Tales of the unexpected: angiocarpous representatives of the Russulaceae in tropical South East Asia.

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.

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 russuloid 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 Russulales 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-orae (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 Elasmomyces 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, 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 reagent, 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 calculated 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, respectively 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 compound microscope. For the details of description and terminology of micromorphological features see Verbeken (1998) and Verbeken & Walleyn (2010).

DNA extraction, PCR amplification and sequencing

Nine gasteroid Lactarius collections were used for the molecular 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 SequencherTM v4.9 (GeneCodes Corporation, Ann Arbor, Michigan, USA). Other sequences were gained following the DNA extraction, PCR and sequencing protocols described by Eberhardt (2002) or Taylor et al. (2006).

Alignment and phylogenetic analyses

Table 1 shows an overview of all specimens and sequences used in the phylogenetic analyses, including GenBank accession numbers. Two alignments were constructed. The first alignment 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 agaricoid 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 placement of the newly described species within Lactarius and its 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 alignments 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 positions (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 positions 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.

Table 1

Specimens and GenBank accession numbers of DNA sequences used in the molecular analyses.

Species	Voucher collection	Origin	ITS accession no.	LSU accession no.	rpb2 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
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

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-reversible model with rate variation across sites and a proportion of invariable sites (GTR+I+G) was used. Rates and model parameters 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 (Ronquist 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 posterior probabilities from the 4 runs on the HPC were compared. An appropriate burn-in value was determined visually using Tracer.

RESULTS

Phylogeny

Fig. 1 shows the obtained ML topology based on the alignment 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 sequestrate milk cap species (indicated in green).

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 fruiting 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.

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.

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.

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. echinellus, 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).

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 interwoven 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 inter-woven palisade 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

KEY TO THE SPECIES

1. Verbeken & Stubbe, sp. nov. — MycoBank MB804182; Fig. 3

Fig. 3

Lactarius pomiolens (holotypus). a. Basidia; b. pseudocystidia; c. basidiospores; d. cystidia; e. peridiopellis. — Scale bar = 10 μm.

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 leatherbrown (FK13–14), buff to ochraceous, irregularly coloured, with patches. Stipe absent. Columella absent. Gleba strongly labyrinthuloid, with small loculi, with some, but very few gelatinous 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 immediately 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, seldomly branched, rather broad and not completely amyloid but with strongly amyloid tranversal bands; surface with amyloid spots between the ridges; plage not distinct, not amyloid. Basidia 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 smell of apples seems a striking character, but more records are needed to evaluate the stability of this feature.

2. Verbeken & Stubbe, sp. nov. — MycoBank MB804180; Fig. 4

Fig. 4

Lactarius saturnisporus (holotypus). a. Basidiospores; b. basidia; c. cystidia; d. pseudocystidia; e. peridiopellis. — Scale bar = 10 μm.

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, indistinct 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 % aqueous 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 striped and bifurcating pattern, mostly unbranching, sometimes branched but never forming a reticulum, edges sharp and mostly 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 Reserve, 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 extremely 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. Stubbe & Verbeken, sp. nov. — MycoBank MB804181; Fig. 5

Fig. 5

Lactarius shoreae (holotypus). a. Basidiospores; b. basidia; c. cystidia; d. peridiopellis. — Scale bar = 10 μm.

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 irregularly 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, subcylindrical 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 intricate, 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. Verbeken & Stubbe, sp. nov. — MycoBank MB804184; Fig. 6

Fig. 6

Lactarius echinellus (holotypus). a. Basidiospores; b. basidia; c. pseudocystidia; d. peridiopellis. — Scale bar = 10 μm.

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 rhizomorphs. Peridium smooth but mostly with several pleats, sometimes with venose wrinkles, forming a thin layer (< 1 mm thick) around the gleba, somewhat translucent revealing loculoid 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 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, Geranium 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 cylindrical, 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. Lactifers very abundant in the gleba. Peridiopellis a loose ixotrichoderm; 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. echinellus 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 angiocarpous 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 bearing isolated spines is Arcangeliella densa (basionym: Elasmomyces 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. Stubbe & Verbeken, sp. nov. — MycoBank MB804183; Fig. 7

Fig. 7

Lactarius echinus (holotypus). a. Basidiospores; b. basidia; c. cystidium; d. pseudocystidia; e. peridiopellis. — Scale bar = 10 μm.

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 subglobose, 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 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. Verbeken & Van de Putte, sp. nov. — MycoBank MB804185; Fig. 8

Fig. 8

Lactarius falcatus (holotypus). a. Basidiospores; b. cystidia; c. basidia; d. pseudocystidia; e. peridiopellis. — Scale bar = 10 μm.

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 characterized 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 yellow 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 sequestrate 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 genus 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 certainy 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 reticulum. 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 informative 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 comparable 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 number 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 conditions are one of the driving forces that enhances the development 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 tropical 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 ecology 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 production 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.