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Research Article
Clitopilus lampangensis (Agaricales, Entolomataceae), a new species from northern Thailand
expand article infoJaturong Kumla‡, Nakarin Suwannarach‡, Witchaphart Sungpalee§, Kriangsak Sri-Ngernyuang§, Saisamorn Lumyong|‡
‡ Chiang Mai University, Chiang Mai, Thailand
§ Maejo University, Chiang Mai, Thailand
| Academy of Science, The Royal Society of Thailand, Bangkok, Thailand

Corresponding author: Saisamorn Lumyong ( scboi009@gmail.com )

Academic editor: Kentaro Hosaka
© 2019 Jaturong Kumla, Nakarin Suwannarach, Witchaphart Sungpalee, Kriangsak Sri-Ngernyuang, Saisamorn Lumyong.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Kumla J, Suwannarach N, Sungpalee W, Sri-Ngernyuang K, Lumyong S (2019) Clitopilus lampangensis (Agaricales, Entolomataceae), a new species from northern Thailand. MycoKeys 58: 69-82. https://doi.org/10.3897/mycokeys.58.36307
Open Access

Abstract

A new species of agaricomycetes, Clitopilus lampangensis, is described based on collections from northern Thailand. This species was distinguished from previously described Clitopilus species by its pale yellow to grayish yellow pileus with the presence of wider caulocystidia. Molecular phylogenetic analyses, based on the data of the internal transcribed spacers (ITS) and the large subunit (LSU) of the nuclear ribosomal DNA, and the second largest subunit of RNA polymerase II (rbp2) genes, also support the finding that C. lampangensis is distinct from other species within the genus Clitopilus. A full description, color photographs, illustrations and a phylogenetic tree showing the position of C. lampangensis are provided.

Keywords

Agaricomycetes, gill mushroom, morphology, phylogeny, tropics

Introduction

The genus Clitopilus was proposed by Kummer (1987) with C. prunulus (Scop.) P. Kummer as the type species. It belongs to the family Entolomataceae of the order Agaricales. This genus is saprotrophic and is widely distributed, especially in northern temperate areas (Singer 1986; Baroni and Watling 1999; Moncalvo et al. 2002; Kirk et al. 2008; Hartley et al. 2009; Crous et al. 2012; Raj and Manimohan 2018). Clitopilus is characterized by basidiocarps that are clitocyboid, omphalinoid or pleurotoid, mostly whitish or occasionally grayish or brownish in color, with pink or pinkish brown spore prints, ellipsoid basidiospores with longitudinal ridges that appear angular in a polar view, and hyphae lack clamp connections (Singer 1986; Noordeloos 1988). There are 30 species of Clitopilus worldwide (Kirk et al. 2008), although there are 201 species names recorded in the Index Fungorum (http://www.indexfungorum.org/Names/Names.asp). The taxa list in the Index Fungorum includes synonyms and misidentifications, as well as some species that are not well documented. Formerly, the genus Clitopilus included Rhodocybe (Moncalvo et al. 2002; Co-David et al. 2009; Vizzini et al. 2011a). However, molecular phylogenetic analyses have provided powerful tools for the identification of Clitopilus, leading to the separation of Clitopilus from Rhodocybe as well as the related genera (Clitocella and Clitopilopsis) (Cooper 2014; Kluting et al. 2014; Raj and Manimohan 2018).

Only six species, Clitopilus apalus (Berk. & Br.) Petch, C. crispus Pat. C. doimaesalongensis Jatuwong, Karun. & K.D. Hyde, C. chalybescens T.J. Baroni & Desjardin, C. peri (Berk. & Br.) Petch and C. prunulus, have been reported in Thailand (Baroni et al. 2001; Chandrasrikul et al. 2011; Kluting et al. 2014; Jatuwong et al. 2017). During an investigation of macrofungi in northern Thailand, we found a population of Clitopilus which we describe here as a new species based on the morphological and molecular characteristics. To confirm its taxonomic status, the phylogenetic relationship of the new species was determined by the ITS and LSU of the rDNA, and the rbp2 genes.

Materials and methods

Sample collection

Basidiocarps were collected in Mae Moh District, Lampang Province, northern Thailand in 2018. Basidiocarps were wrapped in aluminum foil and kept in plastic specimen boxes to be transported to the laboratory. Notes on the macromorphological features and photographs were obtained within 24 h of collection. The specimens were dried at 40–45 °C and deposited at the Herbarium of the Sustainable Development of Biological Resources Laboratory, Faculty of Science, Chiang Mai University (SDBR-CMU), and BIOTEC Bangkok Herbarium (BBH), Pathumthani, Thailand.

