Leucoagaricus houaynhangensis (Agaricaceae), A New Yellowish-green Species from Lao People’s Democratic Republic

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Chiang Mai J. Sci. 2018; 45(3) 1287
Chiang Mai J. Sci. 2018; 45(3) : 1287-1295
http://epg.science.cmu.ac.th/ejournal/
Contributed Paper
Leucoagaricus houaynhangensis (Agaricaceae),
A New Yellowish-green Species from Lao People’s
Democratic Republic
Phongeun Sysouphanthong* [a], Somsanith Bouamanivong [a], Thaviphone Salichanh [a],
Nakhonekham Xaybouangeun [b], Phuping Sucharitakul [c], Maslin Osathanunkul [c] and
Chatmongkon Suwannapoom [d]
[a] Ecology Division , Biotechnology and Ecology Institute, Ministry of Science and Technology,
P.O.Box: 2279, Vientiane Capital, Lao PDR.
[b] National Science Council’s Office, Ministry of Science and Technology, P.O.Box: 2279, Chanthabuly,
Vientiane Capital, Lao PDR.
[c] Department of Biology, Faculty of Science, Chiang Mai University, 239 Huay Kaew Rd., Suthep, Muang,
Chiang Mai Province 50200, Thailand.
[d] School of Agriculture and Natural Resources, University of Phayao, Phayao Province 56000, Thailand.
* Author for correspondence; e-mail: laofungi@gmail.com
Received: 6 October 2016
Accepted: 7 September 2017
ABSTRACT
Leucoagaricus houaynhangensis, a yellowish-green agaric species, is described as a new
species from Laos. It is characterised by light yellow to yellowish-green basidioma, covered
with dark to dark grayish-brown squamules on pileus, free lamellae, subcylindrical and smooth
stipe, membranous and fragile annulus, oblong ovoid and hyaline basidiospore with a germ
pore, clavate and 4-spored basidia, clavate to utriform cheilocystidia with slightly a long apical
appendage, a hymeniderm pileus covering made up of cylindrical elements, a cutis stipe covering
made up of cylindrical elements and absence of clamp-connections in all tissue. Photographs
on field and illustrator line drawing of microscopic characters are given. Two known species,
La. flavovirens J.F. Liang, Zhu L. Yang & J. Xu and La. viridivavus (Petch) T.K.A. Kumar &
Manim., share some morphological similarities with the newly described taxon here, but they
difference by both morphological characters and nrITS DNA.
Keywords: Basidiomycota, diversity ⋅ distribution, lepiotaceous fungi, phylogeny, taxonomy
1. INTRODUCTION
The genus Leucoagaricus (Locq. ex) Singer
belongs to the family Agaricaceae, which
comprises about 90 species that are widely
distributed throughout the world [1-3].
Leucoagaricus is a genus of lepiotaceous fungi;
several recognized macrocharacters including
pluteoid basidiomata, lack of or rarely sulcate
pileus, free lamellae, presence of annulus;
and microcharacters like smooth or rough
basidiospores with or without a germ pore
and dextrinoid, metachromatic, congophilous,
cyanophilous, and absence of clamp-

1288 Chiang Mai J. Sci. 2018; 45(3)
connections in all tissue [2-4]. Locquin [5]
placed Leucoagaricus in genus Leucocopinus as
subgenus Leucoagaricus Locquin without Latin
description, and Singer [2] later provided a
valid Latin diagnosis and gave the genus level
for Leucoagaricus. However, some mycologist
treat it under genus Lepiota sensu lato [6-7],
and some species of Leucoagaricus were placed
into genus Lepiota (Pers.) Gray and Leucocoprinus
Pat. in some study such as Adhikari [8-9],
Berkeley and Broome [10], Dennis [11],
Kumar and Manimohan [12], Manandha
and Adhikari [13], Natarajan et al. [14],
Pegler [15], and Petch [16, 17]. For accepted
classifications of lepiotaceous fungi,
Leucoagaricus is clearly separated from other
genera with independent morphology
[4, 18-19]. However, molecular data
demonstrate that Leucoagaricus is polyphyletic,
and Leucoagaricus and Leucocoprinus are
clustered in phylogenetic analysis [4, 20-21].
