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Research Article
Morphological and molecular identification of two new Marasmiellus species (Omphalotaceae, Agaricales) from Thailand
expand article infoWenhua Lu, Pumin Nutaratat§, Jaturong Kumla, Saowaluck Tibpromma|, Abdallah M. Elgorban, Samantha C. Karunarathna|, Nakarin Suwannarach§
‡ Chiang Mai University, Chiang Mai, Thailand
§ Thaksin University, Phatthalung, Thailand
| Qujing Normal University, Qujing, China
¶ King Saud University, Riyadh, Saudi Arabia
Open Access

Abstract

Marasmiellus (Omphalotaceae, Agaricales) specimens collected in Thailand were investigated based on morphological characteristics and molecular phylogenetic analyses. In the present study, two species are introduced as new to science, namely Marasmiellus thailandicus and M. minutisporus. Phylogenetic analyses were carried out based on the internal transcribed spacer (nrITS) and nuclear ribosomal RNA large subunit (nrLSU) regions, and the results revealed that the two new taxa are distinct species within Marasmiellus. Another specimen was identified as M. scandens and is reported for the first time with morphology and molecular data from Thailand. Descriptions, illustrations, and phylogenetic results are provided. In addition, M. diaphanus and M. colocasiae are proposed as new combinations of Collybiopsis diaphana and Paramarasmius colocasiae, respectively, based on the phylogenetic evidence.

Key words

Basidiomycota, new taxa, omphalioid mushroom, taxonomy, wood-decaying mushroom

Introduction

The genus Marasmiellus Murrill was proposed by Murrill (1915), with the type species M. juniperinus Murrill [treated as Collybiopsis juniperina (Murrill) R.H. Petersen] (Petersen and Hughes 2021). This is one of the well-known and widely distributed genera, which belongs to the family Omphalotaceae Bresinsky, order Agaricales Underw. with a worldwide distribution in the tropics and the subtropics (Moncalvo et al. 2002; Wilson and Desjardin 2005; Blanco-Dios 2015; Sesli et al. 2018). Most species of this genus are saprobes occurring on decaying plant matter, which degrade leaf and woody debris, some species are host-specific, parasitic, and attack various economically important plants (e.g., banana, coconut, and sugar cane) (Singer 1973; Jabeen et al. 2023). Marasmiellus species are characterized by collybioid, omphalioid, or pleurotoid basidiomata, slightly decurrent, pileus white, yellow, pink or brown, convex, lamellae well-developed, intervenose, adnate to decurrent, and smooth, white or hyaline, thin-walled, and ellipsoid to oblong, rarely subcylindrical, fusiform, inamyloid basidiospores, usually with confluent hilar appendage, and pileipellis or stipitipellis with a Rameales-structure (Singer 1973; Pérez-De-Gregorio et al. 2011). Prior to this study, eight Marasmiellus species, including M. albofuscus (Berk. & M.A. Curtis) Singer (Chandrasrikul et al. 2011), M. alliiodorus (Mont.) Singer (Chandrasrikul et al. 2011), M. amygdalosporus Pegler (Chandrasrikul et al. 2011), M. candidus (Fr.) Singer (Seephueak et al. 2018), M. chamaecyparidis (Hongo) Hongo (Chandrasrikul et al. 2011), M. collybioides (Speg.) J.S. Oliveira, M. corticum Singer (Seephueak et al. 2018), and M. paspali (Petch) Singer (Chandrasrikul et al. 2011) have been reported from Thailand.

Some phylogenetic studies on Omphalotaceae based on nrITS and nrLSU in single and multigene analyses have been conducted to solve the stable placement where Gymnopus (Pers.) Gray and Marasmiellus still remain uncertain. Initially, Gymnopus and Marasmiellus were treated as multiple branches, nonmonophyletic groups (Moncalvo et al. 2002; Mata et al. 2004; Wilson and Desjardin 2005; Hughes et al. 2010; Petersen and Hughes 2017). Mata et al. (2004) argued for proposing the broad concept of Gymnopus based on nrITS analysis which places the type species of Marasmiellus within a Gymnopus clade. However, Wilson and Desjardin (2005) argued for keeping Marasmiellus and Gymnopus as distinct genera. In addition, Oliveira et al. (2019) provided consistent evidence for more restricted Gymnopus and a distinct Marasmiellus s. str., which is composed of Gymnopus sect. Vestipedes and members of Marasmiellus sect. Dealbati, Marasmiellus, Rameales, and Stenophylloides.

