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Persoonia 32, 2014: 13–24
www.ingentaconnect.com/content/nhn/pimj http://dx.doi.org/10.3767/003158514X679119
RESEARCH ARTICLE
INTRODUCTION
Sequestrate and angiocarpous basidiomata have developed in
several groups of Agaricomycetes. Various plausible selective
pressures have been proposed to explain this transformation
from agaricoid to gasteroid basidiomata (Miller et al. 2001).
It is often assumed that changing environmental conditions
led to enclosed basidiome morphology and eventually to the
hypogeous gasteroid fruiting bodies, offering protection against
frost and moisture loss from the hymenium and thus preventing
desiccation (Thiers 1984a, Bruns et al. 1989, Bougher & Lebel
2001, Wilson et al. 2011). Arid or seasonally dry climates thus
exert a selection pressure towards a sequestrate fruiting body,
especially in ectomycorrhizal fungi which provide the plants with
extra water through their mycelium and help them to survive the
xeric conditions (Trappe & Claridge 2005, Smith et al. 2006).
The observation that gasteroid and hypogeous gasteroid rus-
suloid taxa are rare or absent in the humid tropics seems to
support this idea (Buyck 1995).
Gasteroid Russulales are indeed particularly well-represented
and well-studied in Australia and New Zealand (Bougher 1997,
Bougher & Lebel 2001, Lebel 2001, 2002, 2003a, b, Lebel &
Castellano 2002) and North America (Zeller & Dodge 1919,
1936, Singer & Smith 1960, Smith 1963, Thiers 1984b, Miller &
Lebel 1999, Desjardin 2003, Smith et al. 2006). Tropical records
seem rare and occasional. Only eight species that are currently
accepted in the Russulales have been described from tropical
Asia. Corner & Hawker (1953) described one Arcangeliella
species and two Elasmomyces species from Malaysia and
Heim (1959) described Elasmomyces densus from Thailand.
In China, Zhang & Yu (1990) described two angiocarp Rus-
sulales species (Gymnomyces lactifer B.C. Zhang & Y.N. Yu
and Martellia ramispina B.C. Zhang & Y.N. Yu) and Tao et al.
(1993) described Martellia nanjingensis B. Liu & K. Tao and
Zelleromyces sinensis B. Liu, K. Tao & Ming C. Chang.
Tropical Africa seems even poorer in sequestrate Russulales
with only Lactarius dolichocaulis (Pegler) Verbeken & U. Eberh.,
L. angiocarpus Verbeken & U. Eberh. and Cystangium capitis-o-
rae (Dring) T. Lebel (Dring & Pegler 1978, Eberhardt & Verbeken
2004, Verbeken & Walleyn 2010).
It is now generally accepted and in many cases molecularly
confirmed that Gymnomyces, Martellia, Cystangium and Elas-
momyces are synonyms of Russula and that Zelleromyces
and Arcangeliella are included in Lactarius (Miller et al. 2001,
Eberhardt & Verbeken 2004, Nuytinck et al. 2004).
This study reports on collections of gasteroid representatives of
Russulales, encountered in tropical forests in the area around
Shinharaja Forest Reserve, Sri Lanka, and around Chiang Mai,
Northern Thailand. None of the collected specimens fits with
previously described taxa, therefore six new species in the
genus Lactarius are described here. Molecular data were used
to ascertain their phylogenetic position and full descriptions and
illustrations are given.
MATERIALS AND METHODS
The study is based on collections made by Kobeke van de Putte,
Annemieke Verbeken and Dirk Stubbe. The studied material is
deposited in the Herbarium Universitatis Gandavensis (GENT).
An overview of the studied specimens, including information on
the collection locality and ecology is given after each species
description section.
Morphological study
Descriptions of macromorphological features are based on
fresh material. Colours were described in daylight conditions
following the colour guide by Kornerup & Wanscher (1978)
and Petersen (1996, indicated as FK in descriptions). Latex
coloration was recorded as it was exuded from the mushroom,
Tales of the unexpected: angiocarpous representatives of the
Russulaceae in tropical South East Asia
A. Verbeken1, D. Stubbe1,2, K. van de Putte1, U. Eberhardt³, J. Nuytinck1,4
Key words
Arcangeliella
gasteroid fungi
hypogeous fungi
Lactarius
Martellia
morphology
phylogeny
Zelleromyces
1 Ghent University, Department of Biology, Research Group Mycology, K.L.
Ledeganckstraat 35, 9000 Ghent, Belgium;
corresponding author e-mail: mieke.verbeken@ugent.be.
2 Scientific Institute of Public Health, Section Mycology and Aerobiology,
Juliette Wytsmanstraat 14, 1050 Brussels, Belgium.
3 Stuttgart State Museum of Natural History, Botany Department, Rosenstein
1, 70191 Stuttgart, Germany.
4 Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Nether-
lands.
Abstract Six new sequestrate Lactarius species are described from tropical forests in South East Asia. Extensive
macro- and microscopical descriptions and illustrations of the main anatomical features are provided. Similarities
with other sequestrate Russulales and their phylogenetic relationships are discussed. The placement of the species
within Lactarius and its subgenera is confirmed by a molecular phylogeny based on ITS, LSU and rpb2 markers.
A species key of the new taxa, including five other known angiocarpous species from South East Asia reported to
exude milk, is given. The diversity of angiocarpous fungi in tropical areas is considered underestimated and driving
evolutionary forces towards gasteromycetization are probably more diverse than generally assumed. The discovery
of a large diversity of angiocarpous milkcaps on a rather local tropical scale was unexpected, and especially the
fact that in Sri Lanka more angiocarpous than agaricoid Lactarius species are known now.
Article info Received: 2 February 2013; Accepted: 18 June 2013; Published: 20 January 2014.
14 Persoonia – Volume 32, 2014
Species Voucher collection Origin ITS LSU rpb2
accession no. accession no. accession no.
