![Micropsalliota Brunneosquamata, a New Species from Thailand Jie Chen [A,B,C], Kevin D](http://data.docslib.org/img/bb83cab136aa3ff9f2192b1a9033dd0b-1.webp)
Chiang Mai J. Sci. 2016; 43(4) 689
Chiang Mai J. Sci. 2016; 43(4) : 689-694 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper
Micropsalliota brunneosquamata, a New Species from Thailand Jie Chen [a,b,c], Kevin D. Hyde [b,c], Ali H. Bahkali [d] and Rui-Lin Zhao* [a] [a] State key laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. [b] School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand. [c] Center of Excellence in Fungal Research, School of Science, Mae Fah Luang University, Tasud, Chiang Rai 57100, Thailand. [d] Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 1145, Saudi Arabia. *Author for correspondence; e-mail: [email protected] Received: 15 January 2015 Accepted: 8 June 2015
ABSTRACT A new species of Micropsalliota, M. brunneosquamata is described and illustrated from northern Thailand based on both morphological and molecular data. The new species is characterized by brownish, erect scales at the centre of the pileus, a stipe covered by concolourous fibrils, and microscopically, by the shape of cheilocystidia which sometimes show obtuse mucro at apex. The new species is compared with morphologically similar species. Phylogenetic analyses of ITS sequences also support its novelty.
Keywords: Agaricaceae, Agaricales, ITS, phylogeny, taxonomy
1. INTRODUCTION The genus Micropsalliota H hn. was small to middle sized basidiomes with a established by von H hnel [1] and emended membranous partial veil, basidiospores by Pegler and Rayner [2] and Heinemann [3]. with an apically thickened endospore and Micropsalliota species are mainly distributed lack of a germ spore, capitate or subcapitate in tropic areas of Africa, Asia and the cheilocystidia and incrusted pileipellis hyphae Americas [4-9]. Zhao et al. provided a which often turn olive-green in ammonia monograph of Micropsalliota from Northern solution [10]. Thailand based on morphological and The present paper is part of our molecular data, and described 23 species, continued research on macrofungi from including 10 new taxa and 13 new records Northern Thailand [10-15], which already for Thailand. Phylogenetic relationships revealed high biodiversity. Micropsalliota within the genus have been investigated using brunneosquamata is introduced here as a new analysis of nrITS and nrLSU sequence datasets species based on morphological and [10]. Micropsalliota comprises species with molecular data. 690 Chiang Mai J. Sci. 2016; 43(4)
2. MATERIALS AND METHODS using primers ITS4/ITS5 following the 2.1 Morphological Character protocol of White et al. [18] with some Examination modification [10]. Amplification failed Collections of marcofungi were made in collections ZRL3073 and ZRL4038. in Northern Thailand from 2004 to 2012 Sequencing was performed on an ABI 3730 [14, 15]. Photographs were taken in the field, XL DNA analyzer (Applied Biosystems) at the odour and colour change on bruising Shanghai Majorbio Bio-Pharm Technology were recorded at the same time. Specimens Co., Ltd, China. The ITS sequence generated were gathered and wrapped in aluminum from this study is available in GenBank under foil to avoid mixing or crushing. The study the accession number of KP316210. of macrocharacters, including chemical testing and further photography of fresh samples 2.2.2 Sequence alignment and were carried out following the methodology phylogenetic analysis described by Largent [16], once returned to In order to investigate the phylogenetic the laboratory. Colour terms followed those position of the new species, representative of Kornerup and Wanscher [17]. Specimens sequences were selected based on the previous were dried using a food drier, sealed in plastic study of Zhao et al. [10]. The original sequence bags, and deposited in the herbarium of generated from this study plus those derived Mae Fah Luang University, Chiang Rai, from GenBank were initially aligned using Thailand (MFLU) and BIOTEC Bangkok T-coffee ver 8.99 [19], then manually adjusted Herbarium (BBH). Duplicate specimens are in BioEdit v. 7.0.4 [21]. deposited in Herbarium Mycologicum A Maximum likelihood (ML) tree was Academiae Sinicae (HMAS), Beijing China. constructed using PhyML ver 3.0 [22] on Microscopic