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Two new unusual
Leucoagaricus
species (Agaricaceae) from
tropical China with blue-green staining reactions
Jun F. Liang
Key Laboratory of Biodiversity and Biogeography,
Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650204, P.R. China, and Research
Institute of Tropical Forestry, Chinese Academy of
Forestry, Guangzhou 510520, P.R. China
Zhu L. Yang
1
Key Laboratory of Biodiversity and Biogeography,
Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650204, P.R. China
Jianping Xu
Center for Environmental Genomics, Department of
Biology, McMaster University, Hamilton, Ontario, L8S
4K1 Canada
Z.W. Ge
Key Laboratory of Biodiversity and Biogeography,
Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650204, P.R. China
Abstract
:Most species of the genus
Leucoagaricus
have been described from temperate regions in North
America and Europe, but little is known about the
genus from tropical areas. In this report we describe
two new species of
Leucoagaricus
,namely
La.
flavovirens
and
La. atroazureus
, from tropical China.
The two species are characterized by turning blue-
green or dark blue where bruised and by unique
phylogenetic placement. Two new combinations,
namely
La. viriditinctus
and
La. caerulescens
, are
proposed. The neotype of
La. viridiflavus
from India
was found to differ from
La. flavovirens
based on
morphology and ITS sequence comparison. A mono-
phyletic group of four bluing species from Old World
tropics is recovered but with poor measures of branch
support.
Key words:
Agaricales, lepiotaceous fungi,
taxonomy, tropics
INTRODUCTION
The name
Leucoagaricus
originally was put forward by
Locquin (1943a, b) as a subgenus of
Leucocoprinus
Pat. but without a Latin description. It subsequently
was elevated to the status of an independent genus
and furnished with a Latin diagnosis by Singer
(1948). The genus
Leucoagaricus
(Locq.) ex Singer
is characterized by these morphological characteris-
tics: an entire or rarely striate pileus margin, free
lamellae, metachromatic spores in cresyl blue, and an
absence of clamp-connections and pseudoparaphyses
(Singer 1986, Vellinga 2001). These features led to
the inclusion of about 90 species within the genus
(Kirk et al. 2008). While this classification has been
accepted by most taxonomists (e.g. Candusso and
Lanzoni 1990, Bon 1996, Akers et al. 2000, Kumar and
Manimohan 2009b), older publications have yet to be
reconciled with a modern concept of
Leucoagaricus
.
As a result confusion remains in the systematics of the
group. For instance in studies by Dennis (1970) and
Pegler (1986) specimens of
Leucoagaricus
species
from tropical areas are treated as belonging to
Lepiota
(Pers. : Fr.) Gray or
Leucocoprinus
.
Based on morphological and molecular data,
several studies have demonstrated that
Leucoagaricus
is polyphyletic (Johnson and Vilgalys 1998, Johnson
1999, Vellinga 2004). Vellinga (2003, 2004) showed
that species of
Leucoagaricus
and
Leucocoprinus
were
phylogenetically clustered but intermixed within a
single clade. Because of the large number of species
in the clade and relatively limited molecular data a
satisfactory solution has not been achieved to resolve
the taxonomy and phylogenetic relationships among
many of the species of
Leucoagaricus
,
Leucocoprinus
,
and
Lepiota
. In this study we follow the majority view
of the separate generic status of
Leucoagaricus
and
Leucocoprinus
. We use morphological characters as
the primary criteria to distinguish these two genera.
Color changes of basidiocarps in response to
bruising and/or drying are important characters in
the taxonomy of lepiotaceous fungi. Some species
display red, blue or green reactions. For example
Lepiota metabola
(Berk. & Broome) Sacc. reacts by
bruising red (Pegler 1986),
L. viriditincta
(Berk. &
Broome) Sacc. (Pegler 1986) and
La. viridiflavoides
Akers & Angels (Akers et al. 2000) reacts by bruising
bluish, and
L. caerulescens
Peck (5
L. virescens
Morgan) exhibits a combination of blue, green and
red bruising reactions. After drying
L. caerulescens
exhibits a bluish tint (Peck 1899, Morgan 1906) but
the tint did not persist in herbarium collections
(Akers et al. 2000).
In the past specimens from China with bluish green
bruising have been identified commonly as
L.
caerulescens
(Teng 1939, Bi et al. 1994, Teng 1996).
However close inspections of these collections indi-
Submitted 12 Feb 2009; accepted for publication 25 Feb 2010.
1
Corresponding author. E-mail: fungi@mail.kib.ac.cn
Mycologia,
102(5), 2010, pp. 1141–1152. DOI: 10.3852/09-021
#2010 by The Mycological Society of America, Lawrence, KS 66044-8897
1141
cate that they belong to different species. Important
features for distinguishing these closely related
species include morphological and chemical features,
such as white or yellow flesh, deep blue or deep green
bruising, a fleeting bluish tint in herbarium collec-
tions and the shape and size of cheilocystidia.