Amylostereum laevigatum olrim409/CBS623.84 AY781246 AF287843 AY218469
Arcangeliella borziana Switzerland AF286204
Switzerland AF373599
Italy JF908775
A. camphorata USA EU644700
USA EU644701
USA EU644702
USA EU834192
USA EU846241
A. crassa USA AY558740
A. sp. Thailand FJ454900
Australia, Tasmania JF960610
USA JX415331
Auriscalpium vulgare AFTOL1897/DAOM128994 DQ911613 DQ911614 AY218472
Echinodontium tinctorium AFTOL455 AY854088 AF393056 AY218482
Lactarius acris EU014 (UPS) Germany DQ421988 DQ421988 DQ421922
L. akahatsu AV2004-141 (GENT) Thailand KF133269 KF133301 KF133333
L. albocarneus AV98-080 (GENT) France KF241545
L. alboscrobiculatus LTH175 (CMU, SFSU, GENT) Thailand EF141538
L. angiocarpus DA00-448 (GENT) Zambia AY606942 AY606970 DQ421921
L. atroviridis AV05-306 (GENT) USA KF133270 KF133302 KF133334
L. auriolla RW1601 (GENT) Sweden KF133257 KF133288 KF133321
L. azonites AV00-124 (GENT) Belgium KF241540
L. baliophaeus AV05-155 (GENT) Malawi GU258277 GU265576 GU258312
L. camphoratus UE04.09.2004 (UPS) Sweden DQ422009 DQ422009 DQ421933
L. chichuensis Wang1236 (HKAS) China KF241541
L. chromospermus AV99-174 (GENT) Zimbabwe KF133260 KF133292 KF133324
L. chrysorrheus UE04.10.2002-8 (UPS) Italy KF133261 KF133293 KF133325
L. citriolens UE20.09.2004-03 (UPS) Sweden DQ422003 DQ422003 DQ421931
L. controversus AV00-117 (GENT) Italy KF241544
L. crassiusculus LTH369 (GENT) Thailand EF560684 KF133303 KF133335
L. cyanescens DS06-058 (GENT) Malaysia GU258278 CU265581 GU258317
L. cyathuliformis UE04.09.2004-2 (UPS) Sweden KF133266 KF133298 KF133330
L. deliciosus JN2001-046 (GENT) Slovakia KF133272 KF133305 KF133337
L. echinellus sp. nov. AV07-169 (GENT) Sri Lanka KF133287 KF133320 KF133352
AV07-175 (GENT) Sri Lanka KF133286 KF133319 KF133351
L. echinus sp. nov. AV07-168 (GENT) Sri Lanka KF133273 KF133306 KF133338
L. falcatus sp. nov. KVP08-038 (GENT) Thailand KF133274 KF133307 KF133339
L. flexuosus UE06.09.2002-1 (UPS) Sweden DQ421992 DQ421992 DQ421925
L. formosus LTH382 (CMU, SFSU, GENT) Thailand EF141549
L. fuliginosus MTB97-24 (GENT) Sweden JQ446111 JQ446180 JQ446240
L. helvus UE08.09.2004-1 (UPS) Sweden KF133263 KF133295 KF133327
L. hispidulus AB152 (GENT) Guinea KF133258 KF133289 KF133322
L. kabansus AV99-205 (GENT) Zimbabwe KF133259 KF133291 KF133323
L. lignyotus UE06.09.2003-5 (UPS) Sweden DQ421993 DQ421993 DQ421926
L. lilacinus RW3774 (GENT) Belgium KF133275 KF133308 KF133340
L. luridus OB11-011 (GENT) Belgium KF241547
L. mairei AV00-118 (GENT) Italy AY336950
L. mammosus UE09.09.2004-5 (UPS) Sweden KF133265 KF133297 KF133329
L. montoyae KD1065 (BSHC) India EF560673 GU265641 GU258380
L. necator AV04-231 (GENT) France KF133276 KF133309 KF133341
L. peckii JN2004-020 (GENT) USA KF133277 KF133310 KF133342
L. pomiolens sp. nov. AV07-159 (GENT) Sri Lanka KF133282 KF133315 KF133347
L. pubescens AV96-931 (GENT) Norway AY336958
UE15.09.2002-2 (UPS) Sweden DQ421996 DQ421996 DQ421929
L. quieticolor UE10.09.2004-1 (UPS) Sweden DQ422002 DQ422002 DQ42930
L. quietus UE16.09.2004 (UPS) Sweden KF133264 KF133296 KF133328
L. romagnesii UE29.09.2002-6 (UPS) France DQ421989 DQ421989 DQ421923
L. rubriviridis DED7312 (SFSU) USA EF685088
L. rufus JN2002-008 (GENT) Norway KF241543
L. saturnisporus sp. nov. AV07-170 (GENT) Sri Lanka KF133283 KF133316 KF133348
DS07-488 (GENT) Sri Lanka KF133284 KF133317 KF133349
DS07-490 (GENT) Sri Lanka KF133285 KF133318 KF133350
L. shoreae sp. nov. AV07-164 (GENT) Sri Lanka KF133278 KF133311 KF133343
L. sphagneti PL2805 (pers. herb. P. Leonard) UK KF133268 KF133300 KF133332
L. spinosulus AT2003068 (UPS) Sweden KF133262 KF133294 KF133326
L. stephensii UK EU784439
RW2930 (GENT) Belgium AY331012
L. subdulcis JV2006-024 (GENT) Belgium KF133279 KF133312 KF133344
L. subplinthogalus AV04-219 (GENT) USA KF241539
L. subsericatus UE11.10.2004-8 (UPS) Sweden DQ422011 DQ422011 DQ421934
L. tenellus DKA3598 (BR) Benin KF133280 KF133313 KF133345
L. thyinos A.Voitk23-08-2004 (GENT) Canada KF133271 KF133304 KF133336
L. torminosus LVL2002-013 (GENT) Belgium AY336959
RW3183 (GENT) Czech Republic KF133281 KF133314 KF133346
L. trivialis UE27.08.2002-17a (UPS) Sweden DQ421991 DQ421991 DQ421924
L. uvidus KVP10-027 (GENT) Russia KF241546
L. vietus UE11.19.2004-1 (UPS) Sweden KF133267 KF133299 KF133331
Table 1 Specimens and GenBank accession numbers of DNA sequences used in the molecular analyses.
15
A. Verbeken et al.: Angiocarpous Russulaceae in South East Asia
Species Voucher collection Origin ITS LSU rpb2
accession no. accession no. accession no.
L. vinaceorufescens JN2007-018 (GENT) Canada KF241542
Lactifluus deceptivus AV04-181 (GENT) USA DQ422020 DQ422020 DQ421935
Lf. edulis AV99-041 (GENT) Zimbabwe DQ421977 DQ421977 DQ421916
Lf. emergens AV99-005 (GENT) Zimbabwe AY606979 KF133290 DQ421919
Lf. gerardii AV05-375 (GENT) USA GU258254 GU265616 GU258353
Lf. longisporus AV99-197 (GENT) /BB 00.1519 (PC) Zimbabwe /Madagascar DQ421971 (AV) DQ421971 (AV) DQ421910 (PC)
Lf. nodosicystidiosus BB97-072 (PC) Madagascar DQ421976 DQ421976 DQ421915
Lf. phlebophyllus BB00-1388 (PC) Madagascar DQ421979 DQ421979 DQ421918
Lf. piperatus UE09.08.2004-6 (UPS) Sweden DQ422035 DQ422035 DQ421937
Lf. vellereus UE20.09.2004-22 (UPS) Sweden DQ422034 DQ422034 DQ421936
Lf. velutissimus AV99-185 (GENT) Zimbabwe DQ421973 DQ421973 DQ421912
Lf. volemus UE09.08.2004-5 (UPS) Sweden DQ422008 DQ422008 DQ421932
Multifurca furcata RH7804 (NY) Costa Rica DQ421994 DQ421994 DQ421927
M. ochricompacta BB02.107 (PC) USA DQ421984 DQ421984 DQ421940
M. zonaria DED7442 (PC) Thailand DQ421990 DQ421990 DQ421942
Russula aeruginea AT2003017 (UPS) Sweden DQ421999 DQ421999 DQ421946
R. albonigra AT2002064 (UPS) Sweden DQ422029 DQ422029 DQ421966
R. camarophylla PAM01081108 (PC) France DQ421982 DQ421982 DQ421938
R. earlei WCRW00-412 (PC) USA DQ422025 DQ422025 DQ421963
R. emetica UE05.10.2003-11 (UPS) Sweden DQ421997 DQ421997 DQ421943
R. firmula AT2004142 (UPS) Sweden DQ422017 DQ422017 DQ421958
R. gracillima UE23.08.2004-14 (PC) Sweden DQ422004 DQ422004 DQ421949
R. heterophylla UE20.08.2004-2 (UPS) Sweden DQ422006 DQ422006 DQ421951
R. illota UE26.07.2002-3 (UPS) Sweden DQ422024 DQ422024 DQ421967
R. lepida HJB9990 (UPS) Belgium DQ422013 DQ422013 DQ421954
R. nigricans UE20.09.2004-07 (PC) Sweden DQ422010 DQ422010 DQ421952
R. ochrospora GD20.07.2004 (UPS) Italy DQ422012 DQ422012 DQ421953
R. parazurea BW06.09.2002-16 /MF01.10.2003 (UPS) Sweden DQ422007 (MF) DQ422007 (MF) DQ421945 (BW)
R. pectinatoides AT2001049 (UPS) Sweden DQ422026 DQ422026 DQ421964
R. persicina UE21.09.2003-01 (UPS) Sweden DQ422019 DQ422019 DQ421960
R. risigallina UE03.07.2003-08 (UPS) Sweden DQ422022 DQ422022 DQ421961
R. vesca AT2002091 (UPS) Sweden DQ422018 DQ422018 DQ421959
R. virescens HJB9989 (UPS) Belgium DQ422014 DQ422014 DQ421955
Stereum hirsutum AFTOL492 AY854063 AF393078 AY218520
Zelleromyces gardneri USA DQ453696
USA JN022500
Z. giennensis Spain AF230900
Z. hispanicus Spain AF231911
Spain AF231912
Spain AF231913
Spain AJ555566
Spain AJ555567
Z. sp. Australia, Tasmania JF960852
Australia, Tasmania JF960853
Australia, Tasmania JF960854
Table 1 (cont.)
but also from a drop placed on a glass slide held over white
paper, and from a drop placed directly on white paper. Pictures
of the basidiocarps will be published on the Russulales News
website (http://www2.muse.it/russulales-news/).
