Three independent evolutionary events of sequestrate Lactifluus species in Australasia.

Three Australian species with sequestrate basidiome forms are recorded for the first time in the genus Lactifluus based on nuclear ITS-LSU and morphological data. These species represent three rare independent evolutionary events resulting in sequestrate basidiomes arising from agaricoid species in three different sections in two subgenera. All three species have highly reduced basidiome forms, and no species with intermediate forms have been found. Lactifluus dendriticus is unique in the genus in having highly branched, dendritic terminal elements in the pileipellis. We provide full descriptions of two species: Zelleromyces dendriticus (= Lactifluus dendriticus comb. nov.) in Lactifluus subg. Lactifluus sect. Gerardii, and Lactifluus geoprofluens sp. nov. in Lf. subg. Lactifluus sect. Lactifluus. A reduced description is provided for the third, Lactifluus sp. prov. KV181 in Lf. subg. Pseudogymnocarpi sect. Pseudogymnocarpi, as it is currently known from a single sequence.

INTRODUCTION

Distinguishing species of Lactarius and Lactifluus is difficult enough, but the loss or reduction of macroscopic features in sequestrate species makes them equally problematic to distinguish between genera. In Lactarius, sequestrate basidiome forms are known from three main subgenera: subg. Plinthogalus, subg. Russularia, and subg. Lactarius (Dring & Pegler 1978, Beaton , Desjardin 2003, Nuytinck , Eberhardt & Verbeken 2004, Verbeken & Walleyn 2010, Wang , Verbeken , b, Sang , Beenken , De Crop , Vidal ). All of the Australasian lactarioid sequestrate forms were historically described in the genera Zelleromyces, Arcangeliella or Gastrolactarius, based on morphology, following Northern Hemisphere circumscriptions of genera. Apart from a regional treatment of species of Zelleromyces by Beaton for Victoria, in which the descriptions and illustrations to six species were provided, Australian species have been little studied. Bougher & Syme (1998) provided a further description of Z. daucinus from Western Australia, Grgurinovic (1997) short descriptions and a key to three South Australian species, and Trappe & Claridge (2003, 2008) descriptions of two more species. However, the diversification of sequestrate basidiome forms in Lactarius in Australasia is known to be high, with eight species currently described and a further 20–25 species remaining to be described (Beaton , Bougher & Lebel 2001, Lebel 2002b, Trappe & Claridge 2003, 2008, Lebel et al. unpub. data). However, no phylogenetic analyses of subgenus or sectional placement has as yet been completed for these taxa or the related epigeal taxa, and species circumscription remains based on morphology.

While conducting research on Australasian sequestrate lactarioid taxa and the Lactifluus clarkeae species complex, DNA sequencing revealed species with affinities to Lactifluus rather than Lactarius. This unexpected finding led us to study their morphological characters in detail, test species delimitation and further examine the phylogenetic relationships between agaricoid and sequestrate Lactifluus species, to determine whether sequestrate forms in Lactifluus are the result of independent evolutionary events, similar to the pattern seen in Lactarius (Verbeken , Sheedy ). Here we provide descriptions where possible and confirm placement of the taxa in different subgenera within Lactifluus.

MATERIALS AND METHODS

Morphology

Several hundred collections from Australasian herbaria and fungaria (MEL, PERTH, BRI, AD, HO, CANB, PDD) and material held at Oregon State Herbarium (OSC) were examined from diverse geographic regions and habitat types across Australia. Detailed coordinate and elevation data for collections mentioned here are available online through the Atlas of Living Australia (https://www.ala.org.au/). Collections matching morphological characters of the known Lactifluus related taxa, or with typical Lactifluus pileipellis structure and cystidia form (approximately 35 collections), were examined in greater detail and sampled for DNA sequencing. Macroscopic characters are described and measured from fresh material, field notes of other collectors, or dried herbarium collections. Measurements taken using dried fungarium material are listed as such and are estimated to be approximately 30 % smaller than measurements taken from fresh specimens. This estimate is based on fresh and dry weights of 30 recent collections of lactarioid sequestrate basidiomes. Colours are described in general terms from field observations in daylight conditions. Habitat, associated plant communities, sporulating season, presence and nature of latex, fresh odour, and taste are based on field notes.

Microscopic characters are described from examination of dried fungarium material. Hand-cut sections were rehydrated in 5 % KOH solution then mounted in congo red to observe the hymenium, trama, and pileipellis. Spore size, shape, ornamentation and amyloidity were observed in lamellae tissue mounted in Melzer’s reagent. Measurements of microscopic characters were taken on an Olympus BX-52 microscope at ×400 or ×1 000 using an Olympus DP-73 camera attachment and measurement tools in cellSens standard (v. 1.16). Microscopic measurements are given as a raw range of length × width with mean ± standard deviation (SD) of n measurements in parentheses. The length/width quotient (Q) of individual spores is presented as the raw range of Q values with mean ± standard deviation (SD) of n measurements in parentheses. Basidia, basidioles, and cystidia measurements are given as length (not including sterigmata) × width at widest point, and width at base or apex. Pseudocystidia, laticiferous hyphae, and hyaline hyphae measurements are given as a raw range of diameters. In highly reduced sequestrate basidiomes there is some difficulty in determining position of cystidial elements, thus in descriptions provided here they are referred to as hymenial cystidia.

