A. sect. Minores based on the combined dataset using the ABGD method. In addition, 22 species-level groups were recognized among 45 specimens for which only ITS sequence data are available. A more accurate species circumscription was performed in some groups of closely related samples exhibiting highly polymorphic ITS sequences including insertions, deletions and heteromorphisms, which are not taken in consideration in phylogenetic analyses. The distribution of putative alleles at such variable positions of the ITS alignment was examined in detail in the three following groups:
Collections NTF63 and ZRL3080 which were recognized as two entities in the ITS dataset, appear to belong to the same putative species. The two sequences differed at six positions, of which four were heteromorphic in ITS sequence of NTF63 but in each case one of the two nucleotides was also present in ZRL3080; indeed, these two samples which differ at only two positions and share putative alleles at four other positions are likely to be the same species.
In a group of nine samples (SCK121, NTF26, LD2012113, SCK099, SCK120, LD2012120, NTSCR1, SCK138, and ZRL3088) of which seven having non-redundant sequences were included in the ITS analysis, NTSCR1 and ZRL3088 formed a clade which might represent a distinct entity in the phylogenetic tree of Fig. 3. Polymorphisms were detected at 13 positions among the nine samples (Table 3). However, at 12 of the 13 polymorphic positions, heteromorphisms were found in one to four samples. Taking into account the heteromorphic positions, NTSCR1 and ZRL3088 do not have any characteristic alleles. Only the sample SCK121 differs from all the other samples by a characteristic allele at position 489. Therefore, we consider that the polymorphism among this group of samples likely reflects allelic diversity within a single species.
Table 3
Polymorphisms at 13 positions within ITS rDNA sequences of nine samples of Agaricus luteopallidus.
Sample Positions in the ITS alignment (657 nts) 109 145 181 198 201 207 231 489 498 511 545 625 630 NTF26 C T A G T C C T T T G T C LD2012113 C T A G T C C T C C A T C SCK099 C T R G K C C T Y Y R T C SCK120 C Y R K K Y M T Y Y R Y Y LD2012120 C Y R K K Y M T Y Y R Y Y NTSCR1 M C R K K T M T Y Y R Y Y SCK138 C C G T G T A T T T G C T ZRL3088 C C G T G T A T T T G C T SCK121 A C A G T T C C T C G T C Note Heteromorphisms: M: A and C; K: G and T; R: A and G; Y: C and T. Characters are in bold types when a nucleotide is shared with the specimen SCK121.
Agaricus luteofibrillosus is represented by six samples (species number 81) within clade A-XI of Fig. 3. They formed a polytomy with a clade containing three samples of the entity numbered 80. The phylogenetic relationships between the two entities remained poorly resolved likely due to insertions, deletions and heteromorphisms. The variability observed at 16 polymorphic positions among the two entities is reported in Table 4. Three samples of A. luteofibrillosus were not included in the phylogenetic analysis because their sequences were redundant. The three samples of entity 80 clearly differ from the 11 samples of A. luteofibrillosus at the four positions 511, 545, 625, and 630. Moreover, the polymorphism at the eight positions 38, 87, 138, 202, 235, 257, 259, and 481 was observed only among the samples of the putative new species 80, while the polymorphism at the four remaining positions 23, 26, 204, and 468 was found only among the samples of A. luteofibrillosus. Knowing that samples differing at more than two positions generally belong to different species in genus Agaricus (Zhao et al. 2011), the clade containing the three samples MS549, MS316, and LD201404 is regarded as a distinct species from A. luteofibrillosus.
Table 4
Characters at 16 polymorphic positions within ITS rDNA sequences of 14 samples of A. fulvoaurantiacus and A. luteofibrillosus.
Sample Positions in the ITS alignment (667 nts) reflecting variability Within A. fulvoaurantiacus Within A. luteo Interspecific 38 87 138 202 235 257 259 481 23 26 204 468 603 632 634 646 A. fulvoaurantiacus MS549 T C C T C A A A T T – A – C C A MS316 C C Y T C K A G T T – A – C C A LD201404 T T C K Y K W R T T – A – C C A A. luteofibrillosus LD201501 T C T T C T A G T T T A G T – G ZRL2110 T C T T C T A G K K – N G T – G ZRL3039 T C T T C T A G T T T G G T – G NTT37 T C T T C T A G T T – G G T – G ZRL2013484 T C T T C T A G T T – R G T – G ZRL2012200 T C T T C T A G T T – R G T – G ZRL2014136 T C T T C T A G T T – A G T – G ZRLWXH3112 T C T T C T A G T T – G G T – G ZRLWXH3183 T C T T C T A G T T – G G T – G ZRL2012121 T C T T C T A G T T – G G T – G ZRL2012359 T C T T C T A G T T – G G T – G Note Heteromorphisms: K: G and T; R: A and G; W: A and T; Y: C and T. N: unidentified nucleotide (A, T, G or C). –: absent nucleotide.
After these adjustments, 81 phylogenetic species were ultimately recognized and numbered in A. sect. Minores (Fig. 3). Among them, 44 are named species including ten newly described in this study and 37 taxa remain unnamed either because their sequences were retrieved from GenBank or because our material and/or morphological/macrochemical information were insufficient.
Geographic distribution of species of A. subg. Minores and A. subg. Minoriopsis
Geographic distribution of the species ordered by subgenera, sections and sectional subclades are summarized in Table 5. We first note that all species of A. subg. Minoriopsis are from the Americas, mostly from tropical or subtropical areas.
Table 5
Geographic distribution of 94 species of Agaricus subg. Minores and A. subg. Minoriopsis.
| Subgenus | Section | Subclade | Number of species | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Total | EURa | GMSa | ASIa | AFRa | AMEa | AUSa | |||
| Minores | Minores | A-I | 4 | 0 | 2 | 1 | 1 | 0 | 0 |
| A-II | 5 | 0 | 1 | 0 | 0 | 0 | 4 | ||
| A-III | 9 | 0 | 8 | 0 | 1 | 0 | 0 | ||
| A-IV | 4 | 0 | 4b | 0 | 0 | 0 | 0 | ||
| A-V | 2 | 0 | 2 | 0 | 0 | 0 | 0 | ||
| A-VI | 3 | 0 | 0 | 0 | 0 | 1 | 2 | ||
| A-VII | 20 | 16 | 2 | 0 | 0 | 2 | 0 | ||
| A-VIII | 3 | 0 | 2 | 1 | 0 | 0 | 0 | ||
| A-IX | 6 | 0 | 0 | 2 | 0 | 1 | 3 | ||
| A-X | 5 | 0 | 4 | 0 | 1 | 0 | 0 | ||
| A-XI | 7 | 0 | 7 | 0 | 0 | 0 | 0 | ||
| Ungrouped | 13 | 3 | 6 | 0 | 1 | 2 | 1 | ||
| Total. (Min.) | 81 | 19 | 38 | 4 | 4 | 6 | 10 | ||
| A. sect. 1 | 4 | 0 | 1 | 1 | 0 | 2 | 0 | ||
| Leucocarpi | 1 | 0 | 1 | 0 | 0 | 0 | 0 | ||
| Minoriopsis | 8 | 0 | 0 | 0 | 0 | 8 | 0 | ||
Notes a EUR = Europe; GMS = the Greater Mekong Subregion; ASI = Asia (China); AFR = Africa; AME = Americas; AUS = Australasia.
b One of the four is also found in Malaysia and in Africa.
In A. sect. Minores most of the species included in this study are from Europe (19) or GMS (38) and among the 24 remaining species, 10 are from Australasia. Among the 19 European species, three are ungrouped and 16 (84 %) are in the same clade (A-VII). Clade A-VII also includes two non-tropical sister species from GMS (Yunnan) [54, 55] and two others from North America [41, 45]. Clade A-VII is a core clade of the section: it contains most of the European species including the type A. comtulus and it does not contain any tropical species. In contrast, 32 of the 38 GMS species of A. sect. Minores, mostly from tropical area in Thailand, are distributed in nine of the 11 subclades, while the six remaining species are ungrouped.
Nine samples belonging to eight secotioid species were included in our studies. One is A. inapertus, a species of A. sect. Arvenses, while the seven remaining species were considered or suspected to belong to A. sect. Minores (Zhao et al. 2011, Lebel & Syme 2012, Lebel 2013, Bates et al. 2016). Our ITS ML tree not only confirms that the seven species belong to A. sect. Minores, but also, as in Lebel (2013), that four Australian species (A. chartaceus, A. lamelliperditus, A. wariatodes and A. cf. wariatodes) are closely related to each other, while the fifth Australian species A. colpetei (‘colpeteii’) is related to the European species A. aridicola. Representatives of these two groups (A. aridicola and A. wariatodes) were included in the MCC tree and appear in Fig. 1 to be in the same moderately supported clade (PP = 0.82). However, the multi-gene ML tree does not confirm this result and the position of the seventh species, A. columellatus from the USA remains uncertain in the ITS ML tree. Therefore, we cannot conclude that the seven secotioid species are closely related, but also this hypothesis cannot be excluded.