The colour changes are important for
classification in lepiotaceous fungi; for
instance, in some species which turn green,
blue or red in ammonia vapour have been
placed in both Leucoagaricus and Leucocoprinus,
and some species with sericeous pileus
covering have placed in genus Sericeomyces
[3, 21-23]. Colour changes are also useful
for classifying at species level; for example,
some species turn blue when they are touched
or dried such as La. flavovirens J.F. Liang,
Zhu L. Yang & J. Xu, La. viriditinctus (Berk. &
Broome) J.F. Liang, Zhu L. Yang & J. Xu and
La. viridiflavoides Akers & Angels [21, 24-26].
In this study, we would like to provide a
new species of Leucoagaricus with uniquely
yellowish-green colour as new for science.
2. MATERIALS AND METHODS
2.1 Collecting and Materials Examination
Three specimens of the new species
were collected from Houay Nhang Preserved
Forest in Xaythany District, Vientiane
Capital (18°05’31.7″N 102°40’34.2″E, 189 m)
[27]. Coordinate of locations, forest types,
soil and substrate were recorded, and then
fresh basidiomata were photographed on
fields. Morphological character notes were
following Vellinga [3]. Colour annotations of
fresh materials were determined based on
the colour charts of Kornerup and Wanscher
[28]. Specimens were dried in a hot air
dryer (30-40 °C) for 24 hours, before being
deposited in the Herbarium National of
Laos (HNL). Microscopic characters were
observed and illustrated from dry specimens
using a microscope. Original colour of spores
was observed in water and 2.5-10% of KOH;
spore wall reaction was observed in melzer’s
reagent, cotton blue and cresyl blue; and line
drawings were observed in Congo red.
Each collection was investigated and 25
basidiospores measured per collection. The
notation (75, 3, 3) indicated that measurements
were made on 75 basidiospores in three
samples in three collections, and the size
average was given in the description while
quotient (Q) of length and width average
quotient were also calculated to indicate
basidiospore shape. The technical term used
for description was following Vellinga [3].
2.2 Phylogenetic Study
DNA was extracted from dried
herbarium collections according to the
instructions of the Biospin Fungus Genomic
DNA Extraction Kit (Bioer Technology Co.,
Ltd., Hangzhou, P.R. China). Three collections
were investigated for sequences of internal
transcribed spacer 1 and 2 (ITS1 & ITS2).
The primers ITS1-F and ITS4 were used for
PCR [29]. The PCR amplified products were
purified and sequenced by Shanghai Sangon
Biological Engineering Technology & Services
Co., Ltd. Sequences were edited and contigs
assembled using SeqMan program (DNAStar,
Madison, WI, USA) and then all new

Chiang Mai J. Sci. 2018; 45(3) 1289
sequences were deposited in GenBank. The
sequences were performed with Basic Local
Alignment Search Tool (BLAST) in National
Center for Biotechnology Information
(NCBI) of USA database (http://www.ncbi.
nlm.nih.gov/genbank/).
Thirty-two sequences related to the
new taxon were obtained from the GenBank,
and a sequence of Agaricus bisporus (J.E. Lange)
Imbach was used as outgroup (Table 1).