Scanning electron microscopy (SEM) of gold-sputtered basidiospores mounted on carbon tape was performed using a Thermo Fisher Scientific XL30 FEG microscope (Waltham, USA) at the University of Melbourne Biosciences Microscopy Unit.

All photographs are based on the type collections unless otherwise stated. Names of fungaria are abbreviated according to Thiers (2011) (http://sweetgum.nybg.org/ih/ — continuously updated). We use the abbreviation ‘Lf.’ to distinguish Lactifluus from Lactarius (‘L.’) when discussing species in these genera.

Molecular studies

Protocols for DNA extraction (Qiagen Plant DNeasy kit, Germantown, USA or the EZNA forensic kit, Omega Bio-tek, Norcross USA, for samples older than 1995), PCR, and sequencing followed those in Lebel & Syme (2012) and Lebel and the references therein. Assembly, manual editing, and preliminary alignment of sequences were performed within Geneious v. 9.1.7 (Biomatters Ltd). Individual alignments for the ITS and LSU were then manually trimmed in BioEdit v. 7.1.3 (Hall 2011) and some final manual editing done in Geneious v. 9.1.7. The concatenated alignment and phylogenetic trees are available from the Landcare Research datastore https://doi.org/10.7931/n4fc-4z93.

Sequences of the ITS and LSU from similar species (based on blast searches), and representative taxa to cover subgenera and sections within Lactifluus were retrieved from GenBank and UNITE (Kõljalg ), to generate a concatenated alignment. This was done with the on-line version of MAFFT v. 7 (Katoh ). Three species of Multifurca were utilised as outgroup. Novel sequences representing collections from Australasia and other regions generated for this study are listed in Table 1 with relevant GenBank accession numbers, and all sequences utilised in analyses. Sequences labelled as provisional taxa “Lf. sp. 1–13” are numbered according to a broader analysis of Australasian species of Lactifluus which will be published elsewhere; we have retained the numbering convention for consistency.

Table 1.

Specimens used in the phylogenetic analysis, including infrageneric taxon, species (as originally identified in the field or as labelled in GenBank), herbarium numbers and typification, country of origin, and ITS/LSU GenBank accession numbers. New sequences generated for this study are indicated in bold. Abbreviations used: AU - Australia, NZ - New Zealand, NCal - New Caledonia, WA - Western Australia, VIC - Victoria, TAS - Tasmania, SA - South Australia, NT - Northern Territory, NSW - New South Wales, QLD - Queensland, FrIsland - Fraser Island.