Micromorphological characters were registered from the dried
specimens. Spores were observed in Melzer’s reagent for
measurements and drawings; other structures in 2–5 % KOH
or Congo-red. For each collection the length and width of at
least 20 spores were measured in side view in Melzer’s rea-
gent, excluding the ornamentation. Measurements are given
as (MINa) [AVa–2×SD] – AVa – AVb – [AVb + 2×SD] (MAXb) in
which AVa = lowest mean value for the measured collections,
AVb = greatest mean value and SD = standard deviation cal-
culated for the measurements of one collection. Q stands for
‘quotient length/width’ and is given as (MINQ)–Qa–Qb–(MAXQ)
in which Qa and Qb stand for the lowest and the highest, re-
spectively mean quotient for the measured specimens. In case
only one collection was available spore measurements are
given as (MINa) [AVa–2×SD] – AVa – [AVa + 2×SD] (MAXa).
Micromorphological features were illustrated with the aid of a
drawing tube attached to an Olympus CX-41 research com-
pound microscope. For the details of description and terminol-
ogy of micromorphological features see Verbeken (1998) and
Verbeken & Walleyn (2010).
DNA extraction, PCR amplification and sequencing
Nine gasteroid Lactarius collections were used for the molecu-
lar analyses. DNA was extracted from dried and fresh fruiting
bodies using the methods described by Nuytinck & Verbeken
(2003) with slight modifications (van de Putte et al. 2010). Three
loci were amplified and sequenced: 1) the internal transcribed
spacer region of the nuclear ribosomal DNA (ITS), using primers
ITS1-F and ITS4 (White et al. 1990, Gardes & Bruns 1993);
2) a part of the nuclear ribosomal large subunit region (LSU),
using primers LR0R and LR5 (Vilgalys & Hester 1990, Rehner
& Samuels 1994); and 3) the region between domains 6 and 7
of the nuclear gene encoding the second largest subunit of RNA
polymerase II (rpb2), using primers bRPB2-6F and fRPB2-7cR
(Liu et al. 1999, Matheny 2005).
Protocols for PCR amplification and sequencing follow Le et al.
(2007), sequencing was also conducted with an ABI 3730XL
or ABI 3700 by MACROGEN (Amsterdam, The Netherlands).
Sequences were assembled and edited with the software Se-
quencherTM v4.9 (GeneCodes Corporation, Ann Arbor, Michi-
gan, USA). Other sequences were gained following the DNA
extraction, PCR and sequencing protocols described by Eber-
hardt (2002) or Taylor et al. (2006).
16 Persoonia – Volume 32, 2014
Alignment and phylogenetic analyses
Table 1 shows an overview of all specimens and sequences
used in the phylogenetic analyses, including GenBank acces-
sion numbers. Two alignments were constructed. The first align-
ment consists of ITS sequences only; it includes all sequestrate
Lactarius, Arcangeliella and Zelleromyces sequences available
from GenBank, the newly discovered sequestrate collections
from Sri Lanka and Thailand, and a broad selection of agari-
coid Lactarius taxa; three Multifurca species were used as the
outgroup. This alignment is used to confirm that our new taxa
are well supported and differ from the sequestrate milk cap
species that have been known and sequenced before. The
second alignment consists of ITS, LSU and rpb2 sequences,
including only specimens for which all three loci are available.
The sampling covers a broader selection of Russulaceae: the
four genera of Russulaceae (Lactarius, Lactifluus, Multifurca
and Russula, see Buyck et al. 2008) are represented. Outgroup
Russulales species are Auriscalpium vulgare Gray, Stereum
hirsutum (Willd.) Pers., Amylostereum laevigatum (Fr.) Boidin
and Echinodontium tinctorium (Ellis & Everh.) Ellis & Everh.
This second alignment is used to study the phylogenetic place-
ment of the newly described species within Lactarius and its
Lactarius fuliginosus MTB97-24
SWEDEN
Lactarius azonites AV00-124
BELGIUM
Lactarius romagnesii UE29.09.2002-6
FRANCE
Lactarius montoyae KD1065
INDIA
Lactarius subplinthogalus AV04-219
USA
Lactarius acris EU014
GERMANY
Zelleromyces gardneri DQ453696
USA
Zelleromyces gardneri JN022500
USA
Lactarius lignyotus UE06.09.2003-5
SWEDEN
Lactarius saturnisporus AV07-170
SRI LANKA
Lactarius saturnisporus DS07-490
SRI LANKA
Lactarius saturnisporus DS07-488
SRI LANKA
Lactarius cyanescens DS06-058
MALAYSIA
Lactarius echinus AV07-168
SRI LANKA
Lactarius shoreae AV07-164
SRI LANKA
Lactarius crassiusculus LTH369
THAILAND
Lactarius echinellus AV07-169
SRI LANKA
Lactarius echinellus AV07-175
SRI LANKA
Lactarius baliophaeus AV05-155
THAILAND
Lactarius angiocarpus DA00-448
ZAMBIA
Lactarius hispidulus AB152
GUINEA
Arcangeliella camphorata EU834192
USA
Arcangeliella camphorata EU644701
USA
Arcangeliella camphorata EU846241
USA
Arcangeliella camphorata EU644702
USA
Arcangeliella camphorata EU644700
USA
‘Arcangeliella’ sp. FJ454900
THAILAND
Arcangeliella sp. JF960610
AUSTRALIA
Lactarius chichuensis Wang1236
CHINA
Lactarius camphoratus UE04.09.2004
SWEDEN
Lactarius helvus UE08.09.2004-1
SWEDEN
Lactarius falcatus KVP08-038
THAILAND
Lactarius quietus UE16.09.2004
SWEDEN
Lactarius chrysorrheus UE04.10.2002-8
ITALY
Lactarius vinaceorufescens JN2007-018
CANADA
Lactarius cyathuliformis UE04.09.2004-2
SWEDEN
Lactarius subdulcis JV2006-024
BELGIUM
Lactarius rufus JN2002-008
NORWAY
Zelleromyces sp. JF960853
AUSTRALIA
Zelleromyces sp. JF960854
AUSTRALIA
Zelleromyces sp. JF960852
AUSTRALIA
Lactarius sphagneti PL2805
UK
Arcangeliella borziana AF286204
SWITZERLAND
Arcangeliella borziana AF373599
SWITZERLAND
Arcangeliella borziana JF908775
ITALY
Lactarius subsericatus UE11.10.2004-8
SWEDEN
Zelleromyces hispanicus AJ555567
SPAIN
Zelleromyces hispanicus AJ555566
SPAIN
Zelleromyces hispanicus AF231913
SPAIN
Zelleromyces hispanicus AF231911
SPAIN
Zelleromyces hispanicus AF231912
SPAIN
Lactarius peckii JN2004-020
USA
100
100
94
70
80
98
82
75
63
100
100
97
73
90
78
54
99
95
97
99
100
71
74
100
96
82
61
65
99
80
66
86
*
Lactarius alboscrobiculatus LTH175
THAILAND
Lactarius pubescens UE15.09.2002-2
SWEDEN
Lactarius pubescens AV96-931
NORWAY
Lactarius torminosus RW3183
CZECH REP.
