![Volvariella thailandensis SDBR-CMUNK0957 (holotype). Basidiomata (a); Basidiospore (b); Basidia (c); Pleurocystidia (d); Cheilocystidia (e); Pileipellis terminal elements (f). Scale bars: (a) = 10 mm; (b) = 5 µm; (c-f) = 10 µm. Additional specimens examined: THAILAND, Chiang Mai province, Chiang Mai University campus, 18 • 48 21 N 98 • 56 52 E, elevation 348 m, on soil in grassland, August 2020, J. Kumla and N. Suwannarach, SDBR-CMUNK0958, gene sequences OM417510 (ITS) and OM386669 (nrLSU). Note: Based on the morphology, the brownish orange to greyish brown pileus and size of V. thailandensis were similar to that of V. aethiops, V. caesiotincta, V. morozovae, V. murinella, V. pseudovolvacea, V. rava, and V. taylorii [9,18,32,36,48,50-52] (Table 2). However, the larger size of the basidiospores and basidia in V. thailandensis differs from](https://www.researchgate.net/profile/Jaturong-Kumla/publication/358851548/figure/fig1/AS:1128541504704513@1646076670676/Volvariella-thailandensis-SDBR-CMUNK0957-holotype-Basidiomata-a-Basidiospore-b_Q320.jpg)
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Citation: Kumla, J.; Suwannarach, N.;
Wannathes, N.; Lumyong, S. Survey
of Volvariella (Agaricales,Basidiomycota)
including Two New Species, V.
neovolvacea and V. thailandensis, from
Northern Thailand. Diversity 2022,14,
161. https://doi.org/10.3390/
d14030161
Academic Editor: Ipek Kurtboke
Received: 28 January 2022
Accepted: 21 February 2022
Published: 24 February 2022
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4.0/).
diversity
Article
Survey of Volvariella (Agaricales,Basidiomycota) including
Two New Species, V. neovolvacea and V. thailandensis, from
Northern Thailand
Jaturong Kumla 1, 2, *, Nakarin Suwannarach 1,2 , Nopparat Wannathes 3and Saisamorn Lumyong 1,2,4
1Research Center of Microbial Diversity and Sustainable Utilization, Chiang Mai University,
Chiang Mai 50200, Thailand; nakarin.su@cmu.ac.th (N.S.); saisamorn.l@cmu.ac.th (S.L.)
2Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
3Microbiology Program, Faculty of Science and Technology, Pibulsongkram Rajabhat University,
Phitsanulok 65000, Thailand; wnopparat@psru.ac.th
4Academy of Science, The Royal Society of Thailand, Bangkok 10300, Thailand
*Correspondence: jaturong.kumla@cmu.ac.th; Tel.: +66-87-192-6527
Abstract:
During the period from 2018 to 2020, a survey of macrofungi in northern Thailand was
conducted and seven specimens of Volvariella were collected. The morphological characteristics
and phylogenetic analyses of the internal transcribed spacers (ITS) and large subunit (nrLSU) of
the nuclear ribosomal DNA (rDNA) sequence data indicated that three specimens belonged to
V.bombycina
and
V.volvacea
. Four other specimens were distinct from all other known species within
the genus Volvariella. We described these specimens as two new species, namely V.neovolvacea and
V. thailandensis
. Comprehensive descriptions, illustrations, line drawings, and a phylogenetic tree are
provided to show the position of the two new species. The comparisons between morphologically
similar and phylogenetically related species are also discussed.
Keywords: gill fungi; phylogeny; saprophytic fungi; taxonomy; tropical area
1. Introduction
The genus Volvariella was first proposed in 1898 with V. argentina as the species type.
Volvariella species are known to be widely distributed throughout Africa, Asia, Australasia,
Europe, and America [
1
–
8
]. They are recognized as saprobes and are mostly found on
the ground in grasslands or forests. They have also been found to grow on dead wood
and on the litter of the forest floor [
7
–
10
]. Volvariella has been characterized by pluteoid
basidiomata, the presence of volva, a pink to pinkish brown spore print, ovoid to elongated
basidiospores with a smooth surface, and by appearing inamyloid [
10
–
12
]. Currently,
V. volvacea
is commercially cultivated as an edible mushroom in Asia, especially in China,
India, the Philippines, and Thailand [
13
,
14
]. Volvariella has been placed within the families
Amanitaceae [15]
,Agaricaceae [
16
], and Pluteaceae [
11
] of the order Agaricales. In 2006, this
genus was placed in the family Pluteaceae based on phylogenetic analyses [
17
]. However, a
later molecular study conducted by Justo et al. [
18
] separated the Volvariella species into two
genera, namely Volvariella and Volvopluteus.Volvopluteus was placed in the family Pluteaceae;
however, Volvariella formed a more distant position from the family Pluteaceae and was
more closely related to genera Cantharocybe and Cuphophyllus in the family Hygrophoraceae.