According to the recent nomenclature, the type species of Marasmiellus (M. juniperinus Murrill) was transferred to Collybiopsis (J. Schröt.) Earle. Petersen and Hughes (2021) considered Marasmiellus as a synonym of Collybiopsis, while the concepts of Collybiopsis between Oliveira et al. (2019) and Petersen and Hughes (2017) are different. In addition, the boundaries among Gymnopus, Marasmiellus, and Collybiopsis would be more blurred, especially between Marasmiellus and Collybiopsis, as well as if these species were transferred to Collybiopsis, then it would be polyphyletic with Rhodocollybia Singer, Paragymnopus J.S. Oliveira, and Lentinula Earle would be a synonym of Gymnopus. Nevertheless, further clarifications are needed for whether Collybiopsis accommodates all members of Marasmiellus. In this study, we treat Marasmiellus following Singer’s (1974, 1986) concept.

The present study aims to describe two new Marasmiellus species and a new report of M. scandens (Massee) Dennis & D.A. Reid collected from Thailand based on morphological characteristics and multigene phylogenetic analyses. In addition, we propose two new combinations of Collybiopsis diaphana and Paramarasmius colocasiae. Descriptions, illustrations, and a phylogenetic tree to show the placement of the taxa are provided.

Materials and methods

Morphological study

Fresh basidiomata were collected from the northern part (Chiang Mai University, Chiang Mai Province) and the southern part (Phatthalung Province) of Thailand. Macromorphological features of the basidiomata were documented and photographed in the field. Color names and codes were determined following Kornerup and Wanscher (1978). The specimens were taken back to the laboratory, dried completely in an electric oven at 45 °C (Hu et al. 2022), and sealed in plastic bags for further micro-morphological characterization. The holotype and other examined specimens were deposited at the Chiang Mai University Biology Department Herbarium (CMUB), Chiang Mai University, Thailand. Freehand sections of the dried specimens were mounted in 5% KOH and Congo red to observe microscopic characteristics, while Melzer’s reagent was used to increase the contrast of structures and the amyloid reaction of basidiospores. The light Eclipse 80i microscope (Olympus, Japan) was used to view features of basidia, basidiospores, cystidia, and hyphae, which were drawn by using the drawing tube attached to the microscope. The sizes of micro-structures were calculated based on at least 50 measurements, and the notations (a–) b–c (–d) describe the basidiospore dimensions, where the range ‘b–c’ represented 90% or more of the measured valued and a and b are the extreme values. The Q refers to the length/width ratio values of all measured basidiospores. Qm refers to the average Q value with standard deviation. ‘L’ refers to the number of complete lamellae, and ‘I’ refers to the number of lamellulae between every two complete lamellae. All the line drawings of the microstructures were made freehand based on the rehydrated materials and finally modified using Adobe Illustrator 2019.

DNA extraction, PCR amplification, and sequencing

The genomic DNA was extracted from fresh specimens using the DNA Extraction Mini Kit (FAVORGEN, China) according to the manufacturer’s instructions. Primer pairs ITS1/ITS4 (nrITS) and LR0R/LR5 (nrLSU) regions were amplified by the polymerase chain reaction (PCR) (Vilgalys and Hester 1990; White et al. 1990), which was performed in a total volume of 20 μL reaction containing 1.0 μL DNA template, 1.0 μL each primer, 10.0 μL 2X Quick Taq® HS DyeMix (TOYOBO, Japan) and 7 μL deionized water. The amplification program was performed with an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30s, annealing at 54 °C for 40 s, and an extension at 72 °C for 1 min, with a final extension at 72 °C for 10 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, and the sequences were automatically determined in the genetic analyzer at 1ST Base Company (Kembangan, Malaysia) with the PCR primers mentioned above. Names of the new taxa were introduced and deposited in MycoBank (https://www.mycobank.org/, assessed on June 12, 2024). All newly generated sequences in the present study were deposited to GenBank (https://www.ncbi.nlm.nih.gov/genbank/, assessed on June 10, 2024).