Morphological and chemical features for specimens
of
L. caerulescens
from China are similar to
L.
caerulescens
from USA,
L. viriditincta
from Sri Lanka
and
La. viridiflavus
(Petch) T.K.A. Kumar & Manim.
from India. Based on comparative studies of available
collections, including the type specimens and other
collections of
L. viriditincta
and
L. caerulescens
,we
propose two new species,
La. flavovirens
sp. nov. and
La. atroazureus
sp. nov., both of which bruise blue or
bluish green. To confirm their autonomy their
internal transcribed spacer (ITS) region and the
nuclear large subunit ribosomal RNA gene (LSU)
sequences were generated and analyzed jointly with
sequences from other molecular phylogenetic studies
of lepiotaceous fungi.
MATERIALS AND METHODS
Specimens and morphological descriptions.—
Collections were
obtained and photographed in the field. Notes were taken
on macromorphological features, and specimens were dried
with an electric drier. Materials are deposited in the
Herbarium of Cryptogams, Kunming Institute of Botany,
Chinese Academy of Sciences (HKAS). The type specimens
and other authentic collections of
L. viriditincta
were
borrowed from the Mycological Herbarium of the Royal
Botanical Gardens, Kew (K), and from National Herbarium
of the Netherlands, Leiden University Branch (L). The
neotype of
La. viridiflavus
was borrowed from University of
Calicut, India (CALI). The type specimen of
L. caerulescens
was borrowed from the Herbarium Biological Survey, New
York State Museum (NYS). Some collections tentatively
identified as
L. caerulescens
Peck were borrowed from the
Herbarium of the Institute of Microbiology of the Chinese
Academy of Sciences (HMAS), Herbarium of Guangdong
Institute of Microbiology (GDGM) and the New York
Botanical Garden (NY). The generic names of
Lepiota
,
Leucoagaricus
and
Leucocoprinus
are abbreviated respective-
ly as
L.
,
La
. and
Lc
. Herbarium abbreviations follow Thiers
(continuously updated).
Sections of specimens were cut by hand and mounted in
5%KOH, Congo red and Melzer’s reagent for microscopic
observations. Basidiospores were mounted in cresyl blue to
test for a metachromatic reaction (Singer 1986). Sizes were
determined for basidia, basidiospores, cheilocystidia and
elements of the pileipellis based on ocular micrometer
measurements for at least 20 elements of each character.
Color designations are from Kornerup and Wanscher
(1981). The abbreviation [n/m/p] indicates that measure-
ments were made on n basidiospores in m samples from p
collections. Dimensions of basidiospores are given using a
notation of the form (a)b–c(d). The range b–c contains a
minimum of 90%of the measured values. Extreme values
are given in parentheses. These abbreviations are used: Q
refers to the length/breadth ratio of basidiospores; Qrefers
to the average Q of all basidiospores plus or minus sample
standard deviation.
Molecular identification.—
Genomic DNA were extracted
from herbarium or silica-dried materials with a CTAB
protocol (Xu et al. 1994). ITS and LSU gene fragments
were amplified with primers ITS1F (Gardes and Bruns
1993) and ITS4 (White et al. 1990) for ITS and primers
LR0R and LR7 for LSU (Vilgalys and Hester 1990). PCR
products were purified with the Bioteke DNA Purification
Kit (Bioteke Corp., Beijing). Sequencing of both strands was
performed with an ABI 3730 DNA analyzer and an ABI
BigDye 3.1 terminator cycle sequencing kit (Shanghai
Sangon Biological Engineering Technology & Services Co.
Ltd, Shanghai, China). These sequences were submitted to
GenBank (accession numbers and detailed information
about each specimen are in SUPPLEMENTARY TABLE I). Aside
from the 31 sequences produced in this study, for broad
comparisons we also included 65 sequences from isolates of
genera
Lepiota
,
Leucoagaricus
,
Leucocoprinus
and related
species (as outgroup) obtained in previous studies (Mueller
et al. 1998, Johnson 1999, Vellinga 2000, Vellinga et al.
2003, Vellinga 2004, Callac and Guinberteau 2005, Vellinga
2007, Ortiz et al. 2008, Liang et al. 2009).
Sequence alignment and phylogenetic analyses.—
DNA se-
quences were edited and aligned with SeqMan (DNA STAR
Package) and Clustal X (Thompson et al. 1997) and
manually checked and modified. Ambiguous positions were
excluded from matrix. Gaps were treated as missing data.
All unambiguous characters and character transformations
were equally weighted.