Therefore, Volvariella should be treated as incertae sedis of the order Agaricales [
18
]. According
to the Dictionary of Fungi by Kirk et al. [
6
] and He et al. [
19
], about 50 species of Volvariella
have been reported worldwide. However, there are 143 records of Volvariella names in the
Index Fungorum (Index Fungorum 2021, accessed on February 2022), including specific
and infraspecific taxa, illegitimate names, invalid names, and names widely accepted
as taxonomic synonyms. These names can include species that have not yet been well-
documented and may also include misidentifications.
Diversity 2022,14, 161. https://doi.org/10.3390/d14030161 https://www.mdpi.com/journal/diversity
Diversity 2022,14, 161 2 of 12
Only eight species of Volvariella, namely V. bombycina,V. cubensis,V. hypopithys,
V. pseudovolvacea
,V. pulla,V. pusilla,V.rostricystidiata, and V. volvacea, have been previ-
ously recorded from Thailand [
6
,
20
,
21
]. Many studies have proposed that Thailand has
proven to be a hot spot for novel species discovery [
22
]. Recently, this has been further
evidenced by the discovery of many new species of macrofungi [
23
–
25
]. In this study, we
found seven Volvariella specimens during our investigations of macrofungi in northern Thai-
land during the period from 2018 to 2020. Amongst these, two new species,
V.neovolvacea
and
V. thailandensis
, were introduced based on studies of their morphological and molec-
ular data. We used a combined analysis of ITS and nrLSU of the rDNA sequence data to
confirm the taxonomic status and infer the phylogenetic position of these two species.
2. Materials and Methods
2.1. Sample Collection
Volvariella was surveyed at Chiang Mai, Mae Hong Son, and Sukhothai provinces,
Thailand during the rainy seasons of the years 2018 to 2020. The collection in Sukhothai
province was granted permission from the Department of National Parks, Wildlife and
Plant Conservation, Bangkok, Thailand (document No. 0907.4/13696). Basidiomata were
collected and wrapped in aluminum foil and kept in plastic boxes. Specimens were dried
in a hot air oven at 45
◦
C until they were completely dried. They were then kept in a
plastic zip-locked bag and deposited in the Herbarium of Bangkok Forest Herbarium (BKF),
Bangkok, Thailand and Sustainable Development of Biological Resources (SDBR-CMU),
Faculty of Science, Chiang Mai University, Thailand. MycoBank numbers are provided [
26
].
2.2. Morphological Observation
Macromorphological data were derived from fresh specimens. Color names and
codes followed Kornerup and Wanscher [
27
]. Micromorphological data were derived
from dried specimens that were rehydrated in 95% ethanol, followed by distilled water,
5% KOH
, Congo red solution, or Melzer’s reagent. Micromorphological characteristics
were examined under a light microscope (Nikon Eclipse Ni U, Tokyo, Japan). Size data of
the anatomical structure (e.g., basidiospores, basidia, and cystidia) are based on at least
50 measurements
of each structure using the Tarosoft (R) Image Frame Work program. For
basidiospore statistics, measurements are presented as (a)b–c–d(e), where ‘a’ and ‘e’ are
the extreme values, ‘b–d’ is the range containing 90% of all values, and ‘c’ is the average.
Q represents
the ratio of length divided by the width of each basidiospore and Q represents
the average Q of all specimens ±standard deviation.
2.3. DNA Extraction, Amplification and Sequencing
Genomic DNA of dry specimens (5 mg) was extracted using a Genomic DNA Ex-
traction Mini-Kit (FAVORGEN, Ping-Tung, Taiwan). The ITS region was amplified by
polymerase chain reaction (PCR) using ITS4 and ITS5 primers [
28
] under the following
thermal conditions: 95
◦
C for 2 min, 30 cycles of 95
◦
C for 30 s, 50
◦
C for 30 s, 72
◦
C for
1 min, and 72
◦
C for 10 min on a peqSTAR thermal cycler (PEQLAB Ltd., Fareham, UK).
The nrLSU region was amplified with LROR and LRO5 primers [
29
] under the following
thermal conditions: 94
◦
C for 2 min, 30 cycles of 95
◦
C for 30 s, 52
◦
C for 30 s,
72 ◦C
for
1 min, and 72 ◦C for 10 min. 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, Düren, 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.