Lactarius torminosus LVL2002-013
BELGIUM
Arcangeliella crassa AY558740
USA
Lactarius mairei AV00-118
ITALY
Lactarius thyinos A.Voitk23-08-2004
CANADA
Lactarius rubriviridis DED7312
USA
Lactarius quieticolor UE10.09.2004-1
SWEDEN
Lactarius akahatsu JN2004-141
THAILAND
Lactarius deliciosus JN2001-046
SLOVAKIA
Lactarius chromospermus AV99-174
ZIMBABWE
Arcangeliella sp. JX415331
USA
Lactarius citriolens UE20.09.2004-03
SWEDEN
Lactarius auriolla RW1601
SWEDEN
Lactarius controversus AV00-117
ITALY
Lactarius stephensii RW2930
BELGIUM
Lactarius stephensii EU784439
UK
Lactarius uvidus KVP10-027
RUSSIA
Lactarius formosus LTH382
THAILAND
Lactarius luridus OB11-011
BELGIUM
Lactarius albocarneus AV98-080
FRANCE
Lactarius trivialis UE27.08.2002-17a
SWEDEN
Lactarius flexuosus UE06.09.2002-1
SWEDEN
Lactarius mammosus UE09.09.2004-5
SWEDEN
Lactarius spinosulus AT2003068
SWEDEN
Zelleromyces giennensis AF230900
SPAIN
Lactarius atroviridis AV05-306
USA
Lactarius necator AV04-231
FRANCE
Lactarius vietus UE11.19.2004-1
SWEDEN
Lactarius pomiolens AV07-159
SRI LANKA
Lactarius kabansus AV99-205
ZIMBABWE
Lactarius tenellus DKA3598
BENIN
Multifurca furcata RH7804
COSTA RICA
Multifurca ochricompacta BB02.107
USA
Multifurca zonaria DED7442
THAILAND
61
84
62
66
100
100
79
82
100
60
100
100
100
0.05
*
Fig. 1 ML tree (RAxML) based on ITS sequences. Bootstrap values are indicated if they
exceed 50 %. Names in orange are the new angiocarpous species described in this paper,
names in green are angiocarpous Lactarius species for which ITS sequences are available
on GenBank. Arcangeliella sp. FJ454900 was obtained by sequencing plant roots; the fruit-
ing body was not observed, and thus it is unclear whether this sequence is actually from an
angiocarpous species. The scale bar represents the number of nucleotide changes per site.
17
A. Verbeken et al.: Angiocarpous Russulaceae in South East Asia
Lactarius deliciosus JN2001-046
Lactarius akahatsu JN2004-141
Lactarius quieticolor UE10.09.2004-1
Lactarius thyinos A.Voitk23-08-2004
Lactarius auriolla RW1601
Lactarius citriolens UE20.09.2004-03
Lactarius pubescens UE15.09.2002-2
Lactarius torminosus RW3183
Lactarius trivialis UE27.08.2002-17a
Lactarius mammosus UE09.09.2004-5
Lactarius flexuosus UE06.09.2002-1
Lactarius lilacinus RW3774
Lactarius spinosulus AT2003068
Lactarius atroviridis AV05-306
Lactarius necator AV04-231
Lactarius vietus UE11.19.2004-1
Lactarius peckii JN2004-020
Lactarius falcatus KVP08-038
Lactarius camphoratus UE04.09.2004
Lactarius helvus UE08.09.2004-1
Lactarius quietus UE16.09.2004
Lactarius chrysorrheus UE04.10.2002-8
Lactarius cyathuliformis UE04.09.2004-2
Lactarius subdulcis JV2006-024
Lactarius sphagneti PL2805
Lactarius subsericatus UE11.10.2004-8
Lactarius angiocarpus DA00-448
Lactarius baliophaeus AV05-155
Lactarius shoreae AV07-164
Lactarius crassiusculus LTH369
Lactarius echinellus AV07-175
Lactarius echinellus AV07-169
Lactarius echinus AV07-168
Lactarius cyanescens DS06-058
Lactarius lignyotus UE06.09.2003-5
Lactarius saturnisporus DS07-490
Lactarius saturnisporus DS07-488
Lactarius saturnisporus AV07-170
Lactarius acris EU014
Lactarius montoyae KD1065
Lactarius romagnesii UE29.09.2002-6
Lactarius fuliginosus MTB97-24
Lactarius pomiolens AV07-159
Lactarius tenellus DKA3598
Lactarius kabansus AV99-205
Lactarius hispidulus AB152
Lactarius chromospermus AV99-174
Multifurca ochricompacta BB02.107
Multifurca zonaria DED7442
Multifurca furcata RH7804
Russula camarophylla PAM01081108
Russula earlei WCRW00-412
Russula albonigra AT2002064
Russula nigricans UE20.09.2004-07
Russula gracillima UE23.08.2004-14
Russula persicina UE21.09.2003-01
Russula emetica UE05.10.2003-11
Russula lepida HJB9990
Russula firmula AT2004142
Russula risigallina UE03.07.2003-08
Russula pectinatoides AT2001049
Russula illota UE26.07.2002-3
Russula parazurea 003BW06.09.2002-16 /MF01.10.2
Russula ochrospora GD20.07.2004
Russula aeruginea AT2003017
Russula virescens HJB9989
Russula heterophylla UE20.08.2004-2
Russula vesca AT2002091
Lactifluus longisporus AV99-197/BB 00.1519
Lactifluus vellereus UE20.09.2004-22
Lactifluus deceptivus AV04-181
Lactifluus emergens AV99-005
Lactifluus velutissimus AV99-185
Lactifluus phlebophyllus BB00-1388
Lactifluus nodosicystidiosus BB97-072
Lactifluus edulis AV99-041
Lactifluus gerardii AV05-375
Lactifluus piperatus UE09.08.2004-6
Lactifluus volemus UE09.08.2004-5
Auriscalpium vulgare AFTOL1897/DAOM128994
Stereum hirsutum AFTOL492
Amylostereum laevigatum olrim409/CBS623.84
Echinodontium tinctorium AFTOL455
100/100
100/100
98/100
100/100
100/100
78/99
100/100
100/100
97/100
100/100
80/99
96/100
100/100
100/100
51/-
90/100
100/100
100/100
69/100
100/100
61/-
85/100
98/100
100/100
98/100
86/100
80/100
52/-
61/100
100/100
100/100
98/100
54/-
94/100
60/99
83/100
69/95
93/100
100/100
100/100
58/-
57/-
100/100
72/-
98/100
91/100
100/100
99/100
92/100
81/100
100/100
99/100
100/100
95/100
100/100
80/96
83/100
80/100
61/-
81/100
100/100
76/-
100/-
0.1
L. s
Plinthogalus
ubg.
L. s
Russularia
ubg.
L. s
Piperites
ubg.
subgenera. Alignments were constructed with the online version
of MAFFTv6 (Katoh & Toh 2008), applying the E-INS-I strategy,
a very slow method recommended for less than 200 sequences
with multiple conserved domains and long gaps. The align-
ments were manually refined in BioEdit v7.0.9.0 (Hall 1999)
and made available in TreeBASE (www.treebase.org, study ID:
S14274). For the second alignment, ambiguously aligned posi-
tions (mainly within ITS1 and 2) were detected using Gblocks
v0.91b (Castresana 2000), specifying less stringent conditions
than default in order to keep gapped sites. Apart from the posi-
tions identified by Gblocks, the intron region of rpb2 was also
deleted from the analyses to avoid the inclusion of ambiguous
alignment. Sequence data were partitioned as follows: 1) ITS
was partitioned into the ribosomal genes 18S (partial) and 5.8S
and the spacer regions ITS1 and ITS2; 2) LSU; and 3) rpb2
was partitioned into codon positions 1, 2 and 3.
Maximum Likelihood (ML) analyses were performed in RAxML
v7.0.3 (Stamatakis 2006), combining a ML search with the
Rapid Bootstrapping algorithm for 1 000 replicates. The model
GTRGAMMA was estimated for each partition separately. The
analyses were first run for the individual loci. Incongruence
between loci was checked by comparing clades with a bootstrap
support of 70 % or higher.
Bayesian Inference (BI) analyses were carried out in MrBayes
v3.2.0 (Ronquist & Huelsenbeck 2003). The general time-rever-
sible model with rate variation across sites and a proportion
of invariable sites (GTR+I+G) was used. Rates and model
para meters were unlinked between all partitions. Two analyses
were run: 1) an analysis on a desktop computer with 2 runs and
1 chain per run was executed for 20 million generations (Ron-
quist et al. 2009); and 2) 4 independent, parallel runs of 1 cold
and 3 heated chains were run for 20 million generations on a
High Performance Computer (HPC) of the Ghent University.