Agaricus bisporus
,
A. campestris
and
Chlorophyllum molybdites
were selected as outgroups. The
dataset was analyzed with Bayesian inference using the
parallel version of MrBayes 3.1.2 with default priors
(Ronquist and Huelsenbeck 2003, Altekar et al. 2004) and
maximum likelihood with the RAxML BlackBox Online
server (Stamatakis et al. 2008).
Bayesian analysis was implemented on a Linux cluster
with Intel Pentium 4 processors under a general-time-
reversible (GTR) model (nst 56) and rate heterogeneity
parameters (gamma-distributed substitution rate and a
proportion of invariable sites) following the Akaike Infor-
mation Criterion (AIC) in Modeltest 3.06 (Posada and
Crandall 1998). Bayesian analyses were run with six chains
for 10 000 000 generations and sampled every 500 genera-
tions. Tree were pooled and used to generate a 50%
majority rule consensus tree with branch lengths. Nodes
that were recovered more than 95%of the time were
considered to have a significant posterior probability (PP).
Maximum likelihood tree generation and bootstrap
analyses were performed with RAxML 2.2.3 (Stamatakis et
al. 2008). One hundred bootstrap analyses were done to
affirm consistency of results. A GTR model of substitution
and GAMMA +P-Invar rate heterogeneity parameters were
invoked. A bootstrap proportion greater than 70%was
considered significant. The results were exported to
Dendroscope for tree viewing (Huson et al. 2007).
1142 MYCOLOGIA
TAXONOMY
Leucoagaricus flavovirens J.F. Liang, Zhu L. Yang & J.
Xu, sp. nov. FIG.1
MycoBank MB515455
Pileus initio campanulatus, deinde convexus vel applana-
tus, albidus, in aeruginosum transit si contundatur, medio
fusco-griseo vel nigello, squamulis fibrillosis, griseis, purpur-
eogriseis vel griseofuscis. Lamellae liberae, flavo-viridae.
Stipes subcylindricus vel sursum attenuatus, annulatus,
glabrus. Caro cremea, pallide flavida vel flavida, in aeru-
ginosum transit si contundatur. Basidiosporae 5.0–7.0 3
3.5–4.5 mm, ellipsoideae vel subamygdaliformes, hyalinae,
dextrinoideae. Basidia clavata, 4-sporigera, raro 2-sporigera.
Cheilocystidia fusoidea, incolorata. Pleurocystidia nulla.
Squamulae pilei ex hyphis cylindricis terminalibus compos-
itae. Fibulae absentes.
Holotypus. J.F. Liang 90c
(HKAS 29580), 650 m, 14
Jul 2005, Gouguyulin, Jianfengling, Ledong County,
Hainan Province, China.
Etymology.
Named because of greenish yellow basi-
diomata.
Basidiomata
(FIG. 1A).
Pileus
1–3 cm diam, when
young campanulate, expanding to plano-convex to
applanate, sometimes center obtusely umbonate;
surface dark gray (4F1) to blackish (1F1) at the
center, elsewhere pale yellow (1B4) but becoming
dark bluish green (24A7–8) when dried, with brown-
ish gray (1E2), gray (4C1) to purplish gray (13D2–3)
or grayish green (28C3–4) appressed to radially
fibrillose squamules, sometimes in some collections
with minute brownish red (11C7–8) squamules;
margin nonstriate.
Context
cream to yellowish or
yellow, turning bluish green where bruised, only
about 0.3 mm thick.
Lamellae
free, crowded, greenish
yellow (1A7–8), greenish yellow to deep green,
bruising bluish green, lamellulae present.
Stipe
2–
3.4 cm 32–3 mm, subcylindrical, attenuate, slightly
curved at the base, smooth, hollow, greenish yellow
but turning bluish green where handled.
Annulus
present, superior, membranous, greenish yellow,
bruising bluish green, often disappearing.
Odor
none.
Flavor
not recorded.
FIG.1.
Leucoagaricus flavovirens
. A. Basidioma. B. Basidiospores. C. Cheilocystidia. D. Pileipellis. All from the holotype.
LIANG ET AL.: NEW
L
EUCOAGARICUS
SPECIES 1143
Basidiospores
(FIG. 1B) [104/4/4] (4.5–)5.0–7.0
(–7.5) 3(3.0–)3.5–4.5(–5.0) mm(Q5[1.22–]1.33–
1.75[–1.88], Q51.56 60.12), ellipsoid to subamyg-
daliform with slightly straight adaxial side in side view,
attenuate, without suprahilar depression and germ
pore, ellipsoid to ovoid in front view, hyaline, smooth,
slightly thick-walled, strongly dextrinoid, congophilic,
metachromatic in cresyl blue, with guttulate contents.