Diversity 2022,14, 161 3 of 12
2.4. Sequence Alignment
Analysis of the sequences was conducted by similarity searches using the BLAST
program available at NCBI (http://blast.ncbi.nlm.nih.gov, accessed on 21 December 2021).
The sequences from this study, previous studies, and the GenBank database (with
≥
60%
query coverage and
≥
85–100% sequence similarity) were selected and listed in Table 1.
Multiple sequence alignment was performed with MUSCLE [
30
]. The alignments were
manually checked. The combined ITS and nrLSU alignment was deposited in TreeBASE
under the study ID number 29067.
Table 1. Details of sequences used in the molecular phylogenetic analysis.
Taxa Strain/Voucher Country GenBank Accession Number
Reference
ITS nrLSU
Cantharocybe virosa SDBR-CMUNK0280 Thailand MG694692 MG694690 [31]
Cuphophyllus yacurensis TQCAM5891 Ecuador NR166388 KY780119 [32]
Volvariella aethiops K(M):195631
United Kingdom
MZ159529 – Unpublished
Volvariella bombycina AJ244 Spain HM562212 HM562256 [18]
Volvariella bombycina SDBR-CMUNK0726 Thailand OM417508 OM373644 This study
Volvariella bombycina xj2018071604 China MT351048 – Unpublished
Volvariella caesinotincta MA54717 Spain HM562211 HM562255 [18]
Volvariella diplasia CBS 355.64 India MH858454 MH870086 [21]
Volvariella dunensis JAC10587 New Zealand MN738630 MN738630 Unpublished
Volvariella dunensis SCM3513 Spain JF415140 – [33]
Volvariella guttulosa TLE 313326 Vietnam – NG068890 [4]
Volvariella hypopithys TO AV137 Italy HM246492 HM246488 Unpublished
Volvariella krizii TPR1516257 Czech Republic – MK770133 [34]
Volvariella krizii BRNM 290000 Czech Republic – MK770133 [34]
Volvariella lepiotospora AJ155 USA HM562214 HM562259 Unpublished
Volvariella lepiotospora FLAS-F-61505 USA MH281874 – Unpublished
Volvariella morozovae TLE 313229 Vietnam MF377507 MF377508 [32]
Volvariella morozovae LE 313323 Vietnam MK882995 – [32]
Volvariella murinella GLM:GLM-F42624 Germany MK412400 MK412400 Unpublished
Volvariella murinella GLM:GLM-F43392 Germany MK412361 – Unpublished
Volvariella neovolvacea TSDBR-CMUNK0758 Thailand OM417503 OM373653 This study
Volvariella neovolvacea SDBR-CMUNK0760 Thailand OM417505 OM417507 This study
Volvariella nivea TGDGM25489 China FJ749127 – [35]
Volvariella niveosulcata TLE 313329 Vietnam MK882996 MK883002 [4]
Volvariella nullicystidiata SP393639 Brazil EU920671 EU920670 [3]
Volvariella perciliata SP393635 Brazil EU920672 EU920668 [3]
Volvariella ptilotricha TLE 313324 Vietnam NR164612 MK882997 [4]
Volvariella pulla TLE 313325 Vietnam NR164613 MK883003 [4]
Volvariella pulla MFLU 19-1534 Thailand MT074696 – [6]
Volvariella pusilla TO AV139 Italy HM246494 HM246479 Unpublished
Volvariella rava TGDGM41955 China KP784686 – [36]
Volvariella reidii F1-1.1 Finland MK770139 MK770131 [34]
Volvariella reidii F1-1.2 Finland MK770140 MK770132 [34]
Volvariella rostricystidiata TMFLU 19-1528 Thailand MT074694 – [6]
Volvariella rostricystidiata MFLU 19-1531 Thailand MT074695 – [6]
Volvariella cf. sathei Hama193 Niger KF926663 – [37]
Volvariella sathei AMH 9436 India JN792550 – [38]
Volvariella strangulata TO AV141 Italy HM246493 HM246484 Unpublished
Volvariella surrecta GLM:GLM-F61563 Germany MK412358 MK412358 Unpublished
Volvariella surrecta AJ55 Spain HM562213 HM562254 [18]
Volvariella taylorii AJ54 Portugal HM562210 HM562260 [18]
Volvariella terrea LUG11010 France JF415141 – [33]
Volvariella thailandensis TSDBR-CMUNK0957 Thailand OM417510 OM417509 This study
Volvariella thailandensis SDBR-CMUNK0958 Thailand OM417510 OM386669 This study
Diversity 2022,14, 161 4 of 12
Table 1. Cont.