TAXONOMY
Here we present descriptions of 13 new taxa (one subgenus, one section, and 11 species from the Greater Mekong Subregion). In addition we propose one new combination and one new species record from Thailand. Generally speaking, within A. sect. Minores, the number of morphological characters which are available for species distinction is scarce, usually with a large level of overlap between closely related species. In some cases, unequivocal identification of individual collections would not be possible without molecular data.
Agaricus subgenus Minoriopsis Linda J. Chen, L.A. Parra, Callac, Angelini & Raspé, subg. nov. — MycoBank MB818040
Facesoffungi number. FoF 02280.
Type. Agaricus martinicensis Pegler, Kew Bull., Addit. Ser. 6: 446. 1983.
Etymology. Referring to the similarities to A. subg. Minores.
Original description and delimitation of Agaricus subg. Minoriopsis — Schäffer’s reaction immediately and strongly positive dark reddish purple, rarely reddish brown and KOH difficult to observe but positive yellow when observable, on the pileus surface of dried specimens. Odour of anise or of bitter almonds when rubbed or cut. Annulus superous, thick at the margin, double, fibrillose squamose or, sometimes with squames radially arranged as a cogwheel near the margin in its lower surface, which, under the microscope is composed only by thin cylindrical hyphae. Cheilocystidia generally simple or with a septum at the base, clavate, pyriform, more or less globose, fusiform, sometimes rostrate or absent in some specimens. Spores lacking a rudimentary apical pore.
Stem age and phylogenetic support — In the MCC tree (Fig. 1 and Table 2), the clade corresponding to A. subg. Minoriopsis has a stem age of 31.02 Ma and is well supported (PP ≥ 0.99). It has 98/90 bootstrap support (ML/MP) in multi-gene phylogenetic analysis (Fig. 2). In the present analyses, A. subg. Minoriopsis includes five to eight species distributed in two sister subclades, which potentially represent respectively two sections since they diverged 22.83 Ma ago.
Agaricus subgenus Minores (Fr.) R.L. Zhao & Moncalvo, Fung. Diversity 78: 257. 2016
Type. Agaricus comtulus Fr. designated by Heinemann, Bull. Jard. Bot. État Bruxelles 26: 42. 1956.
Agaricus [subg. Minores] section 1
Clade B (Clade A2 in Zhao et al. 2016). Four specimens (LD2012129/A. candidolutescens, LAPAM14, LAPAM45 and ZRLWXH3161) cluster together (Fig. 3) in clade B, sister to A. sect. Leucocarpi. Since some important morphological data are lacking, we refrain from describing this section here.
Agaricus [subg. Minores] section Leucocarpi Linda J. Chen & Callac, sect. nov. — MycoBank MB818041
Facesoffungi number. FoF 02281.
Type. Agaricus leucocarpus Linda J. Chen, Callac, R.L. Zhao & K.D. Hyde.
Etymology. The epithet ‘Leucocarpi’ is following the name of the type A. leucocarpus.
Original description and delimitation of Agaricus sect. Leucocarpi — Schäffer’s reaction negative, KOH reaction positive. Surface of basidiomes often flavescent when rubbed. Odour of almonds. Annulus superous, membranous, smooth on both sides. Cheilocystidia present, simple, pyriform or broadly clavate.
Stem age and phylogenetic support — In the MCC tree (Fig. 1 and Table 2), A. sect. Leucocarpi has a stem age of 27.64 Ma and is well supported (PP ≥ 0.99). It has strong bootstrap support (ML/MP) in multi-gene phylogenetic analyses (Fig. 2).
Agaricus leucocarpus Linda J. Chen, Callac, R.L. Zhao & K.D. Hyde, sp. nov. — MycoBank MB818042; Fig. 4, ,55
Agaricus leucocarpus. a. Overall morphology in situ (SCK089); b. appendiculate margin (holotype LD201215); c. lamellae when mature (LD201507); d. overall morphology (LD201226); e. lamellae when young (LD201226).
Microscopic characters of Agaricus leucocarpus. a. Cheilocystidia; b. basidia; c. basidiospores. — Scale bars = 5 μm.
Facesoffungi number. FoF 02282.
Etymology. The epithet ‘leucocarpus’ refers to the white sporocarp of this species.
Pileus 2.5–4 cm diam, 1–3 mm thick at disc; at first parabolic, becoming hemispherical to plano-convex, finally applanate; surface dry, smooth, completely white with light brownish or ochre tinges at the disc. Margin straight, not exceeding the lamellae, often with appendiculate remains of the annulus. Lamellae free, crowded, 3 mm broad, ventricose, with intercalated lamellulae, at first white, turning pinkish when touched, then pink to greyish brown, finally brown. Stipe 40–65 × 2–5 mm (5–9 mm at base), cylindrical with a subbulbous base, fistulose, surface smooth both above and below the annulus, white, flavescent when rubbed or by handing. Annulus simple, superous, membranous, white, fragile. Context firm, white, unchanging when cut. Odour of almonds.
Spores (4.3–)4.5–5 × 3–3.5 μm, (x = 4.7 ± 0.17 × 3.2 ± 0.12 μm, Q = 1.32–1.61, Qm = 1.48 ± 0.02, n = 20), ellipsoid, smooth, brown, thick-walled. Basidia 11–14 × 6.5–8 μm, broadly clavate, hyaline, smooth, 4-spored. Cheilocystidia (11–)18.5–26 × 6–15 μm, simple, pyriform or broadly clavate, hyaline, smooth. Pleurocystidia absent. Pileipellis a cutis composed of hyphae of 4–8 μm wide, cylindrical, not or slightly constricted at the septa, hyaline.
Macrochemical reactions — KOH reaction positive, yellow. Schäffer’s reaction negative on dry specimen.
Habitat — Solitary on soil, in grassland of roadside; or scattered on leaf litter mixed with compost.
Material examined. THAILAND, Chiang Rai Prov., Mae Fah Luang University gate, 27 July 2015, J. Chen, LD201507; Chiang Mai Prov., Tharnthong Lodges, 31 May 2012, J. Chen, LD201215 (holotype MFLU12-0859); Chiang Mai Prov., Tharnthong Lodges, 3 June 2012, J. Chen, LD201226 (MFLU12-0870); Chiang Rai Prov., Bandu, 31 July 2011, S.C. Karunarathna, SCK089 (MFLU11-1283).
Notes — Agaricus leucocarpus is a species morphologically well characterized by its slender, pure white sporocarps, with a brownish tinge at disc, small spores and simple cheilocystidia. Considering its morphology, discoloration when rubbed and the almond smell, it is very likely to be a member of A. sect. Minores. However, it shows negative Schäffer’s reaction, which is in disagreement with A. sect. Minores. Among the other known sections, possibly related to A. sect. Minores, A. sect. Lanosi is characterized by negative Schäffer’s reaction, and A. haematosarcus, is the only species showing pure white sporocarps. But it can easily be distinguished from A. leucocarpus by its woolly pileus and stipe surface, and strong reddening when cut (Heinemann 1956, Parra 2013). Since the attempts at sequencing the type of A. sect. Lanosi failed, in the absence of sequence data from any species of the section, and because the new species does not exhibit any woolly veil, which is a main character of this section, we have no reason to place A. leucocarpus in A. sect. Lanosi. According to the phylogenetic analyses, A. leucocarpus corresponds to clade C, which constitutes A. subg. Minores (Fig. 2) with the two clades A (A. sect. Minores) and B.
Agaricus [subg. Minores] section Minores (Fr.) Henn. in Engler & Prantl, Nat. Pflanzenfam. 1(1**): 238. 1898
≡ Agaricus [unranked] Minores Fr., Hymenomyc. Eur.: 281. 1874.
Type. Agaricus comtulus Fr., designated by Heinemann (1956) 42.
= Agaricus sect. Laeticolores Heinem., Kew Bull. 15(2): 144. 1961.
Type. Agaricus laeticulus Callac, L.A. Parra, Linda J. Chen & Raspé, nom. nov. — MycoBank MB818070.
Etymology. A composite word from the Latin laetus meaning cheerful, pleasant, bright and the suffix -culus denoting diminutive. Thus, laeticulus is ‘the little bright’.
Agaricus laeticulus Callac, L.A. Parra, Linda J. Chen & Raspé, is a replacement name for Agaricus laeticolor Heinem. & Gooss.-Font., Bull. Jard. Bot. État 26: 42. 1956, an illegitimate name because of the existence of the earlier homonym Agaricus laeticolor Lév., Icon. Champ. Paulet: 36. 1855.
Agaricus badioniveus Linda J. Chen, R.L. Zhao & K.D. Hyde, sp. nov. — MycoBank MB818047; Fig. 6, ,77
Agaricus badioniveus (holotype LD2012131). a. Pileus surface; b. lamellae and stipe.