Phylogenetic analyses

The newly generated forward and reverse of sequences from this study were assembled in the BioEdit v. 7.0.5 (Hall 1999), then were subjected to BLASTn searches in the GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi, assessed on 10 June 2024) to check those most related with a high degree of similarity taxa (with ≥ 85% query coverage and ≥ 90–100% percent identity), Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora (CMR UB 2041) was chosen as the outgroup from the family Marasmiaceae, and were aligned by running MAFFT v.7 at the online website server platform (www.ebi.ac.uk/Tools/mafft, assessed on June 10, 2024) (Katoh and Standley 2013) with additional sequences downloaded from GenBank and previous studies (César et al. 2020; Petersen and Hughes 2020) as is shown in Table 1. Gaps and ambiguous regions were automatically removed by trimAL v1.2 (http://trimal.cgenomics.org, assessed on June 10, 2024), and sequences were manually improved in BioEdit whenever necessary. Both sequencing datasets were combined using SequenceMatrix 1.7.8 (Vaidya et al. 2011) and AliView (Larsson 2014). The final FASTA format was converted to PHYLIP and NEXUS format in the Alignment Transformation Environment (ALTER) online program (Glez-Peña et al. 2010). The phylogenetic analyses were carried out by employing Maximum likelihood analysis (ML) and Bayesian analysis (BYPP) methods. The ML was performed on RAxML-HPC BlackBox (v.8.2.4) (Stamatakis 2014) with 1000 rapid bootstrap replicates on the CIPRES Science Gateway v.3.3 (http://www.phylo.org/portal2, assessed on 10 June 2024; Miller et al. 2010), with the GTRGAMMA substitution model. Bayesian analyses of six simultaneous Markov chains were run for 2,000,000 generations, and trees were sampled and printed to output at every 100th generation (resulting in 20,000 total trees) with the GTR+I+G evolution model that was estimated using MrModeltes v. 2.2 (Nylander 2004). Phylogenetic trees were visualized using FigTree v1.4.0 (Rambaut 2009). The reliable bootstrap support values of ML (BS ≥ 60%) and BI (PP ≥ 0.90) were inserted above the nodes.

Table 1.

Names, voucher numbers, countries, and corresponding GenBank accession numbers of the taxa used in the phylogenetic analyses of this study.

Taxa Name Voucher Country GenBank Accession Number
nrITS nrLSU
Collybiopsis biformis TENN58541 USA DQ450054 NA
C. biformis HMJAU61116 China OQ597035 OQ594445
C. brunneogracilis SFSU-AWW01 Indonesia AY263434 NA
C. carneopallida BRNM:747442 Italy OM522632 NA
C. clavicystidiata SFC20180705–84 South Korea OL467252 OL462817
C. confluens HMJAU61120 China OQ597037 NA
C. confluens TENN50524 Sweden DQ450044 NA
C. confluens TENN-F-067864 Germany KP710296 NA
C. diaphana Cesar202 Mexico MT232390 NA
C. diaphana Cesar44 Mexico MT232391 NA
C. furtive SFSU-F-024524h1 USA MN413341 NA
C. furtive SFSU-F-024524h2 USA MN413342 NA
C. istanbulensis KATO fungi 3596 Turkey KX184795 KX184796
C. juniperina TENN59540 USA AY256708 KY019637
C. juniperina TENN-F-58988 Argentina KY026661 KY026661
C. luxurians TENN-F-057910 USA AY256709 AY256709
C. luxurians HMJAU61101 China OQ597045 OQ594455
C. luxurians HMJAU61198 China OQ597046 OQ594456
C. melanopus SFSU AW54 Indonesia OR818034 OR817634
C. melanopus CUH AM093 India KM896875 KP100305
C. orientisubnuda NIBRFG0000500990 Turkey OL467262 OL546546
C. peronata TENN-F-065120 Belgium KY026677 KY026677
C. quercophila TENN-F-69267 Slovakia KY026729 NA
C. quercophila TENN-F-69320 USA KY026736 NA
C. ramealis TENN-F-065146 Belgium MN413346 MW396882
C. ramealis TENN-F-065145 Belgium MN413345 MN413345
C. ramulicola GDGM44256 China KU321529 NA
C. stenophylla TENN-F-051099 USA MN413330 MW396887
C. stenophylla TENN-F-065943 USA MN413331 MW396886
C. ugandensis SFSU-BAP 614 Sao Tome MF100986 NA
C. vellerea SFC20140821-29 South Korea OL467267 OL462810
Gymnopus alkalivirens TENN51249 USA DQ450000 NA
G. brunneiniger XAL-Cesar 49 Mexico MT232389 NG075396
G. efibulatus HGASMF01-7052 China OM970865 OM970865
G. fusipes TENN59300 China AF505777 NA
G. fusipes TENN59217 France AY256710 AY256710
Marasmiellus agrianum NJ201111 Pakistan MZ044839 NA
M. agrianum NJ201112 Pakistan MZ044840 NA
M. alnicola URM90019 Brazil KY302681 KY302682
M. bicoloripes CAL1524 India KY807129 KY817233
M. candidus CBS:252.39 USA MH856003 NA
M. candidus MSM#0017 Pakistan KJ906507 NA
M. celebanticus TO HG2281 Spain JF460781 NA
M. gregarius G0197 Japan NA MK278330
M. griseobrunneus AMH 10117 India MK656132 MK660195
M. griseobrunneus CAL 1752 India MK660191 MK660192
M. griseobrunneus AMH 10118 India MK660194 MK660193
M. lucidus s1 China OP459424 NA
M. lucidus HT10 Japan AB968237 AB968237
M. minutisporus CMUB40054 Thailand PP889931 PP890011
M. minutisporus CMUB40055 Thailand PP889932 PP890012
M. omphaloides PDD:95810 New Zealand HQ533031 NA
M. paspali AHH65 USA EF175515 NA
M. paspali AHH26 USA EF175511 NA
M. pilosus iNat91483993 Cayman Islands OP651730 NA
M. rhizomorphogenus BRNM:715003 South Korea GU319116 GU319120
M. scandens GH-80 Ghana MN794179 NA
M. scandens GH-21 Ghana MN794139 NA
M. scandens KUNCC22-12451 China OP536418 NA
M. scandens CMUB40056 Thailand PP889933 PP890013
M. thailandicus CMUB40052 Thailand PP889929 PP890009
M. thailandicus CMUB40053 Thailand PP889930 PP890010
M. tenerrimus TENN61596H1 USA FJ596840 NA
M. tenerrimus TENN61596H2 USA FJ596841 NA
M. tricolor M01452 Estonia LR872638 NA
M. venosus TNS-F-52281 Japan AB968236 NA
M. violaceogriseus PDD:95788 New Zealand HQ533014 NA
M. volvatus URM 84466 Brazil KC348449 KC348442
M. sacchari CBS:215.32 USA NA MH866745
Moniliophthora perniciosa CMR UB 2041 Brazil AY317136 NA
Paragymnopus foliiphilus TENN-F-68183 USA KY026705 KY026705
P. perforans TENN-F-50318 Sweden KY026623 KY026623
P. perforans TENN-F-50319 Sweden KY026624 KY026624
P. pinophilus TENN-F-69207 USA KY026725 KY026725
Paramarasmius colocasiae SP376044 Brazil GQ452780 NA
Pa. mesosporus TNS-T-48339 Japan OM522625 OM522623
Pa. palmivorus AKD 112/2015 India MG251431 MG251441
Paramycetinis austrobrevipes TENN-F-50135 Australia KY026622 KY026622
Par. caulocystidiatus TENN-F-5405 New Zealand KY026645 NA
Pseudomarasmius efibulatus TENN-F-56187 New Zealand MK268234 NA
Ps. glabrocystidiatus BRNM 718676 Korea NR152899 KF251093
Ps. nidus-avis Cesar36 Mexico MH560576 NA
Ps. pallidocephalus TENN-F-52401 USA KY026635 KY026635
Ps. patagonianus TENN-F-54424 Chile KY352649 NA
Ps. quercophylloides TENN-F-49177 China MK268235 NA
Pusillomyces manuripioides JO674 Brazil MK434210 MK434211
Pu. manuripioides JO1121 Brazil MK434212 MK434213