A complete data set was aligned using MAFFT
version 7.130-win32 [30, 31]. Maximum
Likelihood analysis were executed using
software MEGA version6 [32, 33], and the
settings were: Maximum Likelihood as
statistical method, 1000 bootstrap replications,
using Hasegawa-Lishino-Yano model,
Gamma distributed with Invariable sites
(G+I) as rates among sites, and Nearest-
Neighbor-Interchange (NNI) as Initial Tree
for ML. The evolutionary history was inferred
by using the Maximum Likelihood method
based on the Hasegawa-Kishino-Yano model,
the tree with the highest log likelihood
(-1716.2824) was shown, the percentage of
trees in which the associated taxa clustered
together was shown next to the branches,
initial tree(s) for the heuristic search were
obtained by applying the Neighbor-Joining
method to a matrix of pairwise distances
estimated using the Maximum Composite
Likelihood (MCL) approach, a discrete
Gamma distribution was used to model
evolutionary rate differences among sites
(5 categories (+G, parameter = 0.4248)), the
rate variation model allowed for some sites
to be evolutionarily invariable ([+I], 39.6357%
sites), the tree was drawn to scale with
branch lengths measured in the number of
substitutions per site, the analysis involved
35 nucleotide sequences, and all positions
containing gaps and missing data were
eliminated. There were a total of 371
positions in the final dataset.
Table 1. GenBank accession numbers and geographical origins of taxa used in the phylogenetic
analysis.
Species
Agaricus bisporus
Leucoagaricus atroazureus
Leucoagaricus atroazureus
Leucoagaricus atroazureus
Leucoagaricus cf. atrodisca
Leucoagaricus cf. atrodisca
Leucoagaricus cinerascens
Leucoagaricus cinerascens
Leucoagaricus flavovirens
Leucoagaricus flavovirens
Leucoagaricus holosericeus
Leucoagaricus houaynhangensis
Leucoagaricus houaynhangensis
Leucoagaricus leucothites
Leucoagaricus leucothites
ITS GenBank
accession numbers
FJ223230
EU416297
EU416299
EU416301
GU903304
GU903305
AY176408
AY176410
EU416293
EU416295
GQ329058
KX640915
KX640916
JQ683082
JQ683123
Country of origin
Unknown
China
China
China
USA
USA
USA
USA
China
China
Italy
Laos
Laos
Israel
Ukraine

1290 Chiang Mai J. Sci. 2018; 45(3)
Table 1. Continued.
Species
Leucoagaricus leucothites
Leucoagaricus leucothites
Leucoagaricus naucinus
Leucoagaricus naucinus
Leucoagaricus cf. phaeostica
Leucoagaricus subcretaceus
Leucoagaricus subcretaceus
Leucoagaricus subcretaceus
Leucoagaricus sp.
Leucoagaricus viridiflavus
Leucoagaricus viridiflavus
Lepiotaceae
Lepiotaceae
Lepiotaceae
Lepiotaceae
Lepiotaceae
Lepiotaceae
Lepiotaceae
Lepiotaceae
Leucocoprinus sp.
ITS GenBank
accession numbers
KF316477
KT002154
LNU85315
EU416308
AF079739
GQ329052
GQ329063
KF410815
KR155095
GU574745
KF963609
EF527300
EF527309
EF527314
EF527316
EF527317
EF527326
EF527333
EF527342
KR154960
Country of origin
India
China
USA
China
Panama
Italy
Italy
India
India
Unknown
Japan
Brazil
Brazil
Brazil
Brazil
Brazil
Panama
Panama
Panama
India
3. RESULTS
3.1 Phylogeny Analysis
The BLAST result of two ITS sequences
of La. houaynhangensis (holotype, KX640915:
724 bp; paratypes , KX640916: 402 bp) were
similar.Some sequences related to new taxa
were lower than 89 % such as two sequences
of La. flavovirens J.F. Liang, Zhu L. Yang & J.
Xu ( EU416295, EU416293), two sequences
of La. viridiflavus (Petch) T.K.A. Kumar &
Manim. (GU574745, KF963609), La. leucothites
(Vittad.) Wasser (KF316477, JQ683123).