Agaricus badioniveus (holotype LD2012131). a. Cheilocystidia; b. basidia; c. basidiospores. — Scale bars: c = 10 μm, d–e = 5 μm.
Facesoffungi number. FoF 02283.
Etymology. Refers to the pileus with tawny fibrils on a white background.
Pileus 3.5 cm diam, 3 mm thick at disc; convex and truncated at disc; surface dry, with yellowish brown fibrils, densely at disc, and progressively sparse towards the margin, on a white background. Margin straight, not exceeding the lamellae, with appendiculate remains of the annulus. Lamellae free, crowded, 3 mm broad, with intercalated lamellulae, ventricose, pinkish to brown with time. Stipe 45 × 7 mm (12 mm at base), cylindrical with a bulbous base, surface above the ring smooth, below the ring fibrillose, white, strongly flavescent when bruised. Annulus simple, membranous, superous, white, fragile. Context firm, white, flavescent when cut. Odour of strong almonds.
Spores (5–)5.4–5.8(–6.2) × 3.1–3.5(–3.8) μm, (x = 5.6 ± 0.12 × 3.3 ± 0.11 μm, Q = 1.54–1.86, Qm = 1.67 ± 0.01, n = 20), ellipsoid, smooth, brown, thick-walled. Basidia 15–19 × 6.5–7 μm, clavate to broadly clavate, hyaline, smooth, 4-spored. Cheilocystidia 23–35(–40) × 9–12(–16) μm, abundant, simple, or septate at base, pyriform, clavate or narrowly clavate, with yellowish pigments, smooth. Pleurocystidia absent. Pileipellis a cutis constituted of cylindrical hyphae of 6–9 um wide, not or slightly constricted at the septa, with brownish pigment.
Macrochemical reactions — KOH reaction positive, yellow. Schäffer’s reaction positive, reddish orange on dry specimen.
Habitat — Solitary on soil, in forest.
Material examined. THAILAND, Chiang Rai Prov., Doi Pui site1, 25 July 2012, J. Chen, LD2012131 (holotype MFLU12-0964).
Notes — Agaricus badioniveus is characterized by a pileus surface covered with yellowish brown fibrils, simple cheilocystidia and spores on average size of 5.6 × 3.3 μm.
In gross morphology, A. badioniveus is highly similar to A. megalosporus. However, the latter species has larger sporocarps (the pileus diameter can reach 10 cm) and spores (6 × 3.5 μm on average, Chen et al. 2012). According to phylogenetic results, A. badioniveus is closely related to A. flammicolor, a species easily distinguished by its bright orange colour.
Agaricus brunneolutosus Linda J. Chen, Karun. & K.D. Hyde, sp. nov. — MycoBank MB818048; Fig. 8, ,99
Microscopic characters of Agaricus brunneolutosus. a. Cheilocystidia; b. basidia; c. basidiospores; d. pileipellis. — Scale bars: a = 10 μm, b–c = 5 μm, d = 20 μm.
Agaricus brunneolutosus. a–b. Overall morphology in situ (holotype MS514).
Facesoffungi number. FoF 02284.
Etymology. Refers to the brown yellow colour of the pileus.
Pileus 5.5–8.5 cm diam, 3–5 mm thick at disc; convex to applanate, or uplifted; surface dry, covered with brown fibrils, densely at disc and radially arranged elsewhere, somewhat sparse towards the margin, on a white to yellowish white background. Margin straight, shortly exceeding the lamellae, often with appendiculate remains of the annulus. Lamellae free, crowded, 3–5 mm broad, with intercalated lamellulae, at first white, then pinkish brown, finally dark brown. Stipe 70–110 × 7–10 (10–12 at base) mm, clavate or tapering upwards, fistulose, surface above the ring smooth, below fibrillose, white, flavescent when bruised. Annulus simple, superous, membranous, upper surface smooth, lower surface fibrillose, white, changing to yellowish with time or when rubbed. Odour of almonds. Context firm, discoloration when cut not recorded.
Spores 3.9–4.7(–5.2) × 2.7–3.3 μm, (x = 4.3 ± 0.22 × 2.9 ± 0.14 μm, Q = 1.32–1.65, Qm = 1.48 ± 0.03, n = 20), ellipsoid, smooth, brown, thick-walled. Basidia 13–18 × 5.5–8 μm, clavate to broadly clavate, hyaline, smooth, 4-spored. Cheilocystidia 17–42 × 9–15 μm, abundant, simple, pyriform to broadly clavate, hyaline or with yellowish pigments, smooth. Pleurocystidia absent. Pileipellis a cutis composed of hyphae of 5–13 μm wide, cylindrical, hyaline or with light yellow pigments, smooth, sometimes constricted at the septa; terminal elements observed cylindrical, 13–25 μm wide, with rounded or attenuate apex.
Macrochemical reactions — KOH reaction positive, yellow. Schäffer’s reaction positive, reddish orange on dry specimen.
Habitat — Solitary on soil, in forest dominated by Casternopsis and Lithocarpus.
Material examined. CHINA, Yunnan Prov., Mengsong, 7 July 2012, S.C. Karunarathna, MS514 (holotype MFLU16-0976; isotype HMAS279153); Yunnan Prov., Mengsong, 10 July 2012, S.C. Karunarathna, MS541 (MFLU16-0977, HMAS279154).
Notes — Agaricus brunneolutosus is distinguished by its yellowish white pileus, entirely covered with brown fibrils, small spores on average size of 4.3 × 2.9 μm, large cheilocystidia and the pileipellis hyphae with terminal elements 13–25 μm wide.
Among the members of A. sect. Minores, few species have spores on average shorter than 5 μm: A. comtulus, A. dulcidulus, A. edmondoi, A. entibigae, A. friesianus, A. matrum, and A. pallens. However, A. comtulus (Parra 2013) and A. entibigae (Peterson et al. 2000) have wider spores. The remaining taxa usually have pinkish, reddish pink or reddish purple fibrils on the pileus disc, and are white elsewhere. Additionally, they are well separated by molecular data (Fig. 2). Agaricus brunneolutosus forms a sister clade with A. fulvoaurantiacus and A. luteofibrillosus, however, the two latter species have larger spores (larger than 5 × 3 μm on average).
Agaricus fimbrimarginatus Linda J. Chen, Callac & K.D. Hyde, sp. nov. — MycoBank MB818049; Fig. 10, ,1111
Microscopic characters of Agaricus fimbrimarginatus. a. Cheilocystidia; b. basidia; c. basidiospores. — Scale bars = 5 μm.
Agaricus fimbrimarginatus (holotype LD201250). a. Pileus surface; b. lamellae and stipe.
Facesoffungi number. FoF 02285.
Etymology. Refers to the appendiculate remains on the pileus margin.
Pileus 4 cm diam, 3 mm thick at disc; applanate and slightly depressed at disc; surface dry, with purplish fibrils, densely at disc, radially arranged elsewhere, and sparse towards the margin, on a dirty white background. Margin straight, shortly exceeding the lamellae, with appendiculate remains of the annulus. Lamellae free, crowded, 3 mm broad, with intercalated lamellulae, ventricose, pinkish to brown with time. Stipe 47 × 7–8 mm, cylindrical with a slightly bulbous base, surface above the ring smooth, below the ring fibrillose, white, strongly flavescent when bruised. Annulus simple, membranous, superous, white, fragile. Context firm, white, flavescent when cut. Odour strong of almonds.
Spores (4.4–)4.5–4.9 × (2.9–)3–3.3 μm, (x = 4.7 ± 0.11 × 3.2 ± 0.09 μm, Q = 1.36–1.59, Qm = 1.46 ± 0.01, n = 20), ellipsoid, smooth, brown, thick-walled. Basidia 12–17 × 5–6 μm, clavate to broadly clavate, hyaline, smooth, 4-spored, rarely 2-spored. Cheilocystidia 15–26 × 8–12 μm, simple, pyriform or broadly clavate, with yellowish pigments, smooth. Pleurocystidia absent. Pileipellis a cutis, composed of cylindrical hyphae of 4–9 μm wide, not or slightly constricted at the septa, with brownish pigment.
Macrochemical reactions — KOH reaction positive, yellow. Schäffer’s reaction positive, reddish on dry specimen.
Habitat — Solitary on soil, in grassland along roadside.
Material examined. THAILAND, Chiang Mai Prov., Mae Sa, 25 June 2012, P. Callac & J. Chen, LD201250 (holotype MFLU12-0891).
Notes — Agaricus fimbrimarginatus is characterized by a pileus surface covered with purplish fibrils, simple cheilocystidia and small spores less than 3.5 μm wide.