Morphological studies

Macromorphological data were recorded from fresh specimens. The recording of color names and codes followed Kornerup and Wanscher (1978). Micromorphological data were recorded from dry specimens rehydrated in 95% ethanol followed by distilled water, 3% KOH or Melzer’s reagent. Anatomical features were based on at least 50 measurements of each structure as seen under a light microscope (Olympus CX51, Japan). For spore statistics, Q is the ratio of spore length divided by spore width and Q is the average Q of all specimens ± standard deviation.

Molecular phylogenetic studies

Genomic DNA of dry specimens (1–10 mg) was extracted using a Genomic DNA Extraction Mini-Kit (FAVORGEN, Taiwan). The ITS region of DNA was amplified by polymerase chain reactions (PCR) using ITS4 and ITS5 primers (White et al. 1990), the LSU of rDNA gene were amplified with LROR and LRO5 primers (Vilgalys and Hester 1990), and rbp2 gene was amplified with the bRBP2-6F and bRBP2-7.1R primers (Matheny 2005). The amplification program for these three domains was performed in separated PCR reaction and consisted of an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s (ITS), 52 °C for 45 s (LSU), and 54 °C for 1 min (rpb2), and extension at 72 °C for 1 min on a peqSTAR thermal cycler (PEQLAB Ltd., UK). PCR products were checked on 1 % agarose gels stained with ethidium bromide under UV light. PCR products were purified using a PCR clean up Gel Extraction NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Germany) following the manufacturer’s protocol. The purified PCR products were directly sequenced. Sequencing reactions were performed and the sequences were automatically determined in the genetic analyzer at 1st Base company (Kembangan, Malaysia) using the PCR primers mentioned above. Sequences were used to query GenBank via BLAST (http://blast.ddbj.nig.ac.jp/top-e.html).

For phylogenetic analyses, the sequences from this study, previous studies and the GenBank database were used and provided in Table 1. The multiple sequence alignment was carried out using MUSCLE (Edgar 2004), and the combined ITS and LSU alignment, and rpb2 alignment were deposited in TreeBASE under the study ID 24373 and 24374, respectively. Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI) algorithms, implemented by RAxML v7.0.3 (Stamatakis 2006) and MrBayes v3.2.6 (Ronquist et al. 2012), respectively. Rhodocybe griseoaurantia and R. pallidogrisea were used as outgroup. The best-fit substitution model for BI and ML analyses were estimated by jModeltest 2.1.10 (Darriba et al. 2012) using Akaike information criterion (AIC). For ML analysis, the bootstrap (BS) replicates were set as 1000 and used to test phylogeny (Felsenstein 1985). Clades with bootstrap values (BS) of ≥ 70% were considered significantly supported (Hillis and Bull 1993). For the BI analysis, the Markov chains were run for one million generations, with six chains and random starting trees. The chains were sampled every 100 generations. Among these, the first 2,000 trees were discarded as burn-in, while the postburn-in trees were used to construct the 50% majority-rule consensus phylogram with calculated Bayesian posterior probabilities. Bayesian posterior probabilities (PP) ≥ 0.95 were considered significant support (Alfaro et al. 2003).

Table 1.
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Sequences used for phylogenetic analysis. The newly generated sequences are in bold.