Basidia
13–20 37–11 mm, clavate, four-spored, rarely
two-spored, hyaline, sterigmata short and thin.
Cheilocystidia
(FIG. 1C) (21–)24–45(–48) 3(6–)8–
15(–17) mm, fusiform with a short apical appendage;
apical appendage submoniliform or subcylindrical,
attenuate, often narrowed, occasionally septate; walls
smooth, thin, hyaline in KOH, congophilic.
Pleur-
ocystidia
absent.
Pileipellis
(FIG. 1D) a loosely ar-
ranged cutis of frequently septate cylindrical hyphae,
occasionally lower part of the hyphae encrusted with
brown pigments; terminal elements barely differenti-
ated, 20–76 35–11 mm, subcylindrical, thin-walled,
with pale yellow intracellular pigments.
Clamp con-
nections
absent.
Materials examined
: CHINA: Hainan Province, Ledong
County, Jianfengling (18u239–18u509N, 108u369–109u059E),
Gouguyulin, 650 m, 14 Jul 2005,
J.F. Liang 90c
(HKAS
29580, holotype); same location, 14 Jul 2005,
J.F. Liang 90b
(HKAS 29579); Mingfenggu, 950 m, 13 Jul 2005,
J.F. Liang
90a
(HKAS 29578); Yunnan Province, Jinghong (22u09460N,
100u479370E), Dadugang, on the ground, 1050 m, 6 Jul
2006,
J. F. Liang 308
(HKAS 50024).
Habitat.
Solitary on the ground in tropical rain-
forest or other forests in tropical regions.
Known distribution.
Known only from tropical
China.
Notes. Leucoagaricus flavovirens
is characterized by
the greenish yellowish context, fusiform cheilocystidia
with a short apical appendage, the absence of
pleurocystidia, and the bluish green change of
basidiomata where bruised or when dried.
Among species of
Lepiota
it is not uncommon to
observe bluish green bruising.
Lepiota cyanescens
Beeli
(Beeli 1927),
L. viriditincta
,
L. caerulescens
,
L.
virescens
(Speg.) Morgan (Morgan 1906) and
L.
virescens
Pat. (Patouillard 1924) also display similar
reactions when handled. However these species have
white instead of yellowish to yellow context. In
addition
L. cyanescens
has larger basidiospores than
La. flavovirens
while
L. viriditincta
has broadly clavate
cheilocystidia.
Lepiota caerulescens
and
L. virescens
(Speg.) Morgan, which Morgan (1906) treated as a
single species, have fleeting bluish tints and broadly
clavate cheilocystidia.
Lepiota virescens
Pat. has brown
and pulverulent squamules on the pileus. These
characters distinguish these species from
La. flavo-
virens
.
Our literature review indicates that several de-
scribed species possess similar bruising reactions
and yellow context. These include
L. subcitrophylla
Hongo (Hongo 1956),
L. trichroma
Montoya &
Bandala (Montoya and Bandala 2005),
La. viridi-
flavoides
Akers & Angel and
La. sulphurellus
(Pegler)
Akers (Akers et al. 2000). However
L. subcitrophylla
and
L. trichroma
differ from
La. flavovirens
by having
basally truncate, spurred basidiospores and the
common presence of clamp connections.
La. viridi-
flavoides
and
La. sulphurellus
similarly differ from
La.
flavovirens
by having basidiospores with a germ pore
and abundant pleurocystidia.
Among the published species sharing certain
features with
La. flavovirens
,
La. viridiflavus
(Petch)
T.K.A. Kumar & Manim. deserves some attention.
This species originally was described by Petch (1917)
from Sri Lanka as
L. viridiflava
Petch (non
L.
viridiflava
[Rick 1938] Singer 1975). The original
description appears similar to that of
La. flavovirens
.
The only known macromorphological difference is
the color of the squamules on the pileus of
L.
viridiflava
, which is dark green to green (Petch 1917).
Unfortunately several micromorphological charac-
ters, such as the shape and the size of the cheilocys-
tidia and the structure of squamules on the pileus of
L. viridiflava
, are unknown. There was no citation of
the type specimen or any other representative
collections when Petch described the species as new.
Pegler (1986) listed the species in his agaric flora of
Sri Lanka ‘‘for the sake of completeness’’ but
indicated that no collection of the species could be
found or traced. Kumar and Manimohan (2009b)
designated a neotype for
L. viridiflava
from Kerala,
southern India, and proposed a new combination
La.
viridiflavus
.
Leucoagaricus viridiflavus
differs from
La. flavovirens
by having larger spores with a
truncated apex and an inconspicuous germ pore.