Sample frequency was set at 100. The log probability of the
data given the parameter values and effective sample size
statistics (ESS) of the runs were examined with Tracer v1.5
(Drummond & Rambaut 2007). To check convergence, 1) the
standard deviation of split frequencies across the 2 runs on the
desktop computer was assessed; and 2) topologies and poste-
rior probabilities from the 4 runs on the HPC were compared. An
appropriate burn-in value was determined visually using Tracer.
Fig. 2 ML tree (RAxML) based on ITS, LSU and rpb2 sequences. Bootstrap values and Posterior Probabilities (resulting from Bayesian analysis using the
HPC) are indicated if they exceed 50 % or 95 %, respectively (BS /PP). Names in orange are the new angiocarpous species described in this paper. The scale
bar represents the number of nucleotide changes per site.
18 Persoonia – Volume 32, 2014
RESULTS
Phylogeny
Fig. 1 shows the obtained ML topology based on the align-
ment including only ITS sequences; bootstrap (BS) values are
indicated on the branches. The six new sequestrate Lactarius
species from South East Asia are indicated in orange and are
clearly distinct from the previously known and sequenced se-
questrate milk cap species (indicated in green).
The analysis based on all three sampled loci (ITS, LSU and
rpb2) reveals the position of our South East Asian collections
within the genus Lactarius. Since there was no conflict among
the single loci trees in clades with a bootstrap support of 70 %
or higher, a combined analysis was performed. Fig. 2 shows the
obtained ML topology with BS values and Bayesian posterior
probabilities (PP). ML and both Bayesian phylogenies differ
only in the placement of some terminal, non-gasteroid taxa. All
3 analyses show 3 well-delimited genera in the Russulaceae
(Lactarius BS 92 % - PP 100 %, Multifurca BS 100 % - PP
100 % and Russula BS 90 % - PP 100 %) but fail to support
the monophyly of the genus Lactifluus. Instead, Lactifluus
consistently comes out as paraphyletic and basal to the other
Russulaceae genera. Lactifluus volemus, Lactifluus piperatus
and Lactifluus gerardii are not included in a monophyletic
‘core’-group of Lactifluus (BS 83 % - PP 100 %) represented
by Lactifluus subg. Lactifluus p.p. (excluding section Lactifluus),
subg. Edules and subg. Lactariopsis.
All gasteroid milkcaps included in this study belong to the genus
Lactarius. Lactarius falcatus sp. nov. is member of L. subg.
Russularia, while L. saturnisporus sp. nov., L. echinus sp. nov,
L. echinellus sp. nov. and L. shoreae sp. nov. belong to L. subg.
Plinthogalus. The affinities of L. pomiolens sp. nov. are less
clear. It appears as one of the long, basal branches of the genus
Lactarius for which no subgeneric subdivisions are available.
Taxonomy
All newly proposed species (L. pomiolens, L. echinus, L. echi-
nellus, L. saturnisporus, L. shoreae and L. falcatus) produce
milky exudates or latex. The additional known angiocarpous
species from South East Asia reported to exude milk are also
included in the species key. These are: Arcangeliella lactifera
(B.C. Zhang & Y.N. Yu) J.M. Vidal, A. densa (R. Heim) Singer
& A.H. Sm., Zelleromyces ramispinus (B.C. Zhang & Y.N. Yu)
Trappe, T. Lebel & Castellano, Z. sinensis B. Liu, K. Tao &
Ming C. Chang and Martellia nanjingensis (B. Liu & K. Tao)
J.M. Vidal. Although we also consider these species to be
members of the genus Lactarius, new combinations are not
proposed here because we did not study the type specimens,
or obtained molecular data.
It is striking that all tropical species have a very high spore
ornamentation, either consisting of wings, or of isolated high
spines, while all known Australian species have a much lower
ornamentation that is usually subreticulate or formed of irregular
warts, to sometimes even extremely low resulting in almost
smooth spores, as in Zelleromyces glabrellus (Zeller & C.W.
Dodge) Singer & A.H. Sm. (Zeller & Dodge 1936). The only
angiocarpous milkcap species that have spores with a winged
aspect are Zelleromyces striatus (G. Cunn.) G.W. Beaton,
Pegler & T.W.K. Young and Zelleromyces malaiensis (Corner
& Hawker) A.H. Sm., but the ridges are not exceeding 0.5 µm
height (Pegler & Young 1979, Grgurinovic 1997). The latter
species is also reported from India and Malaysia, but only with
Eucalyptus (Trappe et al. 2002, Desjardin 2003).
KEY TO THE SPECIES
1. Spores winged, reticulate, usually with high ridges....2
1. Spores echinulate, with isolated warts or spines......5
2. Spores lowly ornamented, with small ridges that are
0.5–1.5 µm high ...................... Z. sinensis
2. Spores distinctly winged, with ornamentation that is clearly
exceeding 2 µm high ...........................3
3. Spores > 10 µm......................L. pomiolens
3. Spores < 10 µm............................... 4
4. Spore ornamentation with ridges up to 3–4 µm high, with
distinct transverse striations; peridiopellis a strongly inter-
woven trichopalisade, embedded in a narrow and incrusted
slime-layer ........................L. saturnisporus
4. Spore ornamentation with ridges up to 2.5–3.5 µm high,
lacking striations; peridiopellis a strongly interwoven pali-
sade to trichopalisade, without obvious slime layer.....
.....................................L. shoreae
5. Basidia 4-spored, 2-spored basidia sometimes present 6
5. Basidia exclusively 2-spored.....................8
6. Spores ornamented with irregular warts to short spines,
never more than 1 µm long ................A. densa
6. Spores ornamented with spines up to 2.5 µm long ....7
7. Basidia only 4-spored; spines straight and slender, not
branched ...........................L. echinellus
7. Basidia 2- and 4-spored; spines often branched on top .
.................................. Z. ramispinus
8. Spores on average 12 × 11.5 µm, ornamented with slender
and straight spines up to 4 µm long ........L. echinus
8. Spores on average 10 × 9.3 µm or smaller .......... 9
9. Spores ornamented with spines up to 4 µm long ......
.................................... A. lactifera
9. Spores ornamented with spines that are at most 2 µm long
...........................................10
10 . Spores ornamented with irregular and curved spines up to
2 µm long ............................L. falcatus
10. Spores ornamented with conical to blunt spines up to 1.5
µm long ......................... M. nanjingensis
1. Lactarius pomiolens Verbeken & Stubbe, sp. nov. — Myco-
Bank MB804182; Fig. 3
Holotype. Sri Lanka, near Sinharaja Forest, trail along river, on sandy wet
soil in rainforest with Shorea trapezifolia, Shorea disticta and Dipterocarpus
hispidus, 13 Dec. 2007, Verbeken 07-159 (GENT).
Etymology. With the smell of apples.
Basidiocarp 25–45 mm diam, subglobose, rather regular.
Peridium very slightly tomentose, felty, ochraceous to leather-
brown (FK13–14), buff to ochraceous, irregularly coloured,
with patches. Stipe absent. Columella absent. Gleba strongly
labyrinthuloid, with small loculi, with some, but very few ge-
latinous veins among them, greyish yellow (4B4), a bit more
flesh-coloured, staining dark brown where eaten by insect
larvae, firm in youngest ones, more compressible, rubbery in
older specimens. Latex rather abundant, white, staining im-
mediately sulphur yellow to greenish yellow (1A5–6) on white
paper, slowly changing yellow on the context then apparently
disappearing, when isolated turning golden yellow in a 10 %
aqueous potassium hydroxide solution, not forming a whitish
layer on the gleba when drying. Taste bitter, astringent, not just
dry. Smell very sweet, fruity, like apples, Russula fellea-like.
Spores globose to subglobose, 10.5–12.0–13.3 × 10.2–
11.5–12.8 µm, n = 20, Q = 1.01–1.04–1.13; ornamentation
amyloid, very highly winged; ridges up to 3–4 µm high, sel-
domly branched, rather broad and not completely amyloid but
19
A. Verbeken et al.: Angiocarpous Russulaceae in South East Asia
smell of apples seems a striking character, but more records
are needed to evaluate the stability of this feature.