features, including of the Phylogeny. fr platform (http:// anatomy of lamellae, pileipellis, and partial veil, www.phylogeny.fr/). The HKY85 substitution and features of basidiospores, basidia and model was selected with an estimated cystidia, were examined from dried specimens proportion of invariable sites of 0.413 and following the protocols of Largent [16]. assuming 4 gamma-distributed rate categories. Measurements of microscopic features The gamma shape parameter 0.547 was (spores, basidia and cheilocystidia) are directly estimated from the data. Maximum presented based on at least 20 measurements, parsimony (MP) analysis was performed and include x, the mean of length by width using PAUP* 4.0b10 [23], by heuristic ± SD; Q, the quotient of basidiospore searches with unordered character states, gaps length to width, and Qm, the mean of treated as missing data, random addition of Q-values ± SD. sequences, and the tree bisection-reconnection (TBR) branch swapping. Bootstrap support 2.2 Molecular Phylogenetic Research values (BS) were obtained from 100 and 1000 2.2.1 Nucleic acid preparation and ITS replicates for ML or MP trees. Bayesian amplification analysis was performed with MrBayes 3.1.2 DNA was extracted from dried fungal [24]. Six Markov chains using a GTR+I+G specimens with a commercial DNA model nucleotide substitution detected by extraction kit (E.Z.N.A. Forensic Kit, D3591- MrModeltest 2.2 [25] were run for one million 01, Omega Bio-Tek) according to manufaturer generations and sampled every 100th protocol. The PCR reactions were performed generation, which resulted in 10,000 trees. Chiang Mai J. Sci. 2016; 43(4) 691
Those trees sampled prior to Markov chains uninformative and 134 were parsimony- reaching an average standard deviation of split informative. The phylogenetic trees generated frequency value of 0.01 were discarded as the by ML, MP and Bayesian methods exhibited burn-in, and the remaining trees were used to very similar topologies. Differences are only calculate Bayesian posterior probabilities (PP) noted in the branching pattern of M. allantoidea of the individual clades. Trees were visualized and M. alba. The Maximum Likelihood (ML) in TreeView and exported to graphics tree is shown in Figure 1. In all analyses the programs [26]. outgroup taxa group at the base of the tree. Various species clades received moderate to 3. RESULTS strong support (76-100% BS), however, the 3.1 Phylogenetic Analyses deeper nodes indicating relationships amongst The ITS dataset includes the 5.8S gene species are generally not well-resolved. and contains 33 ITS sequences representing The new species is sister to a well-supported 21 Micropsalliota taxa, Leucoagaricus meleagris clade (99/97/100) comprising M. pusillissima, (Gray) Singer and L. americanus (Peck) Vellinga M. alboserica, M. gracilis, M. subalba, M. alba, were included as outgroup taxa. The final M. allantoidea, M. bifida, M. subarginea, alignment contained 655 characters, of which M. arginophaea and M. pseudoarginea. 463 were constant, 58 were parsimony-
Figure 1. Maximum likelihood tree of Micropsalliota based on ITS sequence data. Leucoagaricus meleagris and L. americanus are outgroup taxa. Bootstrap support values (ML/MP) above 50% and Bayesian posterior probabilities (PP) values above 90% are shown at nodes. The new species M. brunneosquamata is indicated in bold type. “T” refers to sequence from type specimen. 692 Chiang Mai J. Sci. 2016; 43(4)
3.2 Taxonomy 0.03, n = 20], amygdaliform to ellipsoid, Micropsalliota brunneosquamata L.J. Chen, apiculate at apex and base, without germ R.L. Zhao & K.D. Hyde, sp. nov. (Figure 2, pore, light yellowish brown to light brown. 3) Basidia 11-17 × 5.5-7 μm, clavate, hyaline, MycoBank: MB811168 smooth, 4-spored, occasionally 2-spored. Etymology: refers to the brownish scales Cheilocystidia various in shape, 15-42 × 8-23 on the pileus. μm, napiform, ventricose-clavate, rarely Pileus 31-40 mm in diam., convex to clavate, sometimes mucronate to lageniform applanate, subumbonate; cuticle exceeding (obtuse mucro up to 6-18 μm long), hyaline. the lamellae; surface dry, covered by erect Pleurocystidia absent. Pileipellis a cutis composed scales at disc and appressed scales or fibrillose of hyphae of 5-25 μm diam., smooth-walled, squamules elsewhere, reddish-brown (8E6) constricted at the septa, with brown, or dark brown, against whitish background. incrusted, membranous pigments. Annulus Context 2 mm broad, soft, white, unchanging hyphae of (2.5)7.5-12.5 μm in diam, hyaline, when cut. Lamellae free, crowded, ventricose, smooth, similar to pileipellis. Stipitipellis lamellae with 4 series, 3 mm broad, reddish- composed of hyphae similar to pileipellis. grey (8C2) to light brown. Stipe 28-43 × Habit: Solitary in soil of forest. 