Several members of A. sect. Minores resemble A. fimbrimarginatus by exhibiting a reddish brown to purplish brown, fibrillose pileus surface, such as, A. brunneolus, A. dulcidulus, A. gemlii, A. megalosporus and A. patris. However, A. brunneolus, A. gemlii, A. megalosporus and A. patris are easily distinguished by their larger spores (wider than 3.5 μm on average, Chen et al. 2012, Parra 2013). Agaricus dulcidulus differs in its smaller spores (4.3 × 3 μm on average, Parra 2013).
According to the phylogenetic analyses, A. fimbrimarginatus shows close affinities to A. robustulus. However, the latter species differs in its robust sporocarps and larger spores and molecularly has four nucleotides in difference in the ITS sequences, two differences in LSU and with more than 20 differences in tef-1α sequences.
Agaricus flammicolor Linda J. Chen, Callac, R.L. Zhao & K.D. Hyde, sp. nov. — MycoBank MB818050; Fig. 12, ,1313
Microscopic characters of Agaricus flammicolor. a. Cheilocystidia; b. basidia; c. basidiospores. — Scale bars: a = 10 μm; b–c = 5 μm.
Agaricus flammicolor. a. Overall morphology in situ (holotype LD201502), coin = 24 mm diam; b. pileus surface (holotype LD201502); c. lamellae and stipe surface (ZRL2012270).
Facesoffungi number. FoF 02286.
Etymology. The epithet ‘flammicolor’ refers to the orange colour like a flame.
Pileus 4–7 cm diam, 2–4 mm thick at disc, at first parabolic, sometimes truncated at disc, then becoming hemispherical to convex, finally applanate; surface dry, with bright orange fibrils, densely at disc and radially arranged elsewhere, sometimes with fibrils bunching together into finely squamules, on a white background; strongly flavescent when bruised. Margin incurved, shortly exceeding the lamellae, often with appendiculate remains of the annulus. Lamellae free, crowded, 3–4 mm broad, with intercalated lamellulae, first white, then pinkish brown, finally dark brown. Stipe 50–87 × 4–6 mm (8–12 mm at base), clavate or cylindrical with slightly bulbous base, fistulose, surface above the ring smooth, below the ring heavily fibrillose, white, strongly flavescent when rubbed. Annulus simple, superous, membranous, upper surface smooth, lower surface fibrillose, white, except sometimes with orange tinge close to the margin at the lower surface. Odour of almonds. Context firm, white, slightly yellowish at stipe when cut.
Spores 4.4–5.3(–6.2) × 2.5–3.2 μm, (x = 4.9 ± 0.25 × 2.9 ± 0.15 μm, Q = 1.53–1.91, Qm = 1.69 ± 0.04, n = 20), ellipsoid to oblong, smooth, brown, thick-walled. Basidia 12–16 × 5–6 μm, broadly clavate, hyaline, smooth, 4-spored. Cheilocystidia 21–45 × 10–25 μm, abundant, simple, pyriform, broadly clavate or sphaeropedunculate, rarely rostrate or mucronated, with yellowish pigment, smooth. Pleurocystidia absent. Pileipellis a cutis composed of hyphae of 5–7 μm diam, cylindrical, hyaline, smooth, not constricted at the septa.
Macrochemical reactions — KOH reaction positive, bright yellow. Schäffer’s reaction positive, bright orange.
Habitat — Solitary or scattered, on soil, under an Albizia tree.
Material examined. CHINA, Yunnan Prov., Cangyuan county, 11 July 2012, P. Callac & J. Guinberteau, ZRL2012270 (HMAS279148). – THAILAND, Chiang Rai Prov., Mae Fah Luang University, 28 July 2015, J. Chen, LD201502 (holotype MFLU16-0982); Chiang Mai Prov., Thang Thong village, 31 June 2012, J. Chen, LD201225 (MFLU12-0869).
Notes — Agaricus flammicolor is well characterized by a pileus surface covered with bright orange fibrils or fine squamules, spores on average less than 3 μm in width, and with simple and large cheilocystidia containing yellowish pigments. Among the known taxa of A. sect. Minores, species with a pileus surface showing orange tinges are very rare. Agaricus entibigae, originally described from Hawaii, also has a pale orange to brownish orange pileus, but it differs in having its stipe surface base covered with reddish squamules, wider spores (3.8 μm on average) and smaller cheilocystidia (Peterson et al. 2000). According to the phylogenetic results (Fig. 2), A. flammicolor is closely related to A. badioniveus/LD2012131, another new species treated in this study.
Agaricus flavopileatus Linda J. Chen, Karun. & Callac, sp. nov. — MycoBank MB818051; Fig. 14, ,1515
Microscopic characters of Agaricus flavopileatus. a. Cheilocystidia; b. basidia; c. basidiospores. — Scale bars = 5 μm.
Agaricus flavopileatus. a. Overall morphology in situ (holotype MS596); b. lamellae and strong yellowing when bruised on stipe surface (holotype MS596); c. pileus surface (holotype MS596); d. overall morphology in situ (MS603); e. pileus surface (MS603).
Facesoffungi number. FoF 02287.
Etymology. The epithet ‘flavopileatus’ refers to the yellow pileus.
Pileus 4–6 cm diam, 3–4 mm thick at disc; at first parabolic, then hemispherical to plano-convex, truncate or slightly depressed at disc, finally applanate; surface dry, covered with greyish yellow to yellow ochre fibrils or squamules, densely at disc and radially or concentrically arranged elsewhere, on a white to yellowish white background; sometimes squamules are not uniformly distributed on pileus surface. Margin straight, shortly exceeding the lamellae, often with appendiculate remains of the annulus. Lamellae free, crowded, 3–4 mm broad, with intercalated lamellulae, pink to brown, finally chocolate brown. Stipe 30–75 × 4–12 mm, clavate or tapering upwards, with rhizomorphs, fistulose, surface above the ring smooth, below the ring fibrillose, white, strongly flavescent when bruised. Annulus single, membranous, superous, white, upper surface smooth, lower surface fibrillose. Context firm, white. Odour of almonds.
Spores 4.6–5.2(–5.3) × (2.6–)2.7–3.3(–3.4) μm, (x = 4.8 ± 0.13 × 2.9 ± 0.15 μm, Q = 1.42–1.87, Qm = 1.65 ± 0.1, n = 20), ellipsoid, smooth, brown, thick-walled. Basidia 11–17 × 4–6 μm, clavate, hyaline, smooth, 4-spored. Cheilocystidia 14–28 × 5–18 μm, abundant, simple, pyriform to broadly clavate, or sphaeropedunculate, with yellowish pigments, smooth. Pleurocystidia absent. Pileipellis a cutis composed of hyphae of 4–14 μm wide, cylindrical, hyaline or with yellowish brown pigments, smooth, sometimes constricted at the septa.
Macrochemical reactions — KOH reaction positive, yellow. Schäffer’s reaction positive, reddish orange on dry specimen.
Habitat — Solitary or scattered on soil, in forest.
Material examined. CHINA, Yunnan Prov., Mengsong, 21 July 2012, S.C. Karunarathna, MS596 (holotype MFLU16-0984); Yunnan Prov., Mengsong, 22 July 2012, S.C. Karunarathna, MS603 (MFLU16-0983, HMAS279150).
Notes — Agaricus flavopileatus is morphologically well characterized by the white to yellowish white pileus, radially or concentrically covered with greyish yellow to yellowish brown fibrils or squamules, small spores and the simple, pyriform, broadly clavate, or sphaeropedunculate cheilocystidia.
Comparing with other members of A. sect. Minores, which sometimes also have a yellowish to ochre pileus surface, A. flavopileatus can be distinguished as follows: A. azoetes and A. pseudolutosus have larger spores, with an average of 6.37 × 4.78 μm and 5.7 × 4.3 μm, respectively (Peterson et al. 2000, Parra 2013); A. comtulus has wider spores, on average 4.87 × 3.55 μm (Parra 2013), and phylogenetically, it is quite distant from A. flavopileatus (Fig. 2); A. luteoflocculosus differs in having larger spores (5.95 × 4.1 μm on average), the lower side of the annulus is floccose and stipe surface has fibrillose woolly scales (Parra 2013).
Agaricus fulvoaurantiacus Linda J. Chen & Karun., sp. nov. — MycoBank MB818052; Fig. 16, ,1717
Microscopic characters of Agaricus fulvoaurantiacus. a. Cheilocystidia; b. basidia; c. basidiospores. — Scale bars = 5 μm.
Agaricus fulvoaurantiacus. a–b. Overall morphology in situ (holotype LD201404); c. scales on pileus surface (MS316); d. fibrils on pileus surface (MS549); e. annulus (MS549).
Facesoffungi number. FoF 02288.
Etymology. Refers to the tawny orange colour of the pileus.