2. Lactarius saturnisporus Verbeken & Stubbe, sp. nov. —
MycoBank MB804180; Fig. 4
Holotype. Sri Lanka, Kudawa, near Shinharaja Forest Reserve, primary
rainforest with Shorea spp., 14 Dec. 2007, Verbeken 07-170 (GENT).
Etymology. Referring to the spores that are so spectacularly winged that
they are reminiscent of the planet Saturn and its ring system
Basidiocarp 15–25 mm diam, 10–15 mm high, subglobose to
flattened or irregular, sometimes with minute papilla, slightly
rooting. Peridium surface, minutely velutinous, chamois-leather-
like, locally smooth or wrinkled, ochraceous cream coloured,
sometimes with pinkish and purplish tinges. Stipe absent.
Columella absent. Gleba rather soft and compressible, with
labyrinthuloid and rounded loculi, 1–3 per mm, dull cream
coloured to pale or greyish orange (6AB3) sometimes with
pinkish and purplish tinges near the margin, faintly staining
yellow, ultimately becoming pinkish but drying pale fawn, indis-
tinct reaction with ferrous sulphate. Latex scarse to abundant,
whitish hyaline, unchanging or slightly yellowing on the gleba,
staining white paper yellow, becoming yellow in a 10 % aque-
ous potassium hydroxide solution. Taste bitter to astringent,
disagreeable but also somewhat acrid. Smell not remarkable
or somewhat like citrus fruit.
Spores globose to subglobose, 8.0–8.9–9.7 × 7.6–8.3–9.0
µm, n = 20, Q = 1.01–1.07–1.12; ornamentation amyloid, very
highly winged; ridges up to 3–4 µm high with distinct transversly
Fig. 3 Lactarius pomiolens (holotypus). a. Basidia; b. pseudocystidia; c. ba-
sidiospores; d. cystidia; e. peridiopellis. — Scale bar = 10 μm.
c
b
d
a
e
with strongly amyloid tranversal bands; surface with amyloid
spots between the ridges; plage not distinct, not amyloid. Ba-
sidia 40–55 × 5–13 µm, slender and cylindrical to subclavate,
4-spored, thin-walled, hyaline; sterigmata up to 8 µm long.
Pseudocystidia present, irregular, sometimes branching, not
emergent, 5–7 µm diam. Cystidia extremely abundant, mostly
cylindrical, subclavate or clavate and regularly rounded on
top, sometimes fusiform, 25–60 × 10–16 µm, with very dense
needle-like and yellowish brown contents, with walls slightly
refringent to very slightly thickened. Peridiopellis an ixocutis,
composed of intricate, mostly pericline hyphae, 3–5 µm diam,
sometimes with small bulges.
Habitat — Rainforest with Shorea sp. and Dipterocarpus sp.
Specimen examined. Sri Lanka, Kudawa, near Sinharaja Forest, trail along
river, on sandy wet soil in rainforest with Shorea trapezifolia, Shorea disticta
and Dipterocarpus hispidus, 13 Dec. 2007, A. Verbeken GENT AV07-159,
holotype.
Notes — The species is outstanding because of its very
large (average 12 × 11.5 µm) spores and wings. The distinct Fig. 4 Lactarius saturnisporus (holotypus). a. Basidiospores; b. basidia;
c. cystidia; d. pseudocystidia; e. peridiopellis. — Scale bar = 10 μm.
c
b
d
a
e
20 Persoonia – Volume 32, 2014
striped and bifurcating pattern, mostly unbranching, sometimes
branched but never forming a reticulum, edges sharp and most-
ly crenate; surface roughly amyloid and verrucose in between
the ridges; plage not distinct, not amyloid. Basidia 4-spored,
subcylindrical, 45–60 × 10–12 µm, thin-walled, hyaline or with
some oil-drops; sterigmata up to 8 µm long. Cystidia present
in the hymenial cavities, rather abundant, variable in shape,
some clavate, some slightly utriform, 20–45 × 10–15 µm, with
slightly thickened wall, hyaline. Pseudocystidia less abundant,
cylindrical to somewhat tortuous, 4–6 µm diam. Peridiopellis a
strongly interwoven trichopalisade, embedded in a narrow and
incrusted slime-layer, some small globose elements present
but rare, terminal elements usually on top of intricate and short
hyphae; terminal elements cylindric to subclavate, 20–25(45) ×
4–7 µm, some with a prominent needle-like content, thin-walled.
Habitat — Primary tropical forest with Shorea spp.
Specimens examined. Sri Lanka, Kudawa, near Shinharaja Forest Re-
serve, primary rainforest with Shorea spp., 14 Dec. 2007, A. Verbeken GENT
AV07-170, holotype; Kudawa, near Sinharaja Forest Reserve, alongside
Pitakele river with mostly S. trapezifolia and some Dipterocarpus hispidus
stands, half burried in the soil near Shorea spp., 13 Dec. 2007, D. Stubbe
GENT DS07-488, DS07-490.
Notes — The species is easily recognized among most other
known angiocarpous Lactarius species because of the ex-
tremely high wings in the spore ornamentation. With a height of
3–4 µm on relatively small spores, they are so far known, the
highest winged Lactarius spores. They do share this character
with L. pomiolens, which has a similar high ornamentation up
to 4 µm, but remarkably larger spores (see further).
3. Lactarius shoreae Stubbe & Verbeken, sp. nov. — Myco-
Bank MB804181; Fig. 5
Holotype. Sri Lanka, near Shinharaja Forest, primary rainforest with
Shorea spp., 13 Dec. 2007, Verbeken 07-164 (GENT).
Etymology. Referring to the association with the ectomycorrhizal host
Shorea spp.
Basidiocarp 15 mm diam, 10 mm high, irregular. Peridium ir-
regularly shaped, with bulges and folds; pale yellow (2A3), in
some places darker; surface smooth, showing the loculi by
transparency. Stipe absent. Columella absent. Gleba with
rounded and labyrinthuloid, small loculi, buff, pale yellow (4A3).
Latex white, rather abundant, but soon after cutting becoming
hyaline and disappearing. Taste mild, very dry. Smell not very
remarkable, a bit sweetish and rubber-like.
Spores globose to subglobose, 7.9–9.1–10.3 × 7.6–8.7–9.9
µm, n = 20, Q = 1.01–1.04–1.10; ornamentation amyloid, very
highly winged; ridges up to 2.5–3.5 µm high, sharp, mostly
unbranching, sometimes branched without forming a reticulum;
surface roughly amyloid and verrucose in between the ridges;
plage not distinct, not amyloid. Basidia 4-spored, subcylindri-
cal to subclavate, 40–60 × 12–15 µm, thin-walled, hyaline,
sometimes with oil-drops; sterigmata up to 7 µm long. Cystidia
present in the hymenial cavities where they occur dispersed
between the basidia but also locally clustered, hyaline, thin-
walled (occasionally slightly thick-walled parts are observed),
very variable in shape, some fusiform or very narrow, others
irregular and somewhat knotty. Pseudocystidia rare, cylindrical,
4–6 µm diam. Peridiopellis a strongly interwoven palisade to
trichopalisade, with very small globose cells present; terminal
elements partly anticline, but sometimes adpressed and intri-
cate, cylindrical, 10–20 × 4– 6 µm, with thin or slightly thickened
walls.
Habitat — Primary forest with Shorea spp.
Specimen examined. Sri Lanka, Kudawa, near Shinharaja Forest Reserve,
primary rainforest with Shorea spp., 13 Dec. 2007, A. Verbeken GENT AV07-
164, holotype.
Notes — Like the previous species, L. saturnisporus, this
species has rather small (< 10 µm) but highly winged spores.
It differs with L. saturnisporus, however, by the lower wings
(2.5–3.5 µm) without striations, and the lack of a slime layer
in the peridiopellis.
4. Lactarius echinellus Verbeken & Stubbe, sp. nov. — Myco-
Bank MB804184; Fig. 6
Holotype. Sri Lanka, near Sinharaja Forest, 13 Dec. 2007, Verbeken
07-157 (GENT).
Etymology. Latin for small sea urchin or small hedgehog, referring to the
spores that are small and echinate.