Taxa Voucher/strain GenBank accession number Refernces
ITS LSU rpb2
Clitopilus albidus CAL 1320 MF926596 MF926595 MF946579 Raj and Manimohan 2018
CORT:26394WAT – KR869936 KC816906 Largent and Bergemann 2016
M536 – AF261287 – Moncalvo et al. 2002
Clitopilus austroprunulus MEN2009062 KC139085 – – Phillips and Dinis 2012
MEN2009001 KC139084 – – Phillips and Dinis 2012
Clitopilus cf. argentinus MTB480412 – – KC816907 Kluting et al. 2014
Clitopilus chalybescens MFUCC130808 KP938184 – – Jatuwong et al. 2017
MFUCC130809 KP938185 – – Jatuwong et al. 2017
SDBR-CMUUP0039 MK773645 MK764940 MK784129 This study
Clitopilus chrischonensis TOHG 1994 HM623128 HM623131 – Vizzini et al. 2011b
Clitopilus crispus GDGM29931 JQ281489 – – He et al. 2012
CORT:9982 – – KC816910 Kluting et al. 2014
CORT:10027 – – KC816911 Kluting et al. 2014
Clitopilus cystidiatus 26 – GQ289147 GQ289220 Co-David et al. 2009
TOAV130 HM623129 HM623132 – Vizzini et al. 2011b
Clitopilus doimaesalongensis MFUCC130806 KP938183 – – Jatuwong et al. 2017
Clitopilus fusiformis SAAS1038 KY385634 – KY385632 Wang et al. 2017
SAAS1892 KU751777 – KY385633 Wang et al. 2017
Clitopilus giovanellae SF14368 EF413030 EF413027 – Moreno et al. 2007
Clitopilus hobsonii CBS 270.36 FJ770395 – – Hartley et al. 2009
CBS 445.86 FJ770385 – – Hartley et al. 2009
DLL9635 – – KC816913 Kluting et al. 2014
DLL9643 – – KC816913 Kluting et al. 2014
Clitopilus lampangensis SDBR-CMUJK 0147 MK764933 MK764935 MK784127 This study
SDBR-CMUNK 0047 MK764934 MK773856 MK784128 This study
Clitopilus kamaka KA12-0364 KR673433 – – Kim et al. 2015
Clitopilus orientalis CAL 1616 MG345134 MG321558 MG321559 Raj and Manimohan 2018
Clitopilus passeckeriamus CBS299.35 MH855682 MH867198 – Vu et al. 2019
P78 KY962494 KY963078 – Unpublished
Clitopilus paxilloides CORT:5809 – – KC816919 Kluting et al. 2014
Clitopilus peri CORT:10033 – – KC816920 Kluting et al. 2014
CORT:10040 – – KC816921 Kluting et al. 2014
CORT:10041 – – KC816922 Kluting et al. 2014
Clitopilus pinsitus CBS 623.70 MH859879 MH871665 – Vu et al. 2019
Clitopilus prunulus Champ-15 KX449418 – – Pérez-Lzquierdo et al. 2017
CBS 227.93 FJ770408 – – Hartley et al. 2009
Noordeloos 2003-09-14 KR261096 – – Unpublished
COPT:7003 – – KC816925 Kluting et al. 2014
TB9663 – – GU384648 Baroni et al. 2011
TB8229 – – GU384650 Baroni et al. 2011
COPT:REH8456 – – KC816923 Kluting et al. 2014
Clitopilus reticulosporus DC-2010 KC885966 HM164414 HM164416 Vu et al. 2019
Clitopilus scyphoides CBS 127.47 MH856181 MH867707 – Vu et al. 2019
CBS 400.79 FJ770401 – – Hartley et al. 2009
Clitopilus subscyphoides CAL 1325 MF927542 MF946580 MF946581 Raj and Manimohan 2018
Clitopilus venososulcatus CORT:8111 – – KC816930 Kluting et al. 2014
Rhodocybe griseoaurantia CAL 1324 KX083571 KX83574 KX083568 Unpublished
Rhodocybe pallidogrisea CORT 013944 NR154437 – KC816968 Kluting et al. 2014

Results

Phylogenetic analyses

The topology of each single-gene of ITS and LSU, and the combined ITS and LSU phylograms were found to be similar. However, differences were observed in the topology of the rbp2 gene. Therefore, we present only the combined ITS and LSU gene phylogram (Fig. 1), and the single rbp2 gene phylogram (Fig. 2). The combined ITS and LSU sequence dataset consisted of 34 taxa and were comprised of 1774 characters including gaps (ITS: 1–779, LSU: 780–1774). The sequence dataset of rbp2 consisted of 27 taxa and the aligned dataset was comprised of 620 characters that included gaps. The GTR model with gamma rate heterogeneity and invariant sites (GTR+G+I) was the best-fit model used for both ML and BI analyses that were selected by AIC. The average standard deviation of the split frequencies fell to 0.011364 and 0.009837 in the BI analysis of the combined ITS and LSU, and rbp2 sequences, respectively after one million generations. This was observed after the 50% majority consensus phylogram was constructed. The ML analysis of the combined ITS and LSU sequences was based on the parameters estimated under the GTR+I+G model, and the proportion of the invariable sites and the gamma shape parameters were 0.0250 and 0.9320, respectively. Additionally, the tree with log likelihood (-8211.7515) was built after 1000 bootstrapping replications. In the ML analysis of the rbp2 sequence that was based on the GTR+I+G model, the proportion of the invariable sites and the gamma shape parameters were 0.5400 and 1.7960, respectively, while the tree with log likelihood (-3640.1616) was built after 1000 bootstrapping replications.