Taxa Strain/Voucher Country GenBank Accession Number
Reference
ITS nrLSU
Volvariella turcica TOKA-TR17.1 Turkey MK770135 MK770128 [34]
Volvariella turcica OKA-TR17.2 Turkey MK770136 MK770129 [34]
Volvariella volvacea H6 Nigeria KC894929 – [39]
Volvariella volvacea OE-273 India KC14211 – Unpublished
Volvariella volvacea BKF10217 Thailand MN492647 OM373563 This study
Volvariella volvacea SDBR-CMUNK505 Thailand OM417506 OM373623 This study
Superscript “T” represents type species. “–” represents the absence of sequence data in GenBank database.
2.5. Phylogenetic Analyses
Phylogenetic analysis was carried out based on the combined dataset of ITS and nrLSU.
Cantharocybe virosa and Cuphophyllus yacurensis were used as the outgroup.
A phylogenetic
tree was constructed under maximum likelihood (ML) and Bayesian inference (BI) methods.
ML analysis was carried out using RAxML-HPC2 version 8.2.10 [
40
] on the CIPRES web
portal [41]
under the GTRCAT model with 25 categories and 1000 bootstrap
replications [42]
.
BI analysis was performed with MrBayes
v3.2.6 [43]
. The best substitution models for ML
and BI analyses were estimated by Akaike information criterion (AIC) in jModelTest
2.1.10 [
44
]. The best substitution models were GTR+I+G for ITS and nrLSU. For the BI
analysis, six simultaneous Markov chains were run for one million generations with random
initial trees, and every 1000 generations were sampled. The burn-in was set to discard the
first 2000 of the trees, and the remaining trees were used to construct the 50% majority-rule
consensus phylogram with calculated Bayesian posterior probabilities (PP). Branches with
bootstrap support (BS) and PP values greater than or equal to 70%
and 0.95
, respectively,
were considered to be significantly supported [
45
,
46
]. The tree topologies were visualized
in FigTree v1.4.0 [47].
3. Results
3.1. Sample Collection and Morphological Observation
A total of seven specimens of Volvariella were obtained in this study. Morphologi-
cally, the specimen voucher SDBR-CMU505 collected from Chiang Mai province and the
voucher BKF10217 collected from Sukhothai province were found to be similar to the
morphological descriptions of V.volvacea. Notably, SDBR-CMUNK0726 collected from Mae
Hong Son province was initially identified as belonging to V.bombycina. However, the
morphological characteristics of the other four specimen vouchers (SDBR-CMUNK0758,
SDBR-CMUNK0760, SDBR-CMUNK0957, and SDBR-CMUNK0958) collected from Chiang
Mai province differed from the previously known descriptions of Volvariella species. These
four specimen vouchers were representative of a potential new species. Therefore, the
identification was then further confirmed by the multi-gene phylogenetic analyses of ITS
and nrLSU sequences.
3.2. Phylogenetic Results
The sequences of seven specimens were deposited in the GenBank database (Table 1).
The alignment of a combination of ITS and nrLSU genes contained 1989 characters, includ-
ing gaps (ITS: 1–1039 and nrLSU: 1040–1989). RAxML analysis of the combined dataset
yielded the best scoring tree with a final ML optimization likelihood value of
−
13,839.8771.
The matrix contained 1030 distinct alignment patterns with 49.73% undetermined char-
acters or gaps. The estimated base frequencies were recorded as follows: A = 0.2255,
C = 0.2599
,
G = 0.2697
, T = 0.2448; substitution rates AC = 1.1148, AG = 2.3791, AT = 0.9787,
CG = 0.8637
,
CT = 5.0398
,
GT = 1.0000
. The gamma distribution shape parameter alpha
was equal to 0.3033, and the tree length was equal to 4.9110. In addition, the final average
standard deviation of the split frequencies at the end of the total MCMC generations was
Diversity 2022,14, 161 5 of 12
calculated as 0.00826 through BI analysis. Phylograms of the ML and BI analyses were
similar in terms of topology (data not shown). Therefore, the phylogram obtained from the
ML analysis was selected and presented in this study. The phylogram was comprised of
50 specimens of Volvariella and two specimens of the outgroup (Cantharocybe virosa SDBR-
CMUNK0280 and Cuphophyllus yacurensis QCAM5891) (Figure 1). A phylogram clearly
separated our four specimens into two monophyletic clades with high support values
(
BS = 100%
and PP = 1.0). Accordingly, they were clearly distinguished from the previously
known species of Volvariella. Two specimen vouchers, namely SDBR-CMUNK0758 and
SDBR-CMUNK0760 (introduced as V.thailandensis), formed a sister clade to V.sathei with
high support (BS = 100% and PP = 1.0). Moreover, the two specimens in this study, SDBR-
CMUNK957 and SDBR-CMUNK95 (described here as V.neovolvacea), formed a sister clade
to V.thailandensis and V.sathei with 91% BS and 1.0 PP support values. Additionally, the
other obtained specimen vouchers, SDBR-CMUNK505 and SDBR-NW1022, were placed
within the same clade of V. volvacea, while the specimen voucher SDBR-CMUNK0726 was
placed within the clade of V.bombycina.