Several other blue to dark blue species also are
worth noting here. These include
L. caerulea
Beard-
slee (Beardslee 1915),
L. cyanea
Rick,
L. atrocoerulea
Rick (Rick 1920) and
L. azurea
Singer (Singer and
Digilio 1952). However their blue or dark blue
appearance are their original context or squamules
colors, not developed after bruising or drying.
Lepiota
cyanozonata
Longyear (Longyear 1902) is pale blue
but changes to a brownish tan when dried.
Leucoagaricus atroazureus J.F. Liang, Zhu L. Yang &
J. Xu, sp. nov. FIG.2
MycoBank MB515456
Pileus albidus, cremeus vel griseo-aurantiacus, medio
atropurpureo vel brunneo-rubro; margine sordide albida,
striata vel non-striata; squamulis atrobrunneis, atrocyanes-
centibus. Stipes subcylindricus, subglabrus, initio albus vel
1144 MYCOLOGIA
cremeus, deinde pallide-flavus vel brunneus. Caro alba, in
atroazureum transit si contundatur. Basidiosporae 5.5–8.5
33.5–5.0 mm, amygdaliformes. Cheilocystidia angusticlavata
vel clavata. Pleurocystidia nulla. Squamulae pilei ex hyphis
cylindricis terminalibus compositae. Fibulae absentes.
Holotypus. Zhu L. Yang 3695
(HKAS 42670), 16 Jul
2003, Near Xima, Yingjiang County, Yunnan Province,
China.
Etymology.
Named because of the dark blue reaction
of basidiomata where bruised or dried.
Basidiomata
(FIG. 2A).
Pileus
1.6–4.5 cm diam,
convex, slightly umbonate, center black-red (10C8)
to brownish red (11C7–8), elsewhere with whitish to
cream to grayish orange (6B3–5) ground, and
covered with reddish brown (9D7–8) to dark brown
(6F7–8) squamules; margin dirty white, striate in age.
Context
thin to moderately thick, 1–2 mm, white but
becoming dark blue where bruised or dried.
Lamellae
free, white to cream to yellowish (2A2), crowded,
lamellulae present.
Stipe
2–4 cm 32–6 mm, silky-
fibrillose or nearly glabrous, not brittle, attenuate,
occasionally inflated or narrowed toward the base,
fistulous, surface at first white to yellowish (2A2),
becoming grayish orange (6B4–5) when mature,
becoming dark blue when bruised or dried.
Annulus
white but becoming dark blue when dried, edge
brownish red (11C7–8), fragile, superior, about 5 mm
below the apex of stipe, sometimes disappearing.
Odor
none.
Flavor
not recorded.
Basidiospores
(FIG. 2B) [185/9/5] (5.0–)5.5–8.0
(–9.0) 3(3.0–)3.5–5.0(–6.0) mm(Q5[1.25–]1.38–
1.88[–2.00], Q51.61 60.15), amygdaliform in side
view, attenuate toward apex, ovoid in front view,
without germ pore, hyaline, smooth, slightly thick-
walled, dextrinoid, congophilic, metachromatic in
cresyl blue, with guttulate contents.
Basidia
15–23 3
FIG.2.
Leucoagaricus atroazureus
. A. Basidioma. B. Basidiospores. C. Cheilocystidia. D. Pileipellis. All from holotype.
LIANG ET AL.: NEW
L
EUCOAGARICUS
SPECIES 1145
7–11 mm, clavate, four-spored, rarely two-spored,
hyaline.
Cheilocystidia
(FIG. 2C) 22–50(–65) 35–
15 mm, clavate to narrowly clavate, sometimes fusi-
form, thin-walled, hyaline, not abundant.
Pleurocysti-
dia
absent.
Pileipellis
(FIG. 2D) loosely arranged cutis
of cylindrical hyphae; terminal elements barely
differentiated, (17–)21–92(–115) 36–20 mm, subcy-
lindrical, occasionally narrowly clavate or fusiform,
thin-walled, with pale brown intracellular pigments
and often encrusted with brown pigments.
Clamp
connections
absent.
Materials examined
: CHINA: Yunnan Provinve, Yingjiang
County (24u429320N, 97u559550E), near Xima, in forest of
Pinus kesiya
var.
langbianensis
, 1700 m, 16 Jul 2003,
Zhu L.
Yang 3695
(HKAS 42670, holotype); Changning County
(24u499480N, 99u369180E), Bawancun, 2100 m, 17 Jul 2003,
L.F. Zhang 184
(HKAS 42470); Longling County
(24u359230N, 98u419280E), Longxindaxueshan, 2300 m, 30
Aug 2002,
Zhu L. Yang 3371
(HKAS 41440); Hainan
Province, Ledong County, Jianfengling (18u239–18u509N,
108u369–109u059E), 650 m, 14 Jul 2005,
J.F. Liang 113
(HKAS 48453); same location, 14 Jul 2005,
J.F. Liang 110
(HKAS 48450).