Results

Phylogenetic analyses

In the dataset, the combined nrITS and nrLSU sequence dataset consisted of a total number of 86 taxa, and the aligned dataset was comprised of 1593 characters, including gaps (nrITS: 1–709 and nrLSU: 710–1593). The best RAxML tree was obtained with a final ML optimization likelihood value of -15536.515830. The matrix had 780 distinct alignment patterns, with 34.45% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.248165, C = 0.187111, G = 0.251637, T = 0.313087, with substitution rates AC = 1.027131, AG = 5.242142, AT = 1.916708, CG = 0.743477, CT = 6.311829, GT = 1.000000; gamma distribution shape parameter α = 0.681313, tree-Length = 3.382466. Notably, the phylograms of the ML and BI analyses were similar in topology. Therefore, the phylogenetic tree obtained from ML analysis was selected and is presented in this study (Fig. 1).

Figure 1. 

A combined phylogenetic tree was generated from maximum likelihood analysis (RAxML) based on a combined nrITS and nrLSU dataset. Bootstrap values (BS) ≥ 60% from ML analysis (left) and Bayesian posterior probabilities (right) (PP) ≥ 0.90 are shown on the branches. Newly sequenced collections are indicated in red, new combinations are in blue, and the type specimens are denoted by ‘T’.