The phylogeny tree based on Maximum
Likelihood method is given (Figure 1). The
two sequences of La. houaynhangensis were
clustered with bootstrap support (BS) of
100%, and they were related to sequences
of La. viridiflavus and La. flavovirens with BS
of 70%. The species in this clade are
characterized yellow to yellowish-green
basidiomata and turned blue when touched
or dried (Figure 1).

Chiang Mai J. Sci. 2018; 45(3) 1291
Figure 1. Phylogenetic tree based on Maximum Likelihood analysis of nrITS sequences showing
relationship among La. houannhangensis and related species. Bootstrap values ≥ 50 % are indicated
above the branches. GenBank accession numbers are given for each collection. Agaricus bisporus
(J.E. Lange) Imbach was used as an outgroup.

1292 Chiang Mai J. Sci. 2018; 45(3)
3.2 Taxonomy
Leucoagaricus houaynhangensis
Sysouphanthong sp. nov. Figsure 2, 3
MycoBank: MB817806
Holotype: HNL502947
Etymology: The name is referred to the
first collecting site of this species at Houay
Nhang Preserved Forest.
Diagnosis: Basidiomata light green to
yellowish-green and turning blue when dried,
umbonate to plano-convex pileus covered
with dark grey to black granulose to squamules
and with light green appendiculate margin, free
and light green to yellowish-green lamellae,
subcylindrical and yellowish-green stipe with
light green fibrillose remnants, oblong to
ovoid basidiospore with a germ pore,
5.8-7.2 × 3.8-5 μm, clavate to utriform
cheilocystidia with short to long appendages,
18-32 × 8-15 μm, a hymenodermal pileus
covering made up of some cylindrical
elements, a cutis stipe covering made up of
hyaline hyphae and cylindrical elements,
absence of clamp-connections in all tissues.
Pileus 15-35 mm diam, umbonate,
explanding via campanulate to applanate to
plano-convex, with low umbo, with straight
margin; when young granulose, dark grey
(1-30F1) to black, soon breaking up into
concolourous squamules to squamules
fibrillose around granulose umbo toward
margin, on light green to yellowish-green
(30A5-8) background, attached with light
green (30A5) appendiculate margin, fringed
and lamellae exceeding when mature.
Lamellae free, broadly ventricose, light
green to yellowish-green (30A5-6), slightly
crowded, with 5 lamellulae series. Stipe
23-40 × 3-8.5 mm, subcylindrical or wider at
base zone and slightly tapering to apex, with
green to yellowish-green (30A5-6) fibrillose
background. Annulus membranous, rarely
present or often fragile, with green to
yellowish-green (30A5-6) fibrillose remnants
at middle to upper part of stipe. Context in
pileus 2.0-3.0 mm wide, concolourous with
surface; in stipe hollow, concolourous with
surface. Odor and taste not observed.
Spore print white.
Basidiospores [75, 3, 3] l × w= 5.8-7.2 ×
3.8-5 μm, avl × w= 6.5 × 4.3 μm,
Q = 1.4-1.7, Qav= 1.5, in side-view oblong
or ovoid, in frontal view ovoid, oblong,
thick-walled, hyaline, with a germ pore,
dextrinoid, cogophilous, cyanophilous,
metachromatic. Basidia 12-15 × 6-8 μm,
clavate, 4-spored, hyaline. Lamella edge sterile.
Cheilocystidia 18-32 × 8-15 μm, clavate to
utriform, with short to long appendages
(l × w = 5-20 × 2.5-3.5 μm), Pleurocystidia
absent. Pileus covering a hymenoderm made
up of some layers of cylindrical elements,
rarely with narrowly clavate elements,
23-47 × 4-7 μm, hyaline to pale brown-walled,
under layer with hyaline hyphae, cylindrical,
2.5-6.5 μm wide. Stipe covering a cutis made
up of cylindrical elements, 1-2.5 μm wide,
hyaline, with cylindrical hyphae in lower layer,
1-4 μm wide, hyaline. Clamp-connections
absent in all tissues.