Habitat.
Solitary on the ground in tropical and
subtropical forests.
Known distribution.
Known only from southern and
southwestern China.
Notes. Leucoagaricus atroazureus
is characterized by
white context, clavate to narrowly clavate cheilocysti-
dia, absence of pleurocystidia and the dark blue
changes of basidiomata where bruised or dried. The
colors can be observed on herbarium collections.
The species is similar to
L. cyanescens
,
L. viridi-
tincta
,
L. caerulescens
and
L. virescens
Pat. with regard
to bruising and white context. However
L. cyanescens
differs from
La. atroazureus
in having larger and
wider basidiospores (8–10 37–8 mm) (Beeli 1927),
L.
viriditincta
has orange fibrillose squamules on the
pileus and broadly clavate cheilocystidia,
L. caerules-
cens
differs by the broadly clavate cheilocystidia and
fleeting bluish tint and
L. virescens
Pat. has brown and
pulverulent squamules on pileus (Patouillard 1924).
Apart from the above,
L. subcitrophylla
,
L.
trichroma
,
La. sulphurellus
,
La. viridiflavoides
and
La. viridiflavus
also exhibit bluish green bruising, but
these all have yellow or yellowish context. The new
species
La. flavovirens
differs from
La atroazureus
by
its yellow or yellowish context and fusiform cheilocys-
tidia with a short apical appendage.
As mentioned above the blue or dark blue species
(
L. caerulea
,
L. cyanea
,
L. atrocoerulea
and
L. azurea
)
also differ from this new species by original blue or
dark blue in context or squamules (Beardslee 1915,
Rick 1920, Singer and Digilio 1952) while
L.
cyanozonata
turns brownish tan from pale blue when
dry (Longyear 1902).
Leucoagaricus viriditinctus
(Berk. & Broome) J.F.
Liang, Zhu L. Yang & J. Xu, comb. nov. FIG.3
MycoBank MB515457
Basionym:
Agaricus viriditinctus
Berk. & Broome, J.
Linn. Soc. Bot. 11:503. 1871.
;Lepiota viriditincta
(Berk. & Broome) Sacc., Syll. Fung.
5:59. 1887.
5Agaricus pyrocephalus
Berk. & Broome, J. Linn. Soc.
Bot. 11:504. 1871.
;
Lepiota pyrocephala
(Berk. & Broome) Sacc., Syll. Fung.
60. 1887.
Basidiomata
small. Pileus 1–3.5 cm diam, when young
campanulate, expanding to plano-convex to applanate,
center with low umbonate and dark reddish brown to
dark brown, elsewhere with whitish to dirty white ground,
and covered with pale brown, reddish brown to dark
brown squamules; striate or not.
Context
thin, white but
pale blue to dark blue when bruised or dried.
Lamellae
free, white, moderate to crowded; lamellulae present.
Stipe
1.5–6.5 cm 31–4 mm, subcylindrical or attenuate,
occasionally inflated at base, hollow, surface at first white,
dark blue when handled.
Annulus
membranous, disap-
pearing, white but dark blue when dried.
Flavor
sour
(from the original description of L0627034).
Basidiospores
(FIG. 3A) [82/4/4] (6.5–)7.0–8.5 3
4.0–5.0 mm(Q5[1.50–]1.56–2.00[–2.13], Q51.74
60.14), amygdaliform in side view, attenuate toward
the apex, ovoid in front view, without germ pore,
hyaline, smooth, slightly thick-walled, dextrinoid,
congophilic, metachromatic in cresyl blue, with
guttulate contents.
Basidia
16–32 35–13 mm, clavate,
four-spored, rarely two-spored, hyaline.
Cheilocystidia
(FIG. 3B) 10–50 37.5–21 mm, broadly clavate to
pyriform, sometimes sphaeropedunculate, rarely cla-
vate, thin-walled, hyaline.
Pleurocystidia
absent.
Pilei-
pellis
(FIG. 3C) a loosely arranged cutis of cylindrical
hyphae; terminal elements often differentiated, 16–
99(–136) 34–20(–25) mm, subcylindrical, occasion-
ally narrowly clavate, thin-walled, with pale brown
intracellular pigments, and often encrusted with
brown pigments.
Clamp connections
absent.
Materials examined
: Sri Lanka: Kandy Distrrict, Province
Peradeniya, 1869,
Thwaites1153 cum icon
(KM139636,
holotype); same location, Sep 1868,
Thwaites 772
(KM139633, holotype of
A. pyrocephalus
); Indonesia:
Wanariset Forest, East Kali mantan, 50 m,
R.P.J de Kok, F-
61
(L0627034); China, Yunnan Province, Jinghong
(22u09460N, 100u479370E), Dadugang, on the ground,
1050 m, 7 Jul 2006,
J. F. Liang 317
(HKAS 50033).