3.3. Taxonomic Description of New Species
Volvariella neovolvacea
J. Kumla, N. Suwannarach and S. Lumyong, sp. nov. Figure 2.
MycoBank: 842646.
Diagnosis: Similar to V.volvacea but differing in a wider basidiospores.
Etymology: Name derived from the macromorphological similarity to V. volvacea.
Holotype: THAILAND, Chiang Mai province, Chiang Mai University campus,
18◦480600 N
98
◦
57
0
23
00
E, elevation 331 m, on soil in grassland, July 2020, J. Kumla and N. Suwannarach,
SDBR-CMUNK0758.
Gene sequences (from holotype): OM417503 (ITS) and OM373653 (nrLSU).
Pileus 65–80 mm in diameter, convex, umbo and slightly inflexed margin, brownish
orange (5C3) to greyish brown (6E3–7E3), non-hygrophanous, with fibrillose to appressed
squamulose surface; margin decurved to straight, thin, crenulate; context soft, white (4A1)
to light yellow (4A5). Lamellae free, ventricose, close, moderately thin, soft, orange white
(6A2) turning pinkish white (7A2) with age; edge finely fimbriate, concolorous with faces;
lamellulae present, very variable in length, mostly in one tier. Stipe 85–110
×
20–25 mm,
central, tapered from the base to apex, solid, grey (6B1), entirely pubescent; context white
(4A1) to light yellow (4A5); annulus absent. Volva 30–50 mm high, thick, mostly free,
saccate, fragile, white (6A1) to greyish brown (6E3). Spore print pinkish (7A2).
Basidiospores (6.5)6.7–7–7.5(8)
×
(5)5.2–5.5–6(7)
µ
m (n = 50), Q = 1.14–1.45,
Q= 1.28 ±0.1
,
broadly ellipsoid to ellipsoid, thick-walled, hyaline, inamyloid. Basidia
27–40 ×7.5–11 µm
,
narrowly to broadly clavate, hyaline, 4-spored; sterigmata up to 2.5
µ
m long.
Pleurocystidia
45–98 ×23–77 µm
, clavate to broadly lageniform, thin-walled, hyaline.
Cheilocystidia
29–57 ×9–22 µm
, clavate to broadly lageniform, thin-walled, hyaline. Hymenophoral trama
inverse, made up of thin-walled hyaline hyphae,
5–20 µm
wide. Pileipellis a cutis, with ter-
minal elements
37–98 ×8–16 µm
, variable in size, narrowly clavate to clavate, sometimes
with a narrowed or slightly strangulated apex, colorless or with pale brown intracellular
pigment. Stipitipellis a cutis, terminal elements 22–60
×
8–15
µ
m, cylindrical, sometimes
with a narrowed or slightly strangulated apex, colorless or with pale brown intracellular
pigment. Clamp connections absent in all tissue types.
Ecology and distribution: Fruiting solitary or gregarious on soil in grassland. Known
only from northern Thailand.
Additional specimens examined: THAILAND, Chiang Mai province, Chiang Mai
University campus, 18
◦
48
0
7
00
N 98
◦
57
0
23
00
E, elevation 331 m, on soil in grassland, July 2020,
N. Suwannarach and J. Kumla, SDBR-CMUNK0760, gene sequences OM417505 (ITS) and
OM417507 (nrLSU).
Diversity 2022,14, 161 6 of 12
Figure 1.
Phylogram derived from maximum likelihood analysis of 50 specimens of the combined
ITS and nrLSU genes. Cantharocybe virosa and Cuphophyllus yacurensis were used as the outgroup. The
numbers above branches represent bootstrap percentages (left) and Bayesian posterior probabilities
(right). Bootstrap values
≥
75% and Bayesian posterior probabilities
≥
0.90 are shown. The scale bar
represents the expected number of nucleotide substitutions per site. Sequences obtained from this
study are in red. Type species are in bold.
Diversity 2022,14, 161 7 of 12
Figure 2.
Volvariella neovolvacea SDBR-CMUNK0758 (holotype). Basidiomata (
a
);
Basidiospore (b)
;
Basidia (c)
;
Pleurocystidia (d)
; Cheilocystidia (
e
); Pileipellis terminal
elements (f)
. Scale bars:
(a) = 10 mm; (b)=5µm; (c–f) = 10 µm.