Pileus 3.7–7 cm diam, 3–5 mm thick at disc, at first parabolic, then convex or plano-convex, finally applanate; surface dry, with light brownish yellow to brownish orange fibrils, densely at disc and radially arranged elsewhere, or sometimes squamose with appressed squamules or thick scales, against a white background. Margin incurved, shortly exceeding the lamellae, often with appendiculate remains of the annulus. Lamellae free, crowded, 2–5 mm broad, with intercalated lamellulae, first white, then pinkish brown, finally dark brown. Stipe 50–70 × 6–8 mm (11 mm at base), clavate, with numerous rhizomorphs, fistulose, surface above the ring smooth, below the ring with light yellowish brown appressed fibrillose scales, white, strongly flavescent when bruised. Annulus simple, superous, membranous, white, upper surface smooth, lower surface decorated with tiny yellowish flakes, connected with the stipe by cortinate fibrils. Odour of almonds. Context firm, white, flavescent when cut.
Spores (5.2–)5.6–6.1 × 3.5–4.1 μm, (x = 5.8 ± 0.22 × 3.8 ± 0.18 μm, Q = 1.26–1.73, Qm = 1.51 ± 0.01, n = 20), ellipsoid, smooth, brown, thick-walled. Basidia 16–18 × 6–8 μm, clavate to broadly clavate, hyaline, smooth, 4-spored, rarely 2-spored. Cheilocystidia (12–)17–30 × 9–13 μm, abundant, simple, pyriform, broadly clavate or sphaeropedunculate, hyaline or with yellowish pigments, smooth. Pleurocystidia absent. Pileipellis a cutis composed of hyphae of 6–12 μm diam, cylindrical, hyaline or with light yellow pigments, smooth, rarely constricted at the septa.
Macrochemical reactions — KOH reaction positive, bright yellow. Schäffer’s reaction positive, bright orange.
Habitat — Solitary or gregarious on soil, in forest or tea plantations.
Material examined. CHINA, Yunnan Prov., Mengsong, 23 June 2012, S.C. Karunarathna, MS316 (MFLU16-0974, HMAS279151); Yunnan Prov., Mengsong, 11 July 2012, S.C. Karunarathna, MS549 (MFLU16-0978, HMAS279152); Yunnan Prov., Mengsong, 3 July 2014, J. Chen, LD201404 (holotype MFLU16-0980; isotype HMAS279149).
Notes — Agaricus fulvoaurantiacus is well characterized by a pileus surface covered with light brownish yellow to brownish orange fibrils or fibrillose squamules, concolorous fibrillose scales on the lower stipe surface, an annulus with tiny yellowish flakes on the lower surface, spores on average 5.8 × 3.8 μm, and the simple cheilocystidia, hyaline or containing yellowish pigments.
Generally speaking, A. fulvoaurantiacus is very similar to A. luteofibrillosus by having the same appearance of pileus and stipe. However, A. luteofibrillosus has narrower spores (5.8 × 3.2 μm on average) and different cheilocystidia which are sometimes in short chains (see A. luteofibrillosus below) or septate at the base (Li et al. 2016). According to the phylogenetic analyses (Fig. 2), they are closely related. Indeed, A. fulvoaurantiacus differs at four positions in ITS sequences, one position in LSU (except MS316 which is heteromorphic (C and T) at this position), and six positions in tef-1α sequences.
Macromorphologically, A. luteoflocculosus roughly resembles A. fulvoaurantiacus by having the bright yellow fibrillose scales on both pileus and stipe surface. However, it differs by its smaller spores (5.1 × 3.7 μm on average) and the habitat on rotting seaweed of the species Fucus vesiculosus on the sea shore (Parra 2013).
Agaricus luteofibrillosus M.Q. He, Linda J. Chen & R.L. Zhao, Fung. Diversity 78: 126. 2016 — Fig. 18, ,1919
Agaricus luteofibrillosus. a–c. Overall morphology, coin = 24 mm diam; d. annulus and stipe surface; e. flavescent when bruised.
Microscopic characters of Agaricus luteofibrillosus. a. Cheilocystidia; b. basidia; c. basidiospores. — Scale bars = 5 μm.
Pileus 3–10 cm diam, 4–6 mm thick at disc, at first parabolic, then hemispherical or plano-convex, finally applanate or plano-concave, occasionally with a slightly depressed centre; surface dry, initially and uniformly covered with appressed fibrils of a brownish orange tone and more densely at disc, with pileus expansion, the disc remains unbroken, disrupting into subtle squamules or triangular scales appressed or upturned elsewhere, on a yellowish white background, flavescent when rubbed. Margin incurved or straight, not exceeding the lamellae, often with appendiculate remains of the annulus. Lamellae free, crowded, 3–7 mm broad, with intercalated lamellulae, first white, then brownish orange, finally dark brown. Stipe 40–120 × 3–15 (5–25 at base) mm, abruptly bulbous, or rounded with rhizomorphs, fistulose, surface above the ring smooth, below the ring fibrillose woolly of a brownish orange colour, yellowish discoloration when rubbed. Annulus simple, superous, thick when young, with cortinate fibrils connected with stipe, membranous when mature, fragile, smooth on both surfaces, white, sometimes with brownish orange tinge towards to the margin. Odour of almonds. Context firm, white, discolouring slightly yellowish when cut.
Spores (4.7–)5.1–5.9(–6) × 2.8–3.5(–3.8) μm, (x = 5.4 ± 0.22 × 3.2 ± 0.19 μm, Q = 1.44–1.91, Qm = 1.72 ± 0.01, n = 20), ellipsoid, smooth, brown, thick-walled. Basidia 16–20 × 6–8 μm, clavate to broadly clavate, hyaline, smooth, 4-spored, rarely 2-spored. Cheilocystidia 16–22(–30) × 8–15 μm, abundant, simple or sometimes in short chains (in this case, elements measuring 8–11 × 5–9 μm), globose, pyriform, or sphaeropedunculate, rarely clavate, hyaline, smooth. Pleurocystidia absent. Pileipellis a cutis composed of hyphae of 5–12.5 μm diam, cylindrical, hyaline or with light yellow pigment, smooth, occasionally constricted at the septa.
Macrochemical reactions — KOH reaction positive, bright yellow. Schäffer’s reaction positive, bright orange.
Habitat — Caespitose or gregarious on soil, in Fagaceae and Pinaceae mixed forest.
Material examined. THAILAND, Chiang Rai Prov., Doi Mae Salong, 22 June 2015, J.Z. Sun, LD201501 (MFLU16-0981); Chiang Mai Prov., Tong Jown, 3 Aug. 2005, R.L. Zhao, ZRL2110 (BBH19490, HMAS279140); Chiang Mai Prov., Pathummikaram Temple, 8 June 2006, Tim, ZRL3039 (BBH19545, HMAS279155); Chiang Mai Prov., Doi Suthep, 20 June 2010, K. Wisitrassameewong, NTT037.
Notes — Agaricus luteofibrillosus is a species recently described from China. It is morphologically characterized by its yellowish white pileus surface covered with brownish orange squamules or triangular scales and the stipe with concolour fibrils. Our collections match well with the original diagnosis, except for their slightly smaller spores (5.8 × 3.4 μm on average, Li et al. 2016), which can be considered as intraspecific variation. This is the first record of A. luteofibrillosus from Thailand.
Agaricus luteofibrillosus is most similar to A. fulvoaurantiacus in macro-morphology. The differences between the two species are noted in A. fulvoaurantiacus.
Agaricus luteopallidus Linda J. Chen, Karun., R.L. Zhao & K.D. Hyde, sp. nov. — MycoBank MB818053; Fig. 20, ,2121
Microscopic characters of Agaricus luteopallidus. a. Cheilocystidia; b. basidia; c. basidiospores. — Scale bars: a = 10 μm; b–c = 5 μm.
Agaricus luteopallidus. a. Overall morphology in situ (SCK120); b. overall morphology at laboratory (holotype ZRL3088).
Facesoffungi number. FoF 02289.
Etymology. Refers to the pallid yellow colour of the pileus.
Pileus 3–6 cm diam, 2.5–3 mm thick at disc, conico-truncate when young, then convex to hemispherical, finally applanate; surface dry, with pallid yellow to light brownish yellow fibrils, densely at disc, with pileus expansion, outside the unbroken disc, the surface disrupts into finely triangular scales, on a white background; turning yellowish when rubbed. Margin straight, not exceeding the lamellae, often with appendiculate remains of the annulus. Lamellae free, crowded, ventricose, 3–4 mm broad, with intercalated lamellulae, first pink, then pinkish brown, finally dark brown. Stipe 65–95 × 5–11 mm, cylindrical or with slightly bulbous base, with numerous rhizomorphs, fistulose, surface above the ring smooth, below the ring fibrillose, white, strongly flavescent when bruised or by handing. Annulus simple, superous, cortinate when young, membranous when mature, fragile, white. Context firm, white, unchanging when cut. Odour of almonds.