Distribution.
Sri Lanka (Pegler 1986), Indonesia
(Vellinga 2003a), India (Manimohan et al. 1988) and
China.
Notes. Leucoagaricus viriditinctus
is characterized by
white context, broadly clavate to pyriform cheilocysti-
dia, and persistent dark blue changes of basidiomata
when bruised or dried.
1146 MYCOLOGIA
Leucoagaricus viriditinctus
originally was described
as
Agaricus viriditinctus
by Berkeley and Broome
(1871) and then transferred to the genus
Lepiota
by
Saccardo (1887). Pegler (1986) studied the type
specimens of
L. viriditincta
and
L. pyrocephala
(Berk.
& Broome) Sacc. and combined them as
L.
viriditincta
. Most morphological features were similar
between the type specimens of
L. pyrocephala
and
L.
viriditincta
. However cheilocystidia were broadly
clavate to pyriform in the type specimen of
L.
pyrocephala
but could not be found in
L. viriditincta
.
Manimohan et al. (1988) and Kumar and Manimohan
(2009a) reported
L. viriditincta
from southern India
yet with different sizes of basidiospores (5.0–7.0 3
3.5–4.0 mm vs. 7.0–10.0 34.0–5.0 mm). Based on
morphological traits and the molecular phylogenetic
analyses (FIGS. 3, 5), we transfer
L. viriditincta
to
genus
Leucoagaricus
.
Leucoagaricus caerulescens
(Peck) J. F. Liang, Zhu L.
Yang & J. Xu, comb. nov. FIG.4
MycoBank MB515458
Basionym:
Lepiota coerulescens
Peck, Bull. Torrey Bot.
Club 26:63 (1899)
5
Lepiota virescens
(Speg.) Morgan, J. Mycol. 12:245
(1906)
Pileus
1.5–2.0 cm diam, convex, obtuse to slightly
umbonate, center brownish, elsewhere with whitish
ground, and covered with brownish squamules.
Context
thin, whitish.
Lamellae
free, white, crowded;
lamellulae present.
Stipe
3.0–5.0 cm 32–3 mm,
subclavate, hollow, glabrous, brownish.
Basidiospores
(FIG. 4A) [20/1/1] (7.0–)7.5–9.5
(–10.0) 34.0–5.0 mm(Q51.56–2.00, Q51.84 6
0.13), amygdaliform in side view, attenuate toward
apex, ovoid in front view, without germ pore, hyaline,
smooth, slightly thick-walled, dextrinoid, congophilic,
metachromatic in cresyl blue.
Basidia
16–22 37–
12 mm, clavate, four-spored, hyaline.
Cheilocystidia
(FIG. 4B) 21.5–39 310–20 mm, clavate to broadly
clavate, rarely narrowed at the apex, thin-walled,
hyaline.
Pleurocystidia
absent.
Pileipellis
(FIG. 4C) a
loosely arranged cutis of cylindrical hyphae; terminal
elements suberect, sometimes differentiated, 24–51
FIG.3.
Leucoagaricus viriditinctus
. A. Basidiospores. B. Cheilocystidia. C. Pileipellis. A and C from holotype and B
from KM139633.
LIANG ET AL.: NEW
L
EUCOAGARICUS
SPECIES 1147
(–61) 36–11 mm, subcylindrical to narrowly clavate,
thin-walled, with pale brown intracellular pigments,
and often encrusted with brown pigments at base.
Clamp connections
absent.
Materials examined
: USA: Ohio. C.G. Lloyd (NYS, holotype).
Distribution.
Eastern North America.
Notes. Leucoagaricus caerulescens
is characterized by
white context, clavate to broadly clavate cheilocystidia
and the bluish green change of basidiomata when
dried (Peck 1899, Morgan 1906), but the blue tint did
not persist in herbarium collections.
Leucoagaricus caerulescens
originally was described
as
L. coerulescens
[sic] by Peck (1899). Morgan (1906)
considered
L. erythrella
e. (subsp.)
virescens
Speg. with
L. coerulescens
[sic] as conspecific and used the name
L. virescens
(Speg.) Morgan for the species. Based on
the type specimen of
L. coerulescens
and literature
reviews of
L. virescens
, we consider the two specimens
as conspecific.
L. caerulescens
(1899) should have
priority over
L. virescens
(1906), according to
nomenclatural priority (Mcneill et al. 2006). In
addition we propose to transfer the species from the
FIG.4.