Note: Morphologically, the size of the pileus (65–80 mm in diameter) in V. neovolvacea
was clearly larger than those of the brownish orange to greyish brown Volvariella species,
namely V. aethiops (30–50 mm in diameter) [
48
], V. caesiotincta (44 mm in
diameter) [49]
,
V. morozovae
(30–40 mm in diameter) [
32
], V. murinella (30–60 mm in
diameter) [50,51]
,
V. pseudovolvacea
(30–60 mm in diameter) [
9
], V. thailandensis (35–60 mm in diameter), and
V. taylorii
(20–50 mm in diameter) [
51
] (Table 2). In addition, V. bakeri, distributed only in
the USA, has a larger pileus size (100 mm in diameter) than V. neovolvacea [
2
,
52
]. However,
the basidiomata size of V. neovolvacea was similar to
V. pulla
,
V. rostricystidiata
,
V. terastia
,
and
V. volvacea
[
4
,
6
,
9
,
51
]. The smaller size of the basidiospores in V. rostricystidiata and
V. terastia clearly distinguishes them from V. neovolvacea [
6
,
9
]. Additionally, the basid-
iospores of
V. neovolvacea
(6.5–8.0
×
5.0–7.0
µ
m, Q= 1.28) were wider than
V. volvacea
(
6.0–9.0 ×4.7–5.7 µm
,Q= 1.42) [
9
,
15
]. The wider size of pleurocystidia in V. neovolvacea
clearly differed from V. pseudovolvacea,V. pulla,V. rostricystidiata, and V. terastia [4,6].
Diversity 2022,14, 161 8 of 12
Table 2. Comparison of V. neovolvacea and V. thailandensis with the closely related species.
Volvariella
Species Distribution Pileus (mm in
Diameter)
Basidiospore
(µm)
Basidia
(µm)
Pleurocystidia
(µm)
Cheilocystidia
(µm) Reference
V. aethiops France 30–50 7.0–11 ×3.7–7.2 NR
60–100
×
10–25 60–100
×
10–25
[48]
V. bakeri USA 100 6.9–9.3 ×4.6–6.9 21–35 ×7–11 47–71 ×11–31 27–71 ×11–31 [2,52]
V. caesiotincta Europe and
North Africa 44 6.2–7.7 ×4.2–5.4 21–28 ×6.5–9 35–46 ×10–25 34–65 ×9.6–19 [18,49]
V. morozovae Vietnam 30–40 5.0–6.2 ×3.3–4.3
13.5–20
×
7–8.5
45–50 ×18–30 40–50 ×13–18 [32]
V. murinella Europe
and Vietnam 30–60 4.8–5.6 ×4.9–6.6 19–25 ×6–7 20–30 ×10–15 45–65 ×8–15 [50,51]
V. neovolvacea Thailand 65–80 6.5–8.0 ×5.0–7.0 27–40 ×7.5–11 45–98 ×23–77 29–57 ×9–22 This study
V. pseudovolvacea
Sri Lanka 30–60 4.2–5.2 ×2.5–3.5
14–16
×
4.5–6.5
32–45 ×12–16 30–38 ×12–14 [9]
V. pulla Thailand
and Vietnam 40–75 5.5–8.0 ×4.0–5.0 16–19.5 ×7–9 25–56 ×6–32 24–87 ×9–17 [4,6]
V. rava China 25–60 4.0–6.5 ×4.0–5.0 24–30 ×7–10 35–71 ×11–30 55–80 ×15–25 [36]
V. rostricystidiata
Thailand 55–85 5.0–7.0 ×3.5–5.5 17.5–29 ×7–10
41–117
×
10–25
51–80 ×14–39 [6]
V. taylorii
Europe, South
Korea, USA
and Vietnam
20–50 5.0–7.0 ×3.0–4.8 20–24 ×6–8 35–49 ×8–10 28–43 ×9–13 [18,51,52]
V. terastia Sri Lanka 60–110 5.0–6.5 ×4.2–5.2 17–23 ×5.5–7 35–50 ×15–22 32–36 ×10–13 [9]
V. thailandensis Thailand 35–50 5.5–7.0 ×4.0–6.0 15–35 ×6–8 35–77 ×17–30 32–48 ×6–10 This study
V. volvacea Asia and
East Africa 50–100 6.0–9.0 ×4.7–5.7
30–35
×
7.5–9.0 45–120
×
17–30
40–70 ×10–15 [9,51],
This study
“NR” = not reported.