Two specimens in this study, CMUB40054, and CMUB40055, were clustered in a well-supported lineage (1.00 PP/100% BS), introduced as M. minutisporus related to M. pilosus (Dennis) Singer (iNat91483993), and sister to M. griseobrunneus Sharafudheen & Manim (AMH 10117, AMH 10118, and CAL1752) with a support value of 0.99 PP and 84% BS). The second species of this study (CMUB40052 and CMUB40053) formed a separate lineage within the genus Marasmiellus with higher support values (1.00 PP/100% BS) and formed a clade with M. bicoloripes K.P.D. Latha, K.N.A. Raj & Manim (CAL1524) and M. minutisporus cluster, and we introduced it as M. thailandicus. Additionally, one collection (CMUB40056) was clustered with M. scandens strain (GH80 and GH21) with strong statistical support (0.99 PP/100% BS). Noteworthily, M. scandens (KUNCC22-12451) differs from M. scandens (GH21) and (GH80) by 29 base pair differences out of 577, but our strain (CMUB40056) only has two base pair differences out of 570 bases (Fig. 1). Additionally, the phylogenetic tree showed that M. diaphanus César, Bandala & Montoya (Cesar 202 Type, and Cesar 44) belonged to Collybiopsis and formed a sister taxon to C. quercophila (Pouzar) R.H. Petersen (TENN-F-69320 and TENN-F-69267) with high statistical support (1.00 PP/99% BS; Fig. 1). Marasmiellus colocasiae Capelari & Antonín (SP376044, Type) assigned to the Paramarasmius and formed a sister taxon to P. palmivorus (Sharples) Antonín & Kolařík (AKD 112/2015) and clustered with P. mesosporus (Singer) Antonín, K. Hosaka & Kolařík (TNS-F-48339, Type) with strong support value (1.00 PP/98% ML; Fig. 1) in Paramarasmius Antonín & Kolařík branch. Therefore, based on phylogenetic analyses, M. diaphanus is proposed as Collybiopsis diaphana comb. nov., while M. colocasiae is proposed as Paramarasmius colocasiae comb. nov.

Taxonomy

Marasmiellus thailandicus W. Lu, N. Suwannar. & J. Kumla, sp. nov.

MycoBank No: 854272
Figs 2A, B, 3

Type

Thailand • Chiang Mai Province, Chiang Mai University; 18°48'5"N, 98°57'23"E; elevation 337 m; on bark of Lagerstroemia macrocarpa Wall. ex Kurz, 5 August 2023, N. Suwannarach & J. Kumla (CMUB40052). GenBank accession numbers PP889929 (nrITS) and PP890009 (nrLSU).

Etymology

thailandicus” refers to the country Thailand, where the type species was collected.

Diagnosis

Differs from M. candidus by the presence of a reddish gray to dull red, dry, radially wrinkled surface, grooved pileus with distant lamellae, and obovate or ellipsoid spores.

Macrostructures

Basidiomata small-sized, marasmioid. Pileus 6–20 mm diam., hemispherical first, expanding to plano-convex with slightly concave, radially sulcate with age, with involute then deflexed or straight margin, not distinctly hygrophanous, translucently striate up to center, reddish gray (10B2) to dull red (10B3) all over with grayish red (7B3) center, grayish red (7B3) margin, surface smooth, dry, and dull. Lamellae distant, broadly adnate to subdecurrent, sometimes anastomosing, white to cream-colored, with a reddish white (7A2) edge, particularly in young specimens, I = 2–3, L = 11–14. Stipe 4–8 × 1–2 mm, cylindrical, curved, base often slightly swollen, inconspicuous fibrils or scurfy, creamy-white at apex, slightly reddish white (7A2) at the base, dry, pruinose all over, Context thin, fistulose or solid, concolorous with surface. Smell and taste indistinct.

Figure 2. 

Habitat of Marasmiellus species in the present study A, B Marasmiellus thailandicus (CMUB40052, holotype) C Marasmiellus minutisporus (CMUB40054, holotype) D Marasmiellus scandens (CMUB40056, new record). Scale bars: 10 mm (A–D).

Microstructures

Basidiospores (–12)13–16(–17) × 4–5(–6) μm (average = 15 × 5 μm), Q = (2.1)2.5–3.5(–3.75), Qm = 3.05 ± 0.38, sub-cylindrical to elongate with apiculus, inamyloid, thin-walled. Basidia 20–45 × 7–11 μm, 4-spored, clavate, sterigmata up to 5 μm. Cheilocystidia 50–53 × 8–13 μm, clavate. Pleurocystidia absent. Trama hyphae cylindrical, thin-walled, hyaline, inamyloid. Pileipellis, a cutis with transitions to a trichoderm, made up of cylindrical; pigment brown, intracellular and minutely incrusting, hyphal 2–4 μm, negative in Melzer’s reagent. Stipitipellis, a cutis of parallel, somewhat skewed, cylindrical, or clavate-shaped, smooth hyphae, thin-walled, up to 7.5 um wide. Caulocystidia absent. Clamp connection present.

Figure 3. 

Marasmiellus thailandicus (CMUB40052, holotype) A basidiospores B basidia C cheilocystidia D terminal elements of pileipellis E hyphae of stipe. Scale bars: 10 μm (A–E).