Spores (4.5–)5–6 × (3–)3.2–4 μm, (x = 5.4 ± 0.36 × 3.6 ± 0.3 μm, Q = 1.38–1.83, Qm = 1.52 ± 0.02, n = 20), ellipsoid, smooth, brown, thick-walled. Basidia 13–20 × 5.5–7 μm, clavate to broadly clavate, hyaline, smooth, 4-spored, rarely 2-spored. Cheilocystidia 14–28 × 10–22 μm, abundant, simple, rarely in short chains, globose to pyriform or sphaeropedunculate, rarely clavate, with yellowish pigments, smooth. Pleurocystidia absent. Pileipellis a cutis composed of hyphae of 4–11 μm diam, cylindrical, with yellowish membranous pigments, smooth, at times slightly constricted at the septa.
Macrochemical reactions — KOH reaction positive, bright yellow. Schäffer’s reaction positive, bright reddish orange.
Habitat — Solitary, scattered or gregarious on soil, in grassland or rotted litter.
Material examined. THAILAND, Chiang Rai Prov., Parnae Lao Park, 2 Aug. 2006, R.L. Zhao, ZRL3088 (holotype BBH19604; isotype HMAS279147); Chiang Mai Prov., Doi Suthep, 29 June 2010, P. Sysouphanthong, NTF26; Chiang Rai Prov., Mae Fah Luang University park, 3 Aug. 2010, S.C. Karunarathna, NTS-CR01 (MFLU10-0674); 5 Mar. 2011, S.C. Karunarathna, SCK099 (MFLU11-1285); 5 June 2011, S.C. Karunarathna, SCK120 (MFLU11-1287); 5 July 2011, S.C. Karunarathna, SCK121 (MFLU11-1287); Chiang Mai Prov., MRC, 13 May 2011, S.C. Karunarathna, SCK138 (MFLU11-1296); Chiang Rai Prov., Mae Fah Luang University, 20 July 2012, J. Chen, LD2012113 (MFLU12-0950); 21 July 2012, J. Chen, LD2012120 (MFLU12-0956).
Notes — Agaricus luteopallidus is well characterized by having a pileus surface covered with pale yellow to light brownish yellow fibrils or triangular squamules, spores 5.4 × 3.6 μm on average, and the simple cheilocystidia containing yellowish pigments.
In general, several species resemble A. luteopallidus by having a pileus surface with yellowish tinge, and later covered with fibrillose scales, such as A. xantholepis, A. azoetes and A. luteoflocculosus. According to the original diagnosis, A. xantholepis, which has been considered as a synonym of A. brunneolus (Parra 2013), exhibits a distinctively bulbous base up to 15 mm broad and has smaller spores, 4–5.5 × 3 μm (Parra 2013). Agaricus azoetes was originally described from Hawaii and can be easily distinguished from A. luteopallidus by its smaller sporocarps not exceeding 4.5 cm, wider basidiospores (5.7 × 4.3 μm on average), lacking of cheilocystidia and the arid habitats (Peterson et al. 2000). Agaricus luteoflocculosus differs from the new species by the floccose on the lower side of the annulus and fibrillose woolly scales on the stipe surface (Parra 2013).
According to the phylogenetic results, A. luteopallidus is closely related to A. flavopileatus. The latter differs at 6 positions in ITS sequences, and more than 20 positions in tef-1α sequences.
Agaricus patris Linda J. Chen, Callac, K.D. Hyde & R.L. Zhao, sp. nov. — MycoBank MB818054; Fig. 22, ,2323
Agaricus patris. a. Pileus surface (holotype LD201224); b. lamellae and stipe (holotype LD201224); c. pileus surface (ZRL3101); d. section view (ZRL3101).
Microscopic characters of Agaricus patris. a. Cheilocystidia; b. basidia; c. basidiospores. — Scale bars = 5 μm.
Facesoffungi number. FoF 02290.
Etymology. This species honours all the fathers in the world but it is written in singular (patris: of the father) with a plural sense because the plural patrum (of the fathers) is very much alike to matrum an epithet already used in Agaricus.
Pileus 4.5–5 cm diam, 3–4 mm thick at disc; convex to applanate; surface dry, covered with purplish brown to reddish brown or dark purple fibrillose scales, dense at disc and progressively sparse towards the margin, on a greyish white background; no discoloration when rubbed. Margin incurved, then becoming straight, shortly exceeding the lamellae, often with appendiculate remains of the annulus. Lamellae free, crowded, 4 mm broad, with intercalated lamellulae, ventricose, pink to light brown, finally dark brown. Stipe 45–68 × 5–7 mm (8–15 mm at base), cylindrical with a bulbous base, fistulose, surface above the ring smooth, below the ring tomentose, white, flavescent or orange-ochre when rubbed. Annulus simple, membranous, superous, white, fragile. Context firm, white, somewhat flavescent when cut. Odour of almonds.
Spores (5.5–)5.8–6.2(–6.5) × (3.3–)3.5–4.0(–4.2) μm, (x = 6 ± 0.16 × 3.7 ± 0.15 μm, Q = 1.49–1.72, Qm = 1.58 ± 0.01, n = 20), ellipsoid, rarely oblong, smooth, brown, thick-walled. Basidia 14–22 × 6–7 μm, clavate, hyaline, smooth, 4-spored, rarely 2-spored. Cheilocystidia 16–34 × 7–13 μm, simple, clavate to broadly clavate or sphaeropedunculate, hyaline or sometimes with yellowish pigments, smooth. Pleurocystidia absent. Pileipellis a cutis composed of hyphae of 6–13 μm wide, cylindrical, often with brownish pigments, constricted at the septa.
Macrochemical reactions — KOH reaction positive, yellow. Schäffer’s reaction positive, reddish on dry specimen.
Habitat — Solitary, on soil of roadside.
Material examined. THAILAND, Chiang Mai Prov., Mae Pong Nature Trail, 3 June 2012, J. Chen, LD201224 (holotype MFLU2012-0868; isotype HMAS279139); Chiang Mai Prov., MRC, 13 Sept. 2006, R.L. Zhao, ZRL3101 (BBH19617, HMAS279143).
Notes — Agaricus patris is morphologically characterized by having a pileus surface covered with fibrillose scales, of variable colour ranging from purplish brown to reddish brown or dark purple, spores 6 × 3.7 μm on average, and simple cheilocystidia.
Indeed, in view of gross morphology, A. patris is hardly distinguished from many members of the section, such as A. brunneolus, A. dulcidulus, A. gemlii, and A. megalosporus. From average spore size, A. patris can be easily separated from A. dulcidulus, which has the smallest size within the section (4.31 × 3 μm, Parra 2013). Agaricus gemlii differs in its habitat which is in damp Atlantic environments near the coast (Parra 2013). When the collections consist of robust, fleshy specimens with pilei exceeding 7 cm, A. brunneolus and A. megalosporus are easily distinguished from A. patris; otherwise, the sequence data is essential for doubtless identification. According to the phylogenetic results, A. patris is closely related to A. sodalis, a species recently described from Thailand. However, the latter species differs by its pileus surface which is covered with violet brown fibrils, mainly densely arranged at the disc, rare or absent towards the margin and slightly shorter spores (5.4 × 3.6 μm on average, Liu et al. 2015). Phylogenetically, they differ at more than 15 positions in both ITS and tef-1α sequences.
Agaricus purpureofibrillosus Linda J. Chen, R.L. Zhao & K.D. Hyde, sp. nov. — MycoBank MB818055; Fig. 24, ,2525
Microscopic characters of Agaricus purpureofibrillosus. a. Cheilocystidia; b. basidia; c. basidiospores. — Scale bars = 5 μm.
Agaricus purpureofibrillosus (holotype ZRL3080). a. Overall morphology in laboratory; b. section view; c. pileus surface; d. lamellae; e. annulus.
Facesoffungi number. FoF 02291.
Etymology. The epithet ‘purpureofibrillosus’ refers to purplish fibrils on the pileus of this species.
Pileus 2–3 cm diam, 1 mm thick at disc; at first conical, then convex to plano-convex, finally applanate; surface dry, entirely covered with purplish fibrils, dense at disc and more sparse towards the margin, on a white background; strongly flavescent when margin is bruised. Margin straight, shortly exceeding the lamellae, often with appendiculate remains of the annulus. Lamellae free, crowded, 2 mm broad, with intercalated lamellulae, at first white, then pink, brown when mature. Stipe 36–45 × 3–6 mm, cylindrical fistulose, surface both above and below the ring smooth, silky, white, strongly flavescent when rubbed. Annulus simple, membranous, superous, white, fragile. Context firm, white, flavescent when cut. Odour of almonds.
Spores 4.5–5(–5.3) × 2.7–3 μm, (x = 4.9 ± 0.12 × 2.9 ± 0.14 μm, Q = 1.25–1.66, Qm = 1.69 ± 0.02, n = 20), ellipsoid or amygdaliform, smooth, brown, thick-walled. Basidia 16–22 × 6–7 μm, clavate, hyaline, smooth, 4-spored. Cheilocystidia 9–25 × 7–15 μm, abundant, simple or rarely septate at base, pyriform, sphaeropedunculate, or broadly clavate, with yellowish pigments, smooth. Pleurocystidia absent. Pileipellis a cutis composed of hyphae of 6–12.5 μm wide, cylindrical, often with crystalline brownish pigment inside, constricted at the septa.