Our phylogenetic results from the combined ITS and nrLSU sequences confirmed
that V. neovolvacea formed a monophyletic clade, which clearly separated it from the other
morphologically related Volvariella species (Figure 1). The phylogenetic analysis also sup-
ports the determination that V. neovolvacea and V. volvacea are different species. Volvariella
neovolvacea forms a sister taxon to V. sathei. However, the white to yellowish white pileus of
V. sathei clearly separates it from V. neovolvacea [
38
]. The ITS sequence of
V. neovolvacea
is
95.64% and 94.00% similar to V. sathei (JN792550) and
V. cf. sathei
(KF926663), respectively.
Volvariella thailandensis
N. Suwannarach, J. Kumla, and S. Lumyong, sp. nov.
Figure 3.
MycoBank: 842647.
Diagnosis: Similar to V.taylorii but differing in a wider pleurocystidia and narrower
cheilocystidia.
Etymology: Name referred to Thailand, where the new species was found.
Holotype: THAILAND, Chiang Mai province, Chiang Mai University campus,
18◦4802200 N
98
◦
56
0
51
00
E, elevation 348 m, on soil in grassland, August 2020, J. Kumla and N. Suwan-
narach, SDBR-CMUNK0957.
Gene sequences (from holotype): OM417510 (ITS) and OM417509 (nrLSU).
Pileus 35–50 mm in diameter, convex to plano-convex with a low broad umbo, greyish
brown (6E3–7E3) with slightly darker color at the center, non-hygrophanous, with fibrillose
to appressed squamulose surface; margin decurved to straight, thin, crenulate; context
soft, light yellow (4A5). Lamellae free, ventricose, close, moderately thin, soft, pale orange
(6A3) turning brownish orange (6C6) with age; edge finely fimbriate, concolorous with
faces; lamellulae present, very variable in length, mostly in one tier. Stipe 40–65
×
4–5 mm,
central, tapered from the base to apex, solid, greyish brown (6E3) to dark brown (6F5),
slightly paler near the base, entirely pubescent; context light yellow (4A5); annulus absent.
Volva 15–18 mm high, thick, mostly free, saccate, fragile, white (6A1) to greyish brown
(6D4). Spore print pinkish (7A2).
Basidiospores (5.5)6–7–6.5(7)
×
(4)4.4–5–5.5(6)
µ
m (n = 50),
Q = 1.16–1.50
,
Q= 1.30 ±0.1
,
subglobose to broadly ellipsoid, thick-walled, hyaline, inamyloid. Basidia
15–35 ×6–8 µm
,
narrowly to broadly clavate, hyaline, four-spored; sterigmata up to 3
µ
m long.
Pleurocystidia
35–77 ×17–30 µm
, broadly fusiform to lageniform, thin-walled, hyaline.
Cheilocystidia
32–48 ×6–10 µm
, narrowly to broadly clavate, broadly lageniform, thin-walled, hya-
line. Hymenophoral trama inverse, made up of thin-walled hyaline hyphae,
3–10 µm
wide.
Pileipellis
a cutis, with terminal elements 65–150
×
4–16
µ
m, variable in size, nar-
Diversity 2022,14, 161 9 of 12
rowly clavate to clavate, sometimes with a narrowed or slightly strangulated apex, col-
orless or with pale brown intracellular pigment. Stipitipellis a cutis, terminal elements
35–80 ×6–9 µm
, cylindrical, sometimes with a narrowed or slightly strangulated apex, col-
orless or with pale brown intracellular pigment. Clamp connections absent in all
tissue types
.
Figure 3.
Volvariella thailandensis SDBR-CMUNK0957 (holotype). Basidiomata (
a
);
Basidiospore (b)
;
Basidia (
c
); Pleurocystidia (
d
); Cheilocystidia (
e
); Pileipellis terminal elements (
f
). Scale bars:
(a) = 10 mm; (b)=5µm; (c–f) = 10 µm.
Additional specimens examined: THAILAND, Chiang Mai province, Chiang Mai
University campus, 18
◦
48
0
21
00
N 98
◦
56
0
52
00
E, elevation 348 m, on soil in grassland, August
2020, J. Kumla and N. Suwannarach, SDBR-CMUNK0958, gene sequences OM417510 (ITS)
and OM386669 (nrLSU).