Ecology and distribution

Caespitose, in small groups growing on the bark of Lagerstroemia macrocarpa. Known only from the type locality in northern Thailand.

Additional material examined

Thailand, • Chiang Mai Province, Chiang Mai University; 18°48'5"N, 98°57'23"E; elevation 337 m; on bark of Lagerstroemia macrocarpa, 24 August 2023, N. Suwannarach & J. Kumla (CMUB40053). GenBank accession numbers PP889930 (nrITS) and PP889930 (nrLSU).

Marasmiellus minutisporus W. Lu, P. Nutaratat & J. Kumla, sp. nov.

MycoBank No: 854273
Figs 2C, 4

Type

Thailand • Phatthalung Province, Khuan Khanun, Sago Palm (Metroxylon sagu) Forest, 7°44'02"N, 99°59'47"E; elevation 23 m; on decaying leaf and branches of deciduous tree; 6 September 2023; P. Nutaratat, P., Suwannarach & J. Kumla, (CMUB40054). GenBank accession numbers PP889930 (nrITS) and PP890011 (nrLSU).

Etymology

minutisporus” refers to the small basidiospores of this species.

Diagnosis

Differs from M. virgatocutis by the grayish-brown, convex, wrinkled pileus, longer pileus terminal, smaller elongated spores, and caulocystidia.

Macrostructures

Basidiomata small-sized, marasmioid. Pileus 5–11 mm diam., thin, then expanding to applanate, with slightly inflexed, pulvinate when young, then deflexed, finally reflexed, and undulating margin, convex when age with depressed to umbilicate at disc., grayish brown (7D3), often with grey, brown, or dark gray (1F1) at the center, gray at the margin; often radially wrinkled, surface dry, slightly pruinose to tomentose under the lens. Lamellae distant, often more or less reduced, white to sordid beige with a concolorous overall, pruinose edge, I = 1–3, L = 13–16. Stipe 4–9 × 1 mm, cylindrical, often subbulbous at the base, off-white at the apex, fourth to fifth downward, and black or gray at the stipe base, entirely white pruinose, with basal tomentum. Context thin, soft, white, fistulose. Smell and taste none.

Figure 4. 

Marasmiellus minutisporus (CMUB40054, holotype) A basidiospores B basidia C cheilocystidia D elements of pileipellis E hyphae elements of stipe and caulocystidia. Scale bars: 5 μm (A); 10 μm (B–E).

Microstructures

Basidiospores (11)12–15(–16) × (3)4–5 μm (average = 14 × 4 μm), Q = (2.6)2.8–4(4.67), Qm = 3.5 ± 0.56, hyaline, inamyloid, cylindrical to fusiform, thin-walled. Basidia 25–28 × 7–9 μm, 4-spored, sterigmata up to 5 μm long, clavate. Lamella edge sterile. Cheilocystidia 30–33 × 12–15 μm, somewhat clavate to subglobose, Pleurocystidia absent. Pileipellis a cutis made up of 4.0–10 μm wide, inamyloid, inflated or cylindrical hyphae, with scattered suberect to erect, thin-walled, smooth, sometimes terminal elements up to 61–99 × 3.0–10 μm, gradually tapering to an acute or rounded apex. Stipitipellis a cutis composed of cylindrical, inamyloid, parallel, slightly, not incrusted, smooth, thick-walled. Caulocystidia 25–50 × 4–5 μm, adpressed to erect, cylindrical, clavate, smooth, thin-walled. Clamp connection present.

Ecology and distribution

Solitary to caespitose, in small groups growing on decaying leaves and twigs of deciduous trees. Known only from the type locality in southern Thailand.

Additional material examined

Thailand • Phatthalung Province, Khuan Khanun, Sago Palm (Metroxylon sagu) Forest, 7°44'02"N, 99°59'46"E; elevation 23 m; on decaying leaf and twigs of deciduous tree; 7 September 2023; P. Nutaratat, N. Suwannarach & J. Kumla, (CMUB40055). GenBank accession numbers PP889932 (nrITS) and PP890012 (nrLSU).

Marasmiellus scandens (Massee) Dennis & D.A. Reid, Kew Bull. [11](2): 289 (1957)

MycoBank No: 300136
Figs 2D, 5

Macrostructures

Basidiomata small-sized, marasmioid. Pileus 5–10 mm diam., orbicular when young and then convex at age, streaked from disc to margin, margin entire, wavy to irregular, decurved or greatly reflexed, surface dry, smooth, white pruinose, white to grayish orange (5B4–5), grayish orange (5B4) at the margin. Lamellae adnate, subdistant, with 2–3 series of lamellulae, 14–16 major lamellae, unequal, narrow, pale white at face and edge. Smell and taste none. Stipe 4–6 × 1–2 mm, often curved, lateral or central, disc at the base, dry, surface smooth, whitish to pale orange (5A3); Context thick, fistulose, orange white (5A2–3).