Habitat and distribution: solitary,
saprotrophic, on humus soil; widespread in
deciduous forests of Houat Yang Preserved
Forest, Xaythany District, Vienttianr Capital,
Lao PDR.
Material examined: Laos, Vientiane
Capital, Xaythany District, Houay Nhang
Preserved Forest, collected date 12 July
20014, P. Sysouphanthong (PS2014-1448,
HNL502947, holotype); ibidem collected
date 20 August 20014, P. Sysouphanthong
(PS2014-1463, HNL502962, paratypes);
ibidem collected date 2 September 20014,
P. Sysouphanthong (PS2014-1483, HNL
502982, paratypes).

Chiang Mai J. Sci. 2018; 45(3) 1293
Figure 2. Fresh basidiomata of La. houaynhangensis. a-c (HNL502947), d (HNL502962),
e-f (HNL502982).
4. DISCUSSIONS
La. houaynhangensis is commonly
recognized by light green to yellowish-green
whole basidiomata with turning blue when
dried, umbonate pileus covered with dark
grey to black granulose to squamules with light
green appendiculate margin, free lamellae,
subcylindrical stipe with light green fibrillose
remnants, oblong ovoid basidiospore with a
germ pore, clavate to utriform cheilocystidia
with short to long appendages, a hymeniderm
pileus covering, a cutis stipe covering, and
absence of clamp-connections in all tissues.
Since some species of Leucoagaricus with
yellow to yellowish-green basidiomata come
to close to La. houaynhangensis, and they are
often confused on macromorphology. Firstly,
La. flavovirens, a yellow species with dark bluish
green, is the most similar to La. houaynhangensis
on field by dark grey to blackish on pilial
surface with purplish-green to grayish green
to radially fibrillose squamules, free and
greenish-yellow lamellae, greenish yellow
stipe and concolourous on context of pileus
and stipe; however, it differs from La.
houaynhangensis by lack of a germ pore
on basidispore, shorter appendage on
cheilocyctidia, and a cutis pileus covering
[19]; and The BLAST result of nrITS sequence
showed that La. flavovirens was only 83%
related to La. houaynhangensis, and the ML tree
also showed that La. houaynhangensis was clearly
separated from La. flavoverens (Figure 1).
The second yellowish-green species,
La. viridiflavus, are close to La. houaynhangensis
with the same olive green basidiomata with
blushing blue, shape and size of basidiospore
with a germ pore, similar shape and size of
cheilocystidia with a long appendage; but
differs at glabrous pileus without grey or black
squamules and a cutis pileus covering structure,
and with amorphous contents in cheilocystidia
and covered with exudates towards the
apex of appendage which is not found in
La. houaynhangensis [12]. Two nrITS sequences
of La. houaynhangensis were identical with
100% BS, and there were only sequences of
La. viridiflavus and La. Flavoverens come to relate
with low BS (Figure 1).
La. sulphurellus (Pegler) B.P. Akers
La. viridiflavoides B.P. Akers & Angels are
distinguished from La. houaynhangensis by
having cutis pileus covering structures and
presence of pleurocystidia [25, 34].

1294 Chiang Mai J. Sci. 2018; 45(3)
There were some studies of
basidiomycota
in Southeast Asia, numerous yellow-green
species of lepiotaceous fungi are distinguished
from La. houaynhangensis. For example, Lepiota
citrophylla (Berk. & Broome) Sacc. has spurred
basidiospores and a trichodermal pileus
covering [35] and Verrucospora vulgaris Pegler
has angular or verrucose basidiospores
(36-38).
ACKNOWLEDGEMENTS
This study was partially supported by
the project “Ecosystem Classification of Lao
PDR in 2011-2012, Project No. 12 13 3 2
137 37”. The authors also acknowledge the
Center of Excellence in Bioresources for
Agriculture, Industry and Medicine, Faculty of
Science, Chiang Mai University.
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