Leucoagaricus caerulescens
. A. Basidiospores. B. Cheilocystidia. C. Pileipellis. All from holotype.
1148 MYCOLOGIA
FIG. 5. One of 100 RAxML likelihood trees (-In L 19396.165259) based on the combined ITS-LSU dataset. Support values
in boldface are RAxML likelihood bootstrap ($70%). Values in lightface are Bayesian posterior probabilities ($0.95).
LIANG ET AL.: NEW
L
EUCOAGARICUS
SPECIES 1149
genus
Lepiota
to
Leucoagaricus
based on the morpho-
logical features.
PHYLOGENETIC RESULTS
We assembled a combined dataset of ITS and LSU
sequences for 57 taxa. This dataset included 2150
nucleotide sites. The dataset contains all available
sequences of
Leucoagaricus
and
Leucocoprinus
and
three of selected sequences of
Lepiota
in GenBank.
Bayesian and RAxML phylogenetic analyses (FIG.5)
showed strong support (100%ML bootstrap value
and 1.0 posterior probability) for the monophyly of a
clade containing all species of genus
Lepiota
. A clade
with 80%ML bootstrap value and 1.0 posterior
probability containing the two new
Leucoagaricus
species,
La. viriditinctus
,
La. viridiflavus
and other
species of
Leucoagaricus
and
Leucocoprinus
, is shown
as a sister group of
Lepiota
. The species that bruise
blue,
La. viriditinctus
,
La. viridiflavus
,
La. flavovirens
and
La. atroazureus
, form a monophyletic group but
with weak support values. The three species,
La.
viridflavus
,
La. flavovirens
and
La. atroazureus
,are
strongly supported as monophyletic with a 94%ML
bootstrap value.
DISCUSSION
We find that
La. viridiflavus
and
La. viriditincta
fall
into the
Leucoagaricus-Leucocoprinus
clade but not
Lepiota
clade, based on Bayesian and RAxML phylo-
genetic analyses (FIG. 5). It indicates that the two
species belong to the genus
Leucoagaricus
but not
Lepiota
. The two new species,
La. flavovirens
and
La.
atroazureus
, are allied with
La. viridiflavus
and
La.
viriditinctus
. Long branch lengths among the species
that bruise bluish green suggest that other potentially
unknown species might exist and remain to be
identified. At present no DNA sequences from strains
of
La. caerulescens
are available in any public database
to let us determine its phylogenetic position.
The blue-bruising reaction, while not completely
understood, is thought to be caused by a degradation
of psilocin, the oxidization of phenolic compounds by
enzymes within the mushroom cells in genus
Psilocybe
(Singer and Smith 1958, Hofmann et al. 1959). These
compounds have been found in more than 200
species of Basidiomycota, including
Psilocybe
,
Panaeo-
lus
,
Gymnopilus
, and other genera (Guzma´n et al.
1998, Stamets 1996). However these compounds have
not been reported in lepiotaceous fungi, although
lethal amatoxins are reported from several species of
Lepiota
(Is¸ilog˘lu and Watling 1991, Meunier et al.
1995). It is possible that there might be psilocin and
psilocybin in the bluing species of
Lepiota
(Stamets
1996), or in our case
Leucoagaricus
, but no chemical
analysis has been carried out. Of interest, among
lepiotaceous fungi toxic
Chlorophyllum molybdites
also
turn blue when mature or dried, but only pyrrolidine
derivative alkaloides lepiotins A and B and steroidal
derivates in the mushroom have been reported from
this species (Ohta et al. 1998, Yoshikawa et al. 2001).
ACKNOWLEDGMENTS
We are grateful for the help of Drs Else C. Vellinga, Gregory
M. Mueller, Yan-Chun Li and Mrs Mo-Chan Li who offered
advice, suggestions and analytical assistance. We thank Dr B.
Aguirre-Hudson (K), Dr M.E. Noordeloos (L), Dr John H.
Haines (NYS), Dr Tai-Hui Li (HMIGD), Dr Lin Guo
(HMAS), Drs P. Manimohan and T.K.A. Kumar (CALI)
for providing specimens. We are greatly indebted to Dr
Brandon Matheny, University of Tennessee, and an anon-
ymous reviewer for their valuable and critical comments.
This study was supported by the Joint Funds of the National
Natural Science Foundation of China and Yunnan Provin-
cial Government (No. U0836604), by the National Basic
Research Program of China (No. 2009CB522300), by the
National Commonweal Foundation of the Research Insti-
tute of Tropical Forestry, CAF (RITF2007-12), by the
National Science and Technology Support Project
(2006BAD03A0405), by the National Natural Science
Foundation of China (No. 30800004) and by the Ministry
of Science and Technology of China (2008FY110300).
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1152 MYCOLOGIA