Basidiocarp globose to irregularly subglobose and even knotty,
somewhat flattened, 15–35 mm diam, often with short rhizo-
morphs. Peridium smooth but mostly with several pleats,
sometimes with venose wrinkles, forming a thin layer (< 1 mm
thick) around the gleba, somewhat translucent revealing lo-
culoid structure underneath; surface glabrous with chamois-
leather-like patches, dry, often pruinose and whitish in pleats
and dents, predominantly buff to pale ochraceous, reddish
blond to brownish orange (5C4–5), slightly more ochraceous
(FK13–14), pale yellow to pale orange locally (4A3–5A3) with
some small whitish cracks. Stipe absent. Columella absent.
Gleba with very small, round or labyrinthuloid, irregular loculi
(± 3 per mm), firm, hardly compressible, pale yellow to greyish
yellow (4A3–4B4), dark cream coloured, mostly with a pinkish
tinge after exposure, pinkish buff to pale orange (5A3) in older
specimens. Latex white, abundant, thick and sticky, unchanging
Fig. 5 Lactarius shoreae (holotypus). a. Basidiospores; b. basidia; c. cys-
tidia; d. peridiopellis. — Scale bar = 10 μm.
c
b
d
a
21
A. Verbeken et al.: Angiocarpous Russulaceae in South East Asia
or staining the gleba slightly pinkish, drying soon and leaving
a whitish layer on the gleba, not hyaline at all, unchanging in a
10 % aqueous potassium hydroxide solution. Smell distinct but
variable sweetishly rancid or reminiscent of L. azonites, Gera-
nium robertianum, motor oil, boiled rice. Taste mild, immediately
very dry, then mild. Both gleba and peridium unchanging with
ferrous sulphate.
Spores globose to subglobose, 6.8–7.4–7.8–8.6(8.8) ×
(5.8)6.0–6.4–6.6–7.1 µm, n = 40, Q = 1.10–1.16–1.19–1.29;
apiculus 2–4 µm long; ornamentation echinate, composed of
long, isolated spines up to 2.5 µm, rather slender and straight,
sometimes slightly curved, rounded on top, not acute. Basidia
4-spored, some subclavate, some very long and narrowly cy-
lindrical, but mostly irregularly shaped, 25–40(55) × 8–12 µm,
sterigmata up to 5 µm long. Cystidia absent. Pseudocystidia
present, irregular, tortuous to moniliform, 2–4 µm diam. Lacti-
fers very abundant in the gleba. Peridiopellis a loose ixotricho-
derm; terminal elements irregularly shaped and branched, with
intricate finger-like bulges, 10– 20 × 2–10 µm, some locally with
thickened wall.
Specimens examined. Sri Lanka, Kudawa, near Sinharaja Forest Reserve,
patch dominated by Dipterocarpaceae (Shorea congestiflora, S. trapezifolia,
Dipterocarpus hispidus, D. zeylanicus), near S. congestiflora half burried in
the soil, 11 Dec. 2007, D. Stubbe GENT DS07-472, DS07-73, A. Verbeken
GENT AV07-133; Kudawa, near Sinharaja Forest Reserve, alongside Pitakele
river with mostly S. trapezifolia and some D. hispidus stands, half burried
in the soil near S. trapezifolia, 13 Dec. 2007, D. Stubbe GENT DS07-489,
DS07-492, A. Verbeken GENT AV07-157, holotypus; ibid., 17 Dec. 2007, D.
Stubbe GENT DS07-507; Kudawa, near Sinharaja Forest Reserve, primary
rainforest, near Shorea spp., 14 Dec. 2007, D. Stubbe GENT DS07-498,
DS07-499, DS07-500, DS07-169; ibid., 15 Dec. 2007, A. Verbeken GENT
AV07-175; ibid., 16 Dec. 2007, D. Stubbe GENT DS07-505.
Notes — Arcangeliella lactifera (basionym: Gymnomyces
lactifer B.C. Zhang & Y.N. Yu) is a similar species described
from China. It is obviously similar to L. echinus and L. echinel-
lus owing to the spores ornamented with isolated spines. It
shares the 2-spored basidia with L. echinus but the spores are
distinctly smaller: 8–10 µm. Macroscopically the species is also
characterized by globose, subglobose to flattened or irregular
basidiomata without stipe or columella, a pale peridium and
white milky latex. The peridiopellis, however, is described to
be a layer of repent hyphae.
White milky latex is also present in the Chinese angiocar-
pous species Zelleromyces ramispinus (basionym: Martellia
ramispina B.C. Zhang & Y.N. Yu), which differs by the striking
spore ornamentation where 2–2.5 µm high spines have double
or triple forked tips and the peridiopellis which is also a cutis.
Another gasteroid Russulales representative with spores bear-
ing isolated spines is Arcangeliella densa (basionym: Elasmo-
myces densus R. Heim), described from Thailand. The species
has a better developed stipe than the ones proposed here, but
we doubt whether this is a constant feature as intermediates
between sequestrate species with a well-developed stipe and
true angiocarpous species without stipe are possible. A more
important difference is the peridiopellis which is an ixocutis
resulting in a viscid peridium which is ochraceous and zonate.
Heim (1959) suggests a connection with Lactarius species
in L. section Zonarii. Judging from his drawings, the spines
ornamenting the spores are also rather short compared to our
Sri Lanka species.
5. Lactarius echinus Stubbe & Verbeken, sp. nov. — Myco-
Bank MB804183; Fig. 7
Holotype. Sri Lanka, near Sinharaja Forest, primary rainforest with Shorea
spp., 14 Dec. 2007, Verbeken 07-168 (GENT).
Etymology. Latin for sea urchin or hedgehog, referring to the spores that
are large, round and distinctly echinate
Basidiocarp globose to subglobose, 10–15 mm diam. Peridium
light orange to greyish orange (5AB4), smooth, slightly felty,
in some places wrinkled, rugulose or strongly rugulose and
deeply grooved, slightly pinkening after cutting. Stipe absent.
Columella absent. Gleba greyish orange to brownish orange
(5BC5), with very labyrinthuloid loculi. Latex white, very scarse
to rather abundant. Taste mild. Smell distinctly of Geranium
robertianum, but in other specimens not remarkable.
Spores globose to subglobose, 9.6–11.8–12.0–14.0(14.3)
× 9.4–11.2–11.4–13.4 µm, n = 40, Q = 1.01–1.04–1.07–1.15;
apiculus up to 5 µm long; ornamentation amyloid, echinate,
composed of long, isolated spines; spines up to 4 µm long,
rather slender and straight, sometimes slightly curved, rounded
on top, not acute. Basidia 2-spored, some subclavate, but
mostly irregularly shaped, 20–35 × 8–14 µm, sterigmata up to
5 µm long. Cystidia absent. Pseudocystidia present, tortuous to
moniliform, sometimes branched, 2–4 µm diam. Lactifers very
abundant in the gleba. Peridiopellis a palisade to trichopalisade,
embedded in a thin and strongly incrusted slime-layer; terminal
elements usually on a chain of subglobose, small elements or
short hyphal parts; terminal elements clavate to irregularly sub-
globose, 10–17 × 4–14 µm, sometimes with slightly thickened
wall.
Specimens examined. Sri Lanka, Kudawa, near Sinharaja Forest, primary
rainforest with Shorea spp., 14 Dec. 2007, A. Verbeken GENT AV07-168,
holotype; ibid., 16 Dec. 2007, A. Verbeken GENT AV07-178.
Notes — Within the angiocarpous species with echinulate
spore ornamentation, L. echinus is easily charachterized by its
very large spores (average 11.8 × 11.2 µm). Such spores are
exceptionally large for the genus, but were also observed in
L. pomiolens, though clearly different because highly winged.
In Lactarius, the species with the largest spores are species
Fig. 6 Lactarius echinellus (holotypus). a. Basidiospores; b. basidia; c. pseu-
docystidia; d. peridiopellis. — Scale bar = 10 μm.
c
b
d
a
22 Persoonia – Volume 32, 2014
with 2-spored basidia (such as L. acerrimus). In contrast to
L. pomiolens, which has strictly 4-spored basidia, this is the
case here as well, but surprisingly the basidia themselves are
very small. Besides giant spores in L. echinus, some smaller
spores are also observed, probably produced by 4-spored
instead of 2-spored basidia. Four-spored basidia could not be
observed, however a single 1-spored basidium was recorded.