Both the combined ITS and LSU, and the rbp2 phylograms indicated that the sequences were of a new species, C. lampangensis, that had formed a monophyletic clade with high BS (100 %) and PP (1.0) support (Figs 1, 2). A combined ITS and LSU phylogram revealed that the new species was a sister taxon to C. chalybescens. In addition, the rbp2 phylogram indicated that the new species was a sister taxon to C. chalybescens and C. peri.

Figure 1. 

Phylogram derived from maximum likelihood analysis of the combined ITS and LSU region of nuclear rDNA of 34 sequences. Rhodocybe griseoaurantia and R. pallidogrisea were used as outgroup. The numbers above branches represent maximum likelihood bootstrap percentages (left) and Bayesian posterior probabilities (right). Only bootstrap values ≥ 50 % are shown, and the scale bar represents ten substitutions per nucleotide position. The fungal species obtained in this study are in bold.

Figure 2. 

Phylogram derived from maximum likelihood analysis of rpb2 gene of 27 sequences. Rhodocybe griseoaurantia and R. pallidogrisea were used as outgroup. The numbers above branches represent maximum likelihood bootstrap percentages (left) and Bayesian posterior probabilities (right). Only bootstrap values ≥ 50 % are shown, and the scale bar represents ten substitutions per nucleotide position. The fungal species obtained in this study are in bold.

Taxonomy

Clitopilus lampangensis J. Kumla, N. Suwannarach & S. Lumyong, sp. nov.

MycoBank No: 830890
Fig. 3

Diagnosis

Distinguished from other Clitopilus species by its pale yellow to grayish yellow pileus with the presence of caulocystidia, and from C. chalybescens by its wider caulocystidia, longer basidiospores, and lack of grayish blue color change on the pileus and stipe when bruised.

Etymology

‘lampangensis’, referring to Lampang Province, where the holotype was found.

Holotype

THAILAND, Lampang Province, Mae Moh District, (18°24'21"N, 99°42'26"E, elevation 380 m), on ground in a tropical deciduous forest, May, 2018, J. Kumla & N. Suwannarach, SDBR-CMUJK 0147 and BBH 43590 (isotype).

Gene sequence (from holotype)

MK764933 (ITS), MK764935 (LSU) and MK784127 (rbp2).

Basidiocarps small, clitocyboid. Pileus 35–50 mm diam., initially convex or somewhat plano-convex with or without a central depression, becoming deeply umbilicate with age; surface pale yellow (4A3) to greyish yellow (4B5), somewhat velutinous, finely pruinose all over; margin incurved to slightly inrolled, entire or slightly wavy. Lamellae subdecurrent to decurrent, white (1A1), crowded, up to 2.5 mm wide, with lamellulae of 1–3 lengths; edge entire or slightly wavy, concolorous with the sides. Stipe 20–25 × 5–8 mm, central, solid; surface white (1A1) to yellowish white (4A2), finely pruinose all over, densely so towards the apex; base with white cottony mycelium. Odor strong farinaceous. A pale pinkish spore print.

Basidiospores 7.0–9.0 × 3.0–5.0 μm, Q = 1.40–2.33, Q = 1.82 ± 0.27, ellipsoid in polar view, amygdaliform to limoniform in side view, with 6–8 prominent longitudinal ridges, colorless, thin-walled. Basidia 17.0–25.0 × 4.0–8.0 μm, clavate, colorless, thin-walled, 2- and 4-spored; sterigmata up to 4 μm long. Lamella-edge fertile. Pleurocystidia and cheilocystidia absent. Lamellar trama subregular; hyphae 2.5–4.0 μm wide, hyaline, thin-walled. Pileus trama compact, hyaline, cylindrical hyphae 5–10 μm wide. Pileipellis a cutis of loosely interwoven hyphae; 3–5 μm wide, hyaline, thin-walled, and terminal cells; subcylindric or narrowly clavate, 4–8 μm wide. Stipitipellis at stipe apex a layer of repent, hyaline, cylindrical hyphae 4–8 μm wide, thin-walled. Caulocystidia 25.5–42.5 × 8.0–15.0 μm, single or clustered, erect or repent, varying in shape from cylindrical to clavate, hyaline, slightly thick-walled. Clamp connections absent in all tissues.

Figure 3. 

Clitopilus lampangensis SDBR-CMUJK 0147 (holotype). A Basidiocarps B Basidiospores C Basidia D Pileipellis E Caulocystidia. Scale bars: 10 mm (A), 5 μm (B), 10 μm (C–E).