3-5 mm, cylindrical with slightly bulbous base, Material examined: THAILAND, occasionally with rhizomorphs, hollow; Chiang Mai Prov., Mae Pong Nature Trail, surface above ring white, fibrillose, below ring 5 June 2012, collector Jie Chen, Philippe heavily covered by brown fibrils. Annulus Callac, LD201236 (MFLU 120880, holotype), white, superous, fragile. Odor of anise or GenBank accession number: KP316210; seaweed. Chiang Mai Prov., Tung Joaw, 30 June 2006, Macrochemical reactions: KOH reaction collector Le Huyen, ZRL3073 (BBH); reddish brown on surface of pileus, then dark Chiang Mai Prov., Mae Taeng Dist. Highway green. 1095 at 22 km marker, 30 June 2007, collector Basidiospores 5-6 × 3-4 μm, [x = 5.4 ± 0.22 Else Vellinga, ZRL4038 (BBH). × ± ± 3.4 0.21, Q = 1.37-1.83, Qm = 1.61
Figure 2. Morphology of M. brunneosquamata. a. Brownish scales on pileus surface (LD201236 holotype). b. Annulus and stipe characters (LD201236 holotype). c. Context when cut (ZRL3073). d. Sporocarps in laboratory (ZRL3073). a and b photographed by P. Callac. Chiang Mai J. Sci. 2016; 43(4) 693
its violet brown squamules of the pileus, violet tones on the stipe surface, and subcapitate cheilocystidia [10]; M. megaspora R.L. Zhao, Desjardin, K. Soytong & K. D. Hyde has much smaller sporocarps (5-12 mm in diam), larger basidiospore (6-8 × 3.8-4.5 μm), and ventricose-rostrate to pyriform cheilocystidia [10]; M. pilicystis Heinem. differs by purple squamules of the pileus and much larger cheilocystidia, which are clavate or cylindrical with an capitate apex [4]; M. pleurocystidiata Heinem. & Little Flower differs in forming more robust sporocarps up to 130 mm in diam., a pileus surface entirely covered with appressed fibrillose squamules, and the presence of pleurocystidia [3, 10]; M. suthepensis R.L. Zhao, Desjardin, K. Soytong & K.D. Figure 3. Microcharacters of M. Hyde differs in having a reddish-brown brunneosquamata. a. Basidiospores. b. Basidia pileus and clavate cheilocystidia [10]. Molecular and Cheilocystidia. c. Pileipellis. Scale bars: phylogenetic analyses also support M. a, b = 10 μm, c = 5 μm. brunneosquamata as a distinct species.
4. DISCUSSION ACKNOWLEDGEMENTS Micropsalliota brunneosquamata is We are grateful to Dr. Philippe Callac, characterized by brownish, erect scales at Dr. Else Vellinga and Le Huyen for sample the centre of the pileus, the stipe with collecting. This research was supported by concolourous fibrils, and microscopically, by The French Ministry of Foreign and European the particular shape of the cheilocystidia. Affairs (project AGASIA of the regional Micropsalliota brunneola Heinem is the most program Bio-Asie); the National Natural phenotypically similar species because both Science Foundation of China (Project ID: of them have brownish erect squamules at 31000013, 31360014 and 31470152) and the the centre of the pileus and concolourous Thailand Research Fund Grant (BRG fibrils on the surface of the stipe below the 5580009) entitled “Taxonomy, Phylogeny annulus. However, the latter species differs in and Biochemistry of Thai Basidiomycetes”. having smaller sporocarps (up to 30 mm in The authors extend their sincere appreciations diam), smaller spores (4.1-4.9 × 2.9-3.3 μm) to the Deanship of Scientific Research at and brownish cheilocystidia of which the King Saud University for its funding this shape and size were not mentioned by Prolific Research group (PRG-1436 09). Heinemann [4]. Several species exhibit colored fibrils on the stipe surface, for REFERENCES example: M. lateritia var. vinaceipes R.L. Zhao, [1] H hnel F.V., Fragments sur mykologie Desjardin, K. Soytong & K. D. Hyde, a variety XVI, Sitzungsber. Kaiserl. Akad. Wiss. newly described from Thailand, which is Math.-Naturwiss. Cl. Abt. I., 1914; 123: easily distinguished from the new species by 49-155. 694 Chiang Mai J. Sci. 2016; 43(4)
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