6. Lactarius falcatus Verbeken & Van de Putte, sp. nov. — Myco-
Bank MB804185; Fig. 8
Holotype. ThaiLand, Chiang Mai Prov., Mae Tang District, Ban Mae sae
village, 18 June 2008, Van de Putte 08-038 (holo GENT; iso MFU).
Etymology. Latin for sickle-shaped, curved (like the wings of a falcon),
referring to the shape of the spines on the spores.
Basidiocarp globose to subglobose, 17–22 mm diam. Peridium
brown (6E6, but paler) in upper part, part burried in soil paler
brown to buff (4A3, with brown tinge), smooth. Stipe absent.
Columella absent. Gleba cream-coloured (3A3), discolouring
pale greyish brown with light orange pinkish tinge (5A4). Latex
moderately abundant, immediately bright pale yellow (1A4).
Taste unknown. Smell unremarkable.
Spores globose to subglobose, (8.8)9.1–10.1–11.1 × 8.5–
9.3–10.1 µm, n = 40, Q = 1.01–1.07–1.13; apiculus 3–4 µm
long; ornamentation echinate, composed of long, isolated
spines; spines up to 1.5(2) µm long, rather blunt and somewhat
irregular, often curved, rounded on top, seldom acute. Basidia
2-spored, some subclavate, some with remarkable narrower
part in the middle, mostly irregularly shaped, 35–45 × (3)7–10
µm, sterigmata up to 5 µm long. Cystidia absent. Pseudocystidia
present, cylindrical, 2–4 µm diam. Peridiopellis a loose layer
of intricate hyphae, arranged periclinally as well as anticlinally,
no slime-layer present; terminal elements rather regular and
cyindrical.
Specimens examined. ThaiLand, Chiang Mai Prov., Mae Tang District,
Ban Mae sae village, 18 June 2008, K. Van de Putte GENT KVP08-038,
holotypus, MFU08-1214, isotype.
Notes — Within the angiocarpous species with echinulate
spores, L. falcatus is easily recognized by the remarkably
curved spines up to 2 µm long. Macroscopically it is character-
ized by the latex which is white in the beginning but soon turns
bright pale yellow. Martellia nanjingensis differs by the lower
spore ornamentation consisting of conical and blunt spines
which are never curved as in L. falcatus. We assume it also
differs in unchanging latex since a colour change to bright yel-
low is not mentioned in the description (Tao et al. 1993).
DISCUSSION
A striking diversity of sequestrate Russulales was encountered
during these expeditions in tropical South East Asian forests.
Six new species are described here and are phylogenetically
placed in the genus Lactarius. We also found one angiocarpous
Russula species which will be described in a separate paper
(Hampe et al. In prep.). Worldwide, all known species of se-
questrate milkcaps so far belong to the genus Lactarius; none
are described in the genus Lactifluus (Verbeken & Nuytinck In
press). The angiocarpous habit evolved several times in the ge-
nus and has been demonstrated in L. subg. Russularia, L. subg.
Piperites and L. subg. Plinthogalus. The species described here
largely confirm this: L. echinellus, L. echinus, L. saturnisporus
and L. shoreae are included in L. subg. Plinthogalus, L. falcatus
is a representative of L. subg. Russularia. Lactarius pomiolens
has a rather isolated position and cannot be confined with cer-
Fig. 7 Lactarius echinus (holotypus). a. Basidiospores; b. basidia; c. cys-
tidium; d. pseudocystidia; e. peridiopellis. — Scale bar = 10 μm.
c
b
d
a
e
Fig. 8 Lactarius falcatus (holotypus). a. Basidiospores; b. cystidia; c. basidia;
d. pseudocystidia; e. peridiopellis. — Scale bar = 10 μm.
c
b
d
a
e
23
A. Verbeken et al.: Angiocarpous Russulaceae in South East Asia
tainy to one of the existing subgenera. There seems to be a
close relation with the African L. kabansus and L. tenellus, for
which recent phylogenetic studies show that a new infrageneric
group has to be created (Stubbe 2012).
Studying Arcangeliella, Thiers (1984b) stated that there are two
major evolutionary lines: A. borziana and A. densa (Thailand)
have basidiospores ornamented with spines and rods, while
the other line, represented by American and perhaps Australian
species, has spores with either a broken or a complete reticu-
lum. Both types of spore ornamentation are also encountered
here, but it turns out that they do not represent true evolutionary
lines and the spore ornamentation is not a phylogenetic informa-
tive feature at all. It is even striking that in L. subg. Plinthogalus,
a subgenus characterized so far by reticulate to highly winged
spores, two species with isolated spines are occurring.
Wilson et al. (2011) show that the gasteromycete lineages within
the Agaricomycetes might now be diversifying at rates compara-
ble to, or exceeding, those of their nongasteroid relatives. Their
analyses suggest that the net diversification rate of gasteroid
forms exceeds that of nongasteroid forms, and that gasteroid
forms will eventually come to predominate over nongasteroid
forms in the clades in which they have arisen. The low num-
ber of gasteroid forms in the Agaricomycetes as a whole may
reflect the relatively recent origin of many gasteroid lineages.
The even more recent origin of gasteromycetization in the order
Russulales is suggested by several observations. Firstly, the
anatomy of the basidiomes is relatively simple and in no way
comparable to the complex and specialized tissues found in
highly evolved gasteroid groups such as Sclerodermatineae
or Phallomycetidae. Secondly, none of the gasteroid lineages
in Lactarius has evolved into a clade containing a diversity
of species. On the contrary, the gasteroid species appear as
independent and isolated incidents within the phylogeny.
Hibbett et al. (1994) suggested that the genetic mechanisms
resulting in the initial stages of gasteromycetization could be
rather simple. It is generally assumed that dry climatic condi-
tions are one of the driving forces that enhances the develop-
ment of sequestrate fruiting bodies. However, gasteroid species
occur also in Europe and North America in temperate climates,
and the current findings demonstrate a strong presence in tropi-
cal rainforests as well. In Sri Lanka for instance, the number of
known sequestrate Lactarius species now exceeds the number
of known agaricoid species (Pegler 1986, Stubbe 2012). The
exploration rate is rather low and the period of sampling may
play an important role in the number of sequestrate fungi we
encountered, but we assume that angiocarpous mushrooms are
overlooked in these regions and that the phenology and ecol-
ogy of these tropical rainforest angiocarpous species deserve
further investigation. It seems likely to us that more sequestrate
species are to be discovered in rainforest biotopes. Another
impression we have from several expeditions is that produc-
tion of angiocarpous basidiomes does not necessarily coincide
with the seasonality of the majority of macromycetes. During
our expedition in Sri Lanka, agaricoid mushrooms were not
abundant, even scarce. The intensified search efforts lead us
to find a greater number of small and inconspicuously growing
species, among which many false truffles, such as the species
presented in this paper. Perhaps the tropical angiocarpous
species flourish during periods when unfavourable weather
conditions cause too much stress for most agaricoid species.
Another hypothesis is that angiocarpous fructifications are less
susceptible to – or less dependent on – seasonal changes and
are abundant year-round, only to be overlooked during the
fructification season of the other mushrooms. The phenology
and ecology of these tropical rainforest angiocarpous species
deserve further investigation.
Acknowledgements The authors thank Andrus Voitk, Patrick Leonard,
Andy Taylor, André De Kesel and Amadou Bâ for giving access to their
collections and two anonymous reviewers for their valuable comments. DS
was funded by a scholarship of the Institute for the Promotion of Innovation
through Science and Technology (IWT-Vlaanderen). KVdP was supported by
the ‘Bijzonder Onderzoeksfonds Ghent University (BOF)’. We thank the King
Leopold III Foundation for financial support for the expedition in Sri Lanka. We
wish to acknowledge Prof. Kevin Hyde and Mae Fah Luang University, and
Dr. Saisamorn Lumyong and Chiang Mai University for providing a Material
Transfer Agreement for Thai fungal specimens.
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