Figure 5. 

Marasmiellus scandens (CMUB40056, new record) A basidiospores B basidia C cheilocystidia D terminal elements of pileipellis. Scale bars: 5 µm (A–C); 10 µm (D).

Microstructures

Basidiospores 7–9(–10) × (3)3.5–4.5(–6) µm (average = 8 × 4.5 μm), Q = 1.4–1.8(–2), Qm = 1.74 ± 0.11, broadly ellipsoid, smooth, hyaline, inamyloid, thin-walled. Basidia 10–12.5 × 3–4.5 µm, clavate, 4-spored, sterigmata up to 1.5 µm. Cheilocystidia abundant, 15–17(–22) × 8–9(–11) µm, broom-cell type with finger-like excrescences in the upper half, hyaline, thin-walled. Pleurocystidia absent. Pileipellis with poorly Ramealis-structure, trama hyphae 4–9 µm, negative in Melzer’s reagent. Stipitipellis hyphae up to 5 μm wide, smooth, thin-walled, trama not observed, negative in Melzer’s reagent. Clamp connections present.

Ecology and distribution

Caespitose, in small groups on decaying leaf of Metroxylon sagu in southern Thailand (This study), on Aquilaria sinensis (agarwood) trees in China (Zhang et al. 2023), and on the stem of mangosteen in Malaysia (Turner 1971), on cocoa in Australia, Ghana, Caribbean, Papua New Guinea, and Solomon Islands (Amoako-Attah et al. 2020; Ali et al. 2021), on coffee in Fiji, Guinea, and Sierra Leone (Lu et al. 2022).

Material examined

Thailand • Phatthalung Province, Khuan Khanun, Sago Palm (Metroxylon sagu) Forest, 7°44'02"N, 99°59'47"E; elevation 24 m; on decaying leaf of Metroxylon sagu; 3 September 2023; P. Nutaratat, N. Suwannarach & J. Kumla, (CMUB40056). GenBank accession numbers PP889933 (nrITS) and PP890013 (nrLSU).

New combinations

Collybiopsis diaphana (César, Bandala & Montoya) W. Lu, N. Suwannar. & J. Kumla, comb. nov.

MycoBank No: 854271

Marasmiellus diaphanus César, Bandala & Montoya, in César, Montoya, Bandala & Ramos, Mycol. Progr. 19(10): 1022 (2020)

Paramarasmius colocasiae (Capelari & Antonín) W. Lu, N. Suwannar. & J. Kumla, comb. nov.

MycoBank No: 854270

Marasmiellus colocasiae Capelari & Antonín, Cryptog. Mycol. 31(2): 138 (2010)

Discussion

A combination of morphological characteristics and molecular phylogeny inferred from sequence data of the nrITS and nrLSU region revealed two new species of Marasmiellus (M. thailandicus and M. minutisporus) and one known species (M. scandens) in this study. According to the phylogeny results, Marasmiellus is well-clustered in a strongly supported clade (1.00 PP/100% BS) (Fig. 1), which is consistent with the results of previous phylogenetic studies (Sesli et al. 2018; Sharafudheen and Manimohan 2019; César et al. 2020). Furthermore, the phylogenetic tree indicated that M. diaphanus and M. colocasiae belonged to Collybiopsis and Paramarasmius, respectively. Therefore, these two new combinations were introduced as C. diaphana and P. colocasiae.

Morphologically, M. thailandicus is closely related to M. subnigricans (Murrill) Singer, M. candidus, and M. bicoloripes. Marasmiellus subnigricans (11−45 mm diam.) and M. candidus (3–22 mm diam) contain dingy cream and purely white to sordid white (Antonín and Noordeloos 2010; Retnowati 2018). However, M. subnigricans differs from M. thailandicus by having smaller basidia (24−32 × 8−8.8 μm), hygrophanous, subtranslucent to strongly translucent pileus, and longer stipe (13−50 × 1−3 mm), and larger basidiospores (13.6−17.6 × 4−5.6 μm). Marasmiellus candidus has white grooved, large pileus with a tendency to develop pinkish tinges at age, brown and swollen at the base, long, slender neck cheilocystidia, and caulocystidia, as well as large pileus (Antonín and Noordeloos 2010). Marasmiellus bicoloripes is distinguished from M. thailandicus by having smaller basidiospores (4−8 × 3−4 μm) and pileipellis without setae. Phylogenetically, M. thailandicus formed a monophyletic clade and separated other Marasmiellus species with strong statistical support.