Macrochemical reactions — KOH reaction positive, yellow. Schäffer’s reaction positive, reddish orange on dry specimen.
Habitat — Solitary, in soil of roadside.
Material examined. THAILAND, Chiang Mai Prov., MRC, 10 July 2006, T.H. Li, ZRL3080 (holotype BBH19596; isotype HMAS279145); Chiang Rai Prov., Mae Sae, 28 July 2010, N. Tongklang, NTF063 (MFLU).
Notes — Agaricus purpureofibrillosus is morphologically well characterized by its slender sporocarps, a pileus surface entirely covered with purplish fibrils, small spores and simple cheilocystidia.
Among the members of A. sect. Minores, numerous species morphologically resemble A. purpureofibrillosus by sharing a slender sporocarp and purplish fibrillose pileus, such as A. dulcidulus, A. gemlii, A. parvibicolor, and A. purpurellus. However, they can be distinguished on account of the following characters: A. dulcidulus has smaller spores (4.3 × 3 μm on average) and grows under broadleaved trees as Quercus or Carpinus (Parra 2013); A. gemlii differs in its larger spores (5.6 × 3.8 μm on average) and the habitat in damp Atlantic environments near the coast (Parra 2013); A. parvibicolor, a species recently described from Thailand, differs by the finely striate pileus margin and larger spores (5.2 × 3.3 μm on average; Liu et al. 2015); A. purpurellus differs in its wider spores (5.2 × 4 μm on average) and the distinctive habitat in conifer woods (Parra 2013). Otherwise, the molecular data is essential for unequivocal identification.
Agaricus robustulus Linda J. Chen, Callac, L.A. Parra, K.D. Hyde & De Kesel, sp. nov. — MycoBank MB818056; Fig. 26, ,2727
Agaricus robustulus. a. Overall morphology in situ (holotype CA847); b. annulus and stipe (holotype CA847); c. overall morphology in situ (ADK2905); d. section view (ADK2905).
Microscopic characters of Agaricus robustulus. a. Cheilocystidia; b. basidia; c. basidiospores. — Scale bars = 5 μm.
Facesoffungi number. FoF 02292.
Etymology. The epithet ‘robustulus’ refers to the small but robust appearance of the sporocarps of this species.
Pileus 2–6(–8.5) cm diam, 2–3 mm thick at disc; at first parabolic, becoming conico-convex to convex, sometimes with truncated centre, finally applanate; surface dry, with reddish brown or dark golden brown fibrils, densely at disc, soon with pileus expansion, outside the unbroken disc the surface disrupts into triangular scales, concentrically arranged on a dirty white background. Margin incurved, becoming straight when mature, not exceeding the lamellae, often with appendiculate remains of the annulus. Lamellae free, crowded, 4 mm broad, with intercalated lamellulae, subventricose to ventricose, at first white to pink, then light brown, finally dark brown. Stipe 20–40(–95) × 6–10 mm (13–14 mm at base), cylindrical with a bulbous base, fistulose, surface above the ring smooth, below the ring fibrillose, sometimes with camel-coloured appressed scales, white, strongly flavescent when rubbed. Annulus simple, superous, membranous, occasionally somewhat floccose on the below side, white, fragile. Context firm, white, flavescent when cut. Odour of almonds.
Spores 5.4–6.2(–6.6) × 3–4 μm, (x = 5.8 ± 0.25 × 3.7 ± 0.16 μm, Q = 1.47–1.74, Qm = 1.56 ± 0.04, n = 20, Asiatic collections), ellipsoid, rarely oblong, smooth, brown, thick-walled; 4.4–6.1 × 3.1–3.6(–3.8) μm, (x = 5.2 ± 0.43 × 3.3 ± 0.18 μm, Q = 1.37–1.79, Qm = 1.56 ± 0.11, n = 30, African collection), ellipsoid, rarely oblong, smooth, brown, thick-walled. Basidia 12–22 × 6–9 μm, clavate to broadly clavate, hyaline, smooth, 4-spored. Cheilocystidia 16–40(–66) × 14–20(–23) μm, simple, ovoid, pyriform or broadly clavate with a thin base, with yellowish pigments, smooth. Pleurocystidia absent. Pileipellis a cutis composed of cylindrical hyphae 4–13(–15) μm diam, not or slightly constricted at the septa, the thicker the more constricted. With greyish brown diffuse internal pigment. One terminal element observed 8 μm wide with progressively attenuated and rounded apex.
Macrochemical reactions — KOH reaction positive, yellow. Schäffer’s reaction positive, reddish on dry specimen.
Habitat — Solitary or scattered in sandy soil of secondary forest or in park.
Material examined. BÉNIN, Borgou Prov., Wari Maro, 19 Sept. 2000, A. De Kesel, ADK2905 (BR). – CHINA, Yunnan Prov., Lincang, Yongde County, 15 July 2012, Q.H. Yu, ZRL2012357 (HMAS273958). – MALAYSIA, Langkawi Island, 21 Apr. 2013, P. Callac, AK075 (KLU); 22 Apr. 2013, J. Ha, K Yun & P. Callac, MAR145 (KLU). – THAILAND, Chiang Mai Prov., Chiang Mai University, 25 July 2010, J. Guinberteau, CA847 (holotype MFLU16-0973); Chiang Mai Prov., Doi Suthep Pui National Park, 15 Aug. 2009, S.C. Karunarathna, NT055 (MFLU).
Notes — Agaricus robustulus is morphologically well characterized by its fleshy sporocarps, the reddish brown or dark golden brown, fibrillose or squamose pileus, spores with mean of 5.8 × 3.7 μm and simple cheilocystidia.
The average spore size of the new species is slightly different between Asian and African collections which can be considered as intraspecific variation. Several species morphologically resemble A. robustulus by having fleshy sporocarps, fibrillose or squamose pileus and variable colour from reddish brown to purplish brown, such as: A. brunneolus, A. goossensiae, and A. megalosporus. Agaricus goossensiae differs by its larger spores (6.3 × 4.4 μm on average, re-examination of the holotype GF929) and inconspicuous cheilocystidia; A. brunneolus and A. megalosporus are easily separated when their pilei exceed 7 cm diam. Otherwise, the sequence data are crucial for an accurate identification.
DISCUSSION
Advancements in the classification of the genus Agaricus
Zhao et al. (2011) provided evidence for seven strongly supported major tropical clades (TRI to TRVII) in the genus Agaricus that were not represented in the traditional classification mainly based on temperate species of the genus. Zhao et al. (2016) later proposed a new system of classification with taxonomic ranks based on the divergence times. Divergence times of between (18–)20–26 Ma or higher than 30 Ma were used to raise well-supported clades to sectional or subgenus taxonomic ranks, respectively, in Agaricus. As a result, 20 sections and five subgenera were proposed (Zhao et al. 2016). Among those, the following three sections were included in A. subg. Minores: A. sect. Minores, an unnamed section and A. sect. Laeticolores. The latter was so named because A. rufoaurantiacus, which was the single species of the tropical clade TRII that Zhao et al. (2016) included in their multi-gene analysis, had been previously placed in A. sect. Laeticolores by Heinemann (1961). In our multi-gene analyses, we included five species of the tropical clade TRII and also the type specimen of A. laeticulus that is the type of A. sect. Laeticolores. Firstly, our results demonstrate that this clade could not represent A. sect. Laeticolores because the type specimen of A. laeticulus was placed outside of this clade and nested in A. sect. Minores, indicating that Heinemann (1978) erroneously included A. rufoaurantiacus in A. sect. Laeticolores). Secondly, this clade diverged 31.01 Ma ago in our MCC tree and was therefore raised to the subgenus rank as A. subg. Minoriopsis. In addition, the newly described species A. leucocarpus not only diverged 27.54 Ma ago from the other members of the A. subg. Minores, but also, it is morphologically distinct from the species of A. sect. Minores. Therefore, it is excluded from A. sect. Minores and is considered as the type in a new section named A. sect. Leucocarpi.
In conclusion, A. subg. Minores still consists of three sections, but they have changed as follows with respect to the system proposed by Zhao et al. (2016): the new monospecific section A. sect. Leucocarpi is introduced, while the erroneously named A. sect. Laeticolores is excluded and raised to the subgenus rank as A. subg. Minoriopsis. The two remaining sections A. sect. Minores and an unnamed section are unchanged. The genus Agaricus currently comprises six subgenera (Agaricus, Arvenses, Minores, Minoriopsis, Pseudochitonia, and Spissicaules).