Ecology and distribution

Fruiting solitary or gregarious on soil in a tropical deciduous forest. Known only from northern Thailand

Specimens examined

THAILAND, Lampang Province, Mae Moh District, (18°24'20"N, 99°42'3"E, elevation 375 m), on ground in a tropical deciduous forest, May, 2018, N. Suwannarach & J. Kumla, SDBR-CMUNK 0047, GenBank sequence MK764934 (ITS), MK773856 (LSU) and MK784128 (rbp2).

Discussion

The present study has identified a new species of Clitopilus acquired from northern Thailand based on both morphological characteristics and phylogenetic analyses. Clitopilus lampangensis is characterized by its clitocyboid, pale yellow to grayish yellow basidiocarps, pinkish spore-print, ellipsoid basidiospores with longitudinal ridges and hyphae lacking clamp connections. Thus, these morphological characteristics support its placement into the genus Clitopilus (Singer 1986; Noordeloos 1988). Based on the morphology, the pale yellow to grayish yellow pileus of C. lampangensis distinguishes it from the white and grayish pileus of Clitopilus species, with the exceptions of C. catalonicus, C. djellouliae, C. fasciculatus, C. gallaecicus, C. giovanellae, C. incrustatus, C. luteocinnamomeus and C. prunulus, (Kummer 1871; Singer 1942; Noordeloos 1984; Baroni and Halling 2000; Moreno et al. 2007; Ovrebo and Baroni 2007; Vila et al. 2008; Contu et al. 2011; Desjardin et al. 2015). The characteristics of the basidiocarps and size of the basidia, caulocystidia and basidiospores of C. lampangensis were compared with related Clitopilus species (Table 2). The presence of caulocystidia in C. lampangensis clearly distinguishes it from these related species. Moreover, the pileus of C. lampangensis (35–50 mm in diameter) are larger than C. djellouliae (6–18 mm in diameter; Contu et al. (2011)), C. giovanellae (5–15 mm in diameter; Singer (1942) and Moreno et al. (2007)) and C. catalonicus (up to 15 mm in diameter; Vila et al. (2008)). Prior to this study, C. apalus, C. crispus, C. doimaesalongensis, C. chalybescens, C. peri and C. prunulus had been found in Thailand (Baroni et al. 2001; Chandrasrikul et al. 2011; Kluting et al. 2014; Jatuwong et al. 2017). However, C. apalus, C. crispus, C. peri and C. doimaesalongensis differ from C. lampangensis by their white to chalk-white pileus and a lack of caulocystidia (Pegler 1986; Yang 2000; Jatuwong et al. 2017). The larger basidia and basidiospores, and the absence of caulocystidia in C. prunulus clearly differentiate it from C. lampangensis (Kummer 1871; Desjardin et al. 2015) (Table 2). Both C. lampangensis and C. chalybescens have caulocystidia (Baroni et al. 2001; Jatuwong et al. 2017). However, the width of the caulocystidia and the length of the basidiospores of C. chalybescens are narrower and shorter than in C. lampangensis (Table 2) (Baroni et al. 2001; Jatuwong et al. 2017).

Table 2.
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Comparison of Clitopilus lampangensis with the closely related species.