Based on morphological characteristics, M. minutisporus is closely related to M. virgatocutis Robich, Esteve-Rav. & G. Moreno and M. griseobrunneus. However, M. virgatocutis is distinguished by the radially fibrillose-virgate pileus surface and the variable shape of cheilocystidia, which range from clavate to lageniform to molariform. While M. griseobrunneus has a surface of dark brown, the largest pileus (8−38 mm diam.), and observed pleurocystidia compared with M. minutisporus (Sharafudheen and Manimohan 2019). Phylogenetically, M. minutisporus is closely related to M. pilosus. However, M. minutisporus differs from M. pilosus in that it is grayish-brown, convex, wrinkled pileus with a fimbriate margin and the absence of pleurocystidia (Singer 1973).

Marasmiellus scandens was previously reported worldwide (in Asia, Africa, Oceania, and North America) as a pathogen and endophytic form in cocoa and coffee (Amoako-Attah et al. 2020; Ali et al. 2021; Lu et al. 2022; Zhang et al. 2023). In this study, the Thai specimen is similar to the descriptions of Dennis and Reid (1957) from M. scandens, originally described as Marasmius scandens by Massee (1910). However, Thai specimens mostly differed from Chinese specimens (HKAS-124582) (Zhang et al. 2023) in the shape of cheilocystidia with a broom-cell type. This may be influenced by the phenotypic variability across a wide geographic range.

Prior to the present study, eight species of Marasmiellus have been reported based on morphological characteristics in Thailand viz. M. albofuscus, M. alliiodorus, M. amygdalosporus, M. candidus, M. chamaecyparidis, M. collybioides, M. corticum, and M. paspali (Chandrasrikul et al. 2011; Seephueak et al. 2018). Most of them are widely found in Asia (China, Indonesia, and Japan), North America (Hawaii and Florida), South America (Argentina, Brazil, and Chile), the Caribbean (Cuba and the Lesser Antilles), and Oceania (Hongo 1975; Jackson and Firman 1982; Kohler et al. 1997). Some are only known in South America (M. alliiodorus) and West Africa (M. paspali) (Spegazzini 1889; Singer 1962; Pegler 1977; Buadu et al. 2002; Deng et al. 2011; Zhu et al. 2024). Historically, Thai macrofungi have been classified and described based on morphological characteristics. Of them, some are associated with the species previously known only in America, Europe, and other continents (Suwannarach et al. 2022a, b). Therefore, these eight species require further confirmation through newly collected specimens identified based on morphology and phylogeny. Additionally, this finding has increased the number of Marasmiellus species found in Thailand to 11. Finally, this finding is important in stimulating future studies of Marasmiellus in Thailand and contributes to distribution, diversity, phylogeny, and classification in Asia and worldwide.

Acknowledgments

The authors thank Shaun Pennycook for the nomenclatural advice and Russell Kirk Hollis for his help with the English correction.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by Chiang Mai University and National Higher Education, Science Research and Innovation Policy Council, Thaksin University Research Grant Fiscal Year 2023, Thailand. Nakarin Suwannarach and Samantha C. Karunarathna thank CMU Visiting Researcher 2024, Chiang Mai University (Grant number CMUVP038/2567), Thailand. Samantha C. Karunarathna and Saowaluck Tibpromma thank the National Natural Science Foundation of China (No. 32260004), Yunnan Revitalization Talents Support Plan (High-End Foreign Experts and Young Talents Programs), and the Key Laboratory of Yunnan Provincial Department of Education of the Deep-Time Evolution on Biodiversity from the Origin of the Pearl River. The authors extend their appreciation to the Deputyship for Research and Innovation, "Ministry of Education" in Saudi Arabia for funding this research (IFKSUOR3-299-19).

Author contributions

Conceptualization: NS, SCK. Data curation: WL, JK, PN. Investigation: WL, NS, JK, PN, ST, SCK. Project administration: NS, SCK. Software: WL, JK. Supervision: NS, SCK. Writing- review and editing: WL, NS, JK, PN, ST, AME, SCK.

Author ORCIDs

Wenhua Lu https://orcid.org/0000-0001-7283-7596

Pumin Nutaratat https://orcid.org/0000-0001-7924-4822

Jaturong Kumla https://orcid.org/0000-0002-3673-6541

Saowaluck Tibpromma https://orcid.org/0000-0002-4706-6547

Abdallah M. Elgorban https://orcid.org/0000-0003-3664-7853

Samantha C. Karunarathna https://orcid.org/0000-0001-7080-0781

Nakarin Suwannarach https://orcid.org/0000-0002-2653-1913

Data availability

All of the data that support the findings of this study are available in the main text

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