The reaction of Schäffer is among the most pertinent taxonomic characters at the sectional or subgeneric rank in the genus Agaricus. It is useful to identify both new section and new subgenus proposed in this study: it is positive dark reddish purple or reddish brown in dried specimens in the new subg. Minoriopsis, while the positive reaction is orange or red in the phylogenetically related subgenera A. subg. Flavoagaricus and A. subg. Minores except in the new section Leucocarpi for which the reaction is negative.
What are the future prospects? Three putative sections, one in A. sect. Minores and two in A. sect. Minoriopsis, all corresponding to well-supported clades with stem ages earlier than 20 Ma, require further studies with more samples to be described and circumscribed as new sections. About a dozen of ungrouped species of A. sect. Minores also require supplementary studies and more specifically A. huijsmanii that may belong to a clade which diverged more than 20 Ma ago. In the genus Agaricus some species such as A. martineziensis, A. heterocystis, or the entire clade TRIV including A. deserticola, remain unclassified. We did not include these in the analysis because only ITS sequence data were available and preliminary tests based on these data suggested they were not closely related to A. subg. Minores.
It can be noted that, except the clade TRIV, the six other well-supported tropical clades reported by Zhao et al. (2011) are now included in the new classification. TRI is A. sect. Brunneopicti within A. subg. Pseudochitonia (Chen et al. 2015, Zhao et al. 2016); TRII is A. subg. Minoriopsis (this study); TRIII is A. sect. Amoeni within A. subg. Spissicaules (Zhao et al. 2016); TRV, TRVI, and TRVII diverged too recently to be raised to sectional rank and thus remain in A. sect. Minores (Zhao et al. 2016, this study).
Reconstruction of Agaricus section Minores and evolutionary considerations
Phylogenetic reconstruction has been made so far in the genus Agaricus for five sections (Arvenses, Bivelares, Brunneopicti, Nigrobrunnescentes, and Xanthodermatei) based on ITS sequence data (Challen et al. 2003, Kerrigan et al. 2005, Thongklang et al. 2014, Chen et al. 2015, Gui et al. 2015, Parra et al. 2015). The successive studies of Zhao et al. (2011), Lebel (2013) and Zhao et al. (2016) included 25–30 species of A. sect. Minores roughly distributed in 5–7 major subclades. The present study includes 81 species distributed in 11 major subclades.
Delimitation of A. sect. Minores has always been problematic. Here, we present a section with estimated mean stem and crown ages of 30.06 and 24.19 Ma, respectively. This means that clades diverging between 24.19 Ma and 20 Ma could be also ranked at the sectional rank in the system of classification that we adopted with the condition they form a strongly supported clade. However, one early divergent clade is not well-supported in Fig. 1. It includes elements which are not grouped in the other analyses: [59] A. huijsmanii (Europe), [57] ZRL3102 (Thailand), and [27-28-29] the clade A-VI which is a curiosity since it includes A. campbellensis from a subantartic island and A. sp. GAL 5812 from arctic tundra in Alaska (Geml et al. 2008).
Only two regions are relatively well represented in our study: Europe with 19 species and Greater Mekong Subregion with 38 species. Completely different phylogenetic patterns are observed in these two regions. In Europe 16 species belong to the same clade A-VII and the three remaining samples are ungrouped. In Greater Mekong Subregion 32 species are distributed in 9 of the 11 clades. This difference neither results from the larger number of species from Greater Mekong Subregion, nor from the fact that Thailand (tropical) and Yunnan (subtropical) have been regrouped since the 27 species reported from Thailand are distributed in six clades and the 14 species reported from Yunnan (three are both in Yunnan and in Thailand) are also distributed in six clades. Therefore, compared to Europe, Greater Mekong Subregion is remarkable both by its species richness and by its phylogenetic diversity. Fig. 1 shows very well that the Greater Mekong Subregion diversity results from multiple species diversification that have occurred over the past 24 Myr, while most of the species today present in Europe result from a major diversification event that occurred relatively recently. The estimated stem and crown ages of the clade TRVII were 14.30 and 10.63 Ma, respectively. This diversification might have followed the middle Miocene climatic optimum (15 Ma), likely accompanying the re-installation of the temperate vegetation in Europe (Pound et al. 2012). More investigations are required to establish to which extent, the species of the A-VII clade should be specifically adapted to temperate climates. In Fig. 3, with a broader sampling, two North American species and two species from Yunnan are also found in clade A-VII, but still no typically tropical species. Our sampling is not sufficient in non-European temperate regions to determine where this diversification occurred, but it is likely that climatic changes in Europe were favourable for its extension.
Seven secotioid species were included in the analysis. It was expected to establish whether they are related or how many times the gasteroid morphology, considered an adaption to xeric conditions, appeared independently. We did not found strong evidence for either, but only some indications that most species might be related. Presently, we cannot reject the hypothesis they would have a common ancestor.
Species diversity in Agaricus section Minores
The present study is far from comprehensive, however, it includes all of the tropical and temperate species of A. sect. Minores with ITS sequence data available in GenBank. In total, 81 phylogenetic species are recognized worldwide. Sequence data have not been obtained for the following dozen of species:
– six that we failed to sequence: one only known from Estonia (A. luteoflocculosus) and five from Hawaii (A. azoetes, A. cheilotulus, A. entibigae, A. kiawetes, and A. xeretes).
– two from Japan (Imai 1938) that are not traceable and lack a designated holotype (A. comptulellus and A. semotellus).
– The four remaining species are A. johnstonii from tropical North America (Murrill 1918), A. nothofagorum and A. singeri from tropical South America (Heinemann 1962, 1986, 1990, 1993) and A. heinemanniensis from India (Natarajan & Purushothama 1996).
Because of the rather brief descriptions generally given in the past and our inability to re-examine the type specimens of these species, we conservatively accept these as good species in A. sect. Minores. Therefore, A. sect. Minores may comprise at least 93 species.
Our results suggest that the species diversity of A. sect. Minores is largely underestimated. This is partly due to the fact that many areas, especially tropical regions, are underexplored. Secondly, before the application of molecular techniques (primarily DNA sequencing), species were typically identified and clumped by gross morphology. However, species diversity can be masked by a lack of discriminant morphological differences between cryptic species (Bickford et al. 2007). As a consequence, potentially valuable good species may have been misidentified as known taxa. This occurred for A. marisae, which was considered as A. heinemannianus until sequence data proved its novelty (Parra 2013). This is also the case in the present study for an unnamed species of A. subg. Minoriopsis represented by the samples HAI10186 and HAI10371 from North Carolina (USA), previously misidentified as A. comtulus (Didukh et al. 2005). Indeed, according to our phylogenetic analyses of hundreds of collections of the genus Agaricus, none of the European taxa except A. subrufescens are conspecific with tropical taxa of Southeast Asia. Therefore, the multiple records in literature of species like A. purpurellus from various regions including Africa, South Asia and tropical South America (Heinemann 1961, 1962, 1980, 1986, 1993) may appear doubtful and remains to be confirmed by sequencing specimens from these regions.
In A. sect. Minores, species richness appears much higher in tropical areas since 21 species are recognized throughout Europe while 27 species or putative species are recorded mainly from northern Thailand in the present study. Three of the 27 species have been also recorded from subtropical areas of Yunnan (China), while 11 other species have been recorded in Yunnan, but not in Thailand. This makes a total of 38 species reported from only two countries of the Greater Mekong Subregion. It is a good indication of the potential high species diversity in this area which also includes Cambodia, Lao, Myanmar, and Vietnam.
The distribution range of these species is unknown but it could be relatively broad for few of them such as A. robustulus, which has been reported from Africa, Malaysia, and Thailand. However, we did not find any conspecific record between samples from Europe, Greater Mekong Subregion, and Australasia which represent 83 % (67/81) of the species included in this study. It can be reasonably expected that at least 200 species of A. sect. Minores could occur worldwide.
Edibility of species of A. sect. Minores is generally unknown and they are not consumed because they are small-sized in general and hard to identify. However, to our knowledge intoxication has never been reported by any species. They have a pleasant odour and A. brunneolus, the largest European species of the section, sometimes abounds and is locally consumed (Cappelli 2011). In Greater Mekong Subregion, some medium-sized or attractive fleshy species such as A. megalosporus and A. robustulus should be tested for their edibility.
Acknowledgments
The authors are grateful to P. Sysouphanthong, N. Thongklang, and J. Guinberteau, and to the team of the Mushroom Research Centre of the University of Malaya who helped with field work or provided us fungal specimens. This research was supported by The French Ministry of Foreign Affairs (project AGASIA of the regional program Bio-Asie); the National Natural Science Foundation of China (Project ID: 31470152 and 31360014), the Innovative Group of Edible Mushrooms Industry of Beijing (Project ID:BAIC05-2017), the Key Research and Development Program from Government of Guangxi Zhuang Autonomous Region (Project ID: 2016AB05317) and the Thailand Research Fund Grant (BRG 5580009) entitled ‘Taxonomy, Phylogeny and Biochemistry of Thai Basidiomycetes’.
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