Note: Based on the morphology, the brownish orange to greyish brown pileus
and size of V. thailandensis were similar to that of V. aethiops,
V. caesiotincta
,
V. morozovae
,
V. murinella
,
V. pseudovolvacea
,V. rava, and V. taylorii [
9
,
18
,
32
,
36
,
48
,
50
–
52
] (Table 2). How-
ever, the larger size of the basidiospores and basidia in V.thailandensis differs from
Diversity 2022,14, 161 10 of 12
V. pseudovolvacea [9]
. The longer and shorter sizes of basidiospores in V. thailandensis
clearly distinguishes it from V. murinella [
50
,
51
] and V. aethiops [
48
], respectively. Ad-
ditionally, the pleurocystidia (
35–77 ×17–30 µm
) of V. thailandensis were found to be wider
than of the
V. taylorii
(
35–49 ×8–10 µm
) found in Europe, South Korea, the USA, and
Vietnam [18,51,52]
. The wider cheilocystidia in V. caesiotincta (34–65
×
9.6–19
µ
m) [
18
,
49
],
V. morozovae (40–50 ×13–18 µm) [32]
,V.pulla (24–87
×
9–17
µ
m) [
4
,
6
],
V. rava
(55–80
×
15–25 µm) [36]
, and
V. taylorii (28–43 ×9–13 µm) [18,51,52]
clearly distinguishes them from
V. thailandensis (32–48 ×6–10 µm).
The phylogenetic analyses of the combined ITS and nrLSU sequences confirmed that
V. thailandensis formed a monophyletic clade, which clearly separated it from the other
Volvariella species (Figure 1). Volvariella thailandensis is closely related to V. neovolvacea
and V. sathei. However, it is morphologically quite different from V. sathei which has
white to yellowish white pileus [
38
]. Moreover, V. thailandensis is characterized by smaller
basidiomata and wider cheilocystidia than V. neovolvacea (Table 2).
4. Discussion
Volvariella is widely distributed in both temperate and tropical areas throughout
the world [
2
,
5
–
8
,
53
]. Traditionally, morphological characteristics have been used in the
identification of Volvariella species [
5
,
7
,
9
,
11
]. However, identification can be difficult as
some species have similar features. Thus, effective identification can be limited by certain
morphological characteristics as well as the different environmental conditions that affect
those morphological characteristics. Over the last two decades, molecular phylogeny has
been an essential tool in the identification of Volvariella species [
3
,
4
,
6
,
10
,
17
–
19
,
21
,
32
–
39
].
Thus, the current classification of the genus Volvariella has been based on the combined
data of the morphological characteristics and molecular data.
In 2011, six Volvariella species (V.bombycina,V.cubensis,V.hypopithys,V.pseudovolvacea,
V.pusilla, and V.volvacea) have been recorded by mycologists in Thailand according to
their morphological characteristics. However, in this regard, there is a lack of available
molecular data [
20
]. Molecular data of V.volvacea from Thai specimen has been reported
by
Vu et al. [21]
. In 2021, a new species (V.rostricystidiata) and a new record (V.pulla)
of Volvariella were reported from Thailand by Niego et al. [
6
]. In this study, two new
species of Volvariella (V.thailandensis and V.neovolvacea) and two previously known species
(
V. bombycina
and V.volvacea), collected from northern Thailand, were identified based
on their morphological characteristics and phylogenetic analyses. Thus, the number of
Volvariella species recorded in Thailand has risen to a total of ten species, of which six species
were confirmed by morphological and molecular evidence. Nevertheless, four Volvariella
species listed by Chandrasrikul et al. [
20
] require further confirmation by molecular data.
To our knowledge, our discovery is considerably important in terms of stimulating a deeper
investigation of macrofungi in Thailand, and will help researchers better understand the
distribution and ecology of Volvariella.
Author Contributions:
Conceptualization, J.K., N.S. and N.W.; methodology J.K. and N.S.; software,
J.K.; validation, J.K., N.S., N.W. and S.L.; formal analysis, J.K. and N.W.; investigation, J.K., N.S.
and N.W.; resources, J.K., N.S. and N.W.; data curation, J.K., N.S. and N.W.; writing—original draft
preparation, J.K. and N.S.; writing—review and editing, J.K. and N.S.; project administration, J.K., N.S.
and N.W.; supervision, J.K., N.S., N.W. and S.L. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was supported by Chiang Mai University, Thailand.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The DNA sequence data obtained from this study have been deposited
in GenBank under accession numbers; ITS (MN492647, OM417503, OM417505, OM417506, OM417508,
Diversity 2022,14, 161 11 of 12
OM417510 and OM417513) and nrLSU (OM373563, OM373623, OM373644, OM373653, OM386669,
OM417507 and OM417509).
Acknowledgments:
The authors are grateful to the staff of Si Satchanalai National Park for their
excellent field assistance and to Russell Kirk Hollis for kind help in the English correction.
The authors
are also grateful to Surapong Khuna and Netethip Khamkiti for phylogram preparation.
Conflicts of Interest: The authors declare no conflict of interest.
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