(Infrageneric) taxon	Species	Herbarium or collection number	Country	GenBank accession numbers
				ITS	LSU
Multifurca	Multifurca ochricompacta	BB02107	n/a	DQ421984	DQ421984
	Multifurca sp.	MEL238568	AU	MW134734	MW128106
	Multifurca stenophylla	CWD584	AU	JX266628	JX266633
	Multifurca zonaria	FH12-009	Thailand	KR364083	KR364212
subg. Gymnocarpi sect. Gymnocarpi	Lactifluus foetens	ADK3688 BR	Benin	KR364022	KR364149
	Lactifluus gymnocarpus	EDC12-047 GENT	Cameroon	KR364065	KR364194
	Lactifluus tanzanicus	TS1277	Tanzania	KR364037	n/a
subg. Gymnocarpi sect. Luteoli	Lactifluus brunneoviolascens	AV13-038 GENT	Italy	KR364123	KR364246
	Lactifluus caliendrifer	KW378 GENT Holotype	Thailand	MK517655	n/a
	Lactifluus luteolus	AV05-253 GENT	USA	KR364016	KR364142
	Lactifluus russulisporus	REH9398 NY Holotype	AU	KR364097	KR364229
subg. Gymnocarpi sect. Nebulosi	Lactifluus chiapanensis	V.M.Bandala 4374A GENT	Mexico	GU258297	GU265580
	Lactifluus guadeloupensis	RC_Guad11-023 LIP Holotype	Guadeloupe	KP691412	KP691421
subg. Gymnocarpi sect. Panuoidei	Lactifluus panuoides	G128	Guyana	KJ786647	KJ786551
subg. Gymnocarpi sect. Phlebonemi	Lactifluus aff. phlebonemus	EDC12-023 GENT	Cameroon	KR364062	KR364191
subg. Gymnocarpi sect. Tomentosi	Lactifluus albens	MEL2231695 Type	AU_WA	MW134740	MW128112
	Lactifluus aurantioruber	MEL2257827	AU_TAS	MW134745	MW128115
	Lactifluus clarkeae	MEL2101947 Epitype	AU_SA	MW134754	MW128120
	Lactifluus clarkeae	PDD102596	NZ	MW134770	MW128130
	Lactifluus flocktonae	MEL2238290 Epitype	AU_VIC	JX266621	JX266637
	Lactifluus psammophilus	MEL2238407 Type	AU_VIC	MW134791	MW128144
	Lactifluus pseudoflocktonae	MEL2238269 Holotype	AU_VIC	MW134801	MW128151
	Lactifluus sp. 1	environmental sample CMMy30M1	New Caledonia	KY774240	n/a
	Lactifluus sp. 5	PGK13-130 Nothofagus	New Caledonia	KP691436	KR605507
	Lactifluus sp. 6	environmental sample KT-26 Tristaniopsis	New Caledonia	LC271308	n/a
subg. Lactariopsis sect. Albati	Lactifluus vellereus	UE20.09.2004-22 UPS	n/a	DQ422034	DQ422034
subg. Lactariopsis sect. Edules	Lactifluus edulis	FN05-628 GENT	Malawi	KR364020	KR364147
subg. Lactariopsis sect. Lactariopsis	Lactifluus annulatoangustifolius	BB00-1518 PC	Madagascar	AY606981	KR364253
	Lactifluus velutissimus	JD886	Congo	KR364075	KR364204
subg. Lactariopsis sect. Neotropicus	Lactifluus venezuelanus	RC_Gaud11-017 LIP	Guadeloupe	KP691411	KP691420
subg. Lactifluus sect. Allardii	Lactifluus allardii	AV05-286 GENT	USA	KF220015	KF220124
subg. Lactifluus sect. Ambicystidiati	Lactifluus ambicystidiatus	KUN_F88179	China	KR908670	KR908672
subg. Lactifluus sect. Gerardii	Lactifluus auriculiformis	AV12-050 GENT Holotype	Thailand	KR364086	KR364216
	Lactifluus bhandaryi	TENN 051830 Holotype	Nepal	KR364111	n/a
	Lactifluus conchatulus	LTH457 GENT Isotype	Thailand	GU258296	GU265659
	Lactifluus coniculus	DS07-496 GENT Holotype	Sri Lanka	GU258236	GU265594
	Lactifluus coniculus	DS07-497 GENT	Sri Lanka	GU258237	GU265595
subg. Lactifluus sect. Gerardii	Lactifluus dendriticus comb. nov .	MEL2063377	AU_VIC	MW471118	MW471115
	Lactifluus dendriticus comb. nov .	MEL2292167_CANB748620	AU_VIC	MW471119	MW471116
	Lactifluus dendriticus comb. nov .	MEL2326248	AU_VIC	MW471120	n/a
	Lactifluus dendriticus comb. nov .	MEL2126553	AU_VIC	MW471121	n/a
	Lactifluus dendriticus comb. nov .	MEL2061947	AU_NSW	MW471122	n/a
	Lactifluus fuscomarginatus	GO2010-144	Mexico	KC152157	n/a
	Lactifluus genevievae	G.Gates_D.Ratkowsky 17-2-2005	AU	GU258294	GU265657
	Lactifluus gerardiellus	KW386 GENT Holotype	Thailand	KX889845	KX889844
	Lactifluus gerardii	AV05-375 GENT	USA	GU258254	GU265616
	Lactifluus gerardii	Desjardin3630	USA	GU258220	n/a
	Lactifluus igniculus	LE262983 Type	Vietnam	JX442759	n/a
	Lactifluus indicus	CAL 1282 Holotype	India	KU145119	KU145121
	Lactifluus leae	AV-RW04-90 GENT	Thailand	GU258244
	Lactifluus leae	FH12-13 GENT	Thailand	KF432957
	Lactifluus leonardii	P.Leonard 35607	AU	GU258295	GU265658
	Lactifluus leonardii	G.Gates 29-1-2002	AU	GU258304	GU265664
	Lactifluus limbatus	DS06-230 GENT	Malaysia	GU258222	GU265578
	Lactifluus limbatus	DS06-247 GENT	Malaysia	GU258223	GU265579
	Lactifluus midnapurensis	CAL 1516 Holotype	India	KY785175	KY785177
	Lactifluus ochrogalactus	E.Nagasawa 80-102 TMI Type	Japan	GU258280
	Lactifluus parvigerardii	KUN_F61367 Holotype	China	JF975641	JF975642
	Lactifluus petersenii	AV05-267 GENT	USA	GU258282	GU265643
	Lactifluus pulchrellus	KW304_FH12-037 GENT Holotype	Thailand	KR364092	KR364223
	Lactifluus raspei	EDC14-517 Holotype	Thailand	KX889849	n/a
	Lactifluus reticulatovenosus	Horak 6472 GENT Holotype	Indonesia	GU258286	GU265649
	Lactifluus robustus	K16053113	China	KY353803	KY353806
	Lactifluus robustus	