Taxa Origin Pileus Basidia Caulocystidia Basidiospores
C. lampangensis a Thailand 35–50 mm in diameter, pale yellow to greyish yellow 17.0–25.0 × 4.0–8.0 μm, 2–4 streigmata 25.5–42.5 × 8.0–15.0 μm Ellipsoid, 7.0–9.0 × 3.0–5.0 μm, 6–8 longitudinal ridges
C. chalybescens b, c Thailand 15–90 mm in diameter, white, yellowish white to greyish blue 15.0–21.0 × 5.1–8.0 μm, 4 streigmata 16.0–32.0 × 5.0–7.0 μm Ellipsoid, 5.3–7.5 × 3.6–5.0 μm, 8–10 longitudinal ridges
C. peri d,e India, Sri Lanka, Thailand 8–22 mm in diameter, white 16.0–18.0 × 5.0–7.0 μm, 4 streigmata Absent Ellipsoid, 6.7–8.5 × 3.0–4.0 μm, 6–9 longitudinal ridges
C. prunulus f,g Netherlands, Thailand, United State 25–80 mm in diameter, white, yellowish white to grayish or yellow cream 25.0–47.0 × 7.0–12.0 μm, 4 streigmata Absent Ellipsoid, 9.0–14.0 × 4.5–8.0 μm, 6–8 longitudinal ridges
C. fasciculatus h Netherlands, 20–70 mm in diameter, pale brown Sizes were not reported, 4 streigmata Absent Ellipsoid, 4.5–6.3 × 3.0–4.0 μm, 3–6 longitudinal ridges
C. gallaecicus i Spain 80–90 mm in diameter, creamy, ochre to ochre-brown 20.0–35.0 × 8.5–10.5 μm, 4 streigmata Absent Ellipsoid, 8.0–14.5 × 4.5–7.5 μm, 3–6 longitudinal ridges
C. incrustatus j Costa Rica, United State 80–90 mm in diameter, grayish brown 16.0–24.0 × 7.0–8.0 μm, 4 streigmata Absent Ellipsoid, 5.0–6.5 × 3.0–4.0 μm, 3–6 longitudinal ridges
C. djellouliae k France 6–18 mm in diameter, light yellowish brown 22.0–32.0 × 7.5–8.5 μm, 4 streigmata Absent Ellipsoid, 6.0–9.0 × 4.0–6.0 μm
C. giovanellae l,m Italy, Spain 5–15 mm in diameter, grayish to light brown 14.0–22.0 × 6.5–9.5 μm, 4 streigmata Absent Ellipsoid, 5.0–8.0 × 3.0–4.0 μm
C. luteocinnamomeus n Panama 15–45 mm in diameter, ochre to light cinnamon-brown 19.0–27.0 × 6.0–7.0 μm, 4 streigmata Absent Subglobose to ellipsoid, 4.5–6.0 × 3.5–5.0 μm
C. catalonicus o Panama Up to 15mm in diameter, light yellowish brown 32.0–40.0 × 6.4–8.0 μm, 4 streigmata Absent Ellipsoid, 5.3–7.5 × 3.7–4.5 μm

aThis study, bBaroni et al. (2001), cJatuwong et al. (2017), dPegler (1986), eKluting et al. (2014), fKummer (1871), gDesjardin et al. (2015), hNoordeloos (1984), iBlanco-Dios (2013), jBaroni and Halling (2000), kContu et al. (2011), lSinger (1942), mMoreno et al. (2007), nOvrebo and Baroni (2007) and oVila et al. (2008).

The phylogenetic analyses of the combined ITS and LSU, and rpb2 sequences confirmed that C. lampangensis formed a monophyletic clade which clearly separated it from the other Clitopilus species. Clitopilus lampangensis forms a sister taxon to C. chalybescens and C. peri. Clitopilus peri differs from C. lampangensis by its smaller white basidiocarps (8–22 mm in diameter) and the absence of caulocystidia (Pegler 1986). Additionally, the different morphological characteristics that exist between C. lampangensis and C. chalybescens have been mentioned above.

Therefore, a combination of the morphological characteristics and the molecular analyses strongly support recognition of a new fungus species. This discovery is considered important in terms of stimulating a deeper investigation of macrofungi in Thailand, and will help researchers to better understand the distribution and ecology of Clitopilus.

Key to Clitopilus species known from Thailand

1 Pileus white to chalk-white colors 2
– Pileus white or with other colors 5
2 Stipe ≥ 3 mm thick 3
– Stipe < 3 mm thick C. peri
3 Basidia < 8 μm wide 4
– Basidia ≥ 8 μm wide, basidiospores 6.8–9.2 × 4.1–5.5 μm C. doimaesalongensis
4 Basidia up to 25 μm, basidiospores 6–8.5 × 4.5–5.5 μm C. apalus
– Basidia up to 30 μm, basidiospores 5.5–9 × 4–6 μm C. cripus
5 Pileus white to pale grayish or yellowish cream colors 6
– Pileus pale yellow to greyish yellow colors, caulocystidia present, basidiospores 7.0–9.0 × 3.0–5.0 μm C. lampangensis
6 Basidia ≥ 25 μm long, caulocystidia absent, basidiospores 8.0–12.0 × 4.0–6.5 μm C. prunulus
– Basidia < 25 μm long, caulocystidia present, basidiospores 5.3–7.5 × 3.6–5.0 μm C. chalybecens

Acknowledgements

This work was supported by grants from Chiang Mai University and Center of Excellence on Biodiversity (BDC), Office of Higher Education Commission (BDC-PG3-161005), Thailand. We are grateful to staff of Mae Moh Forestry Industry Organization for their excellent field assistance, and Dr. Eric H.C. McKenzie for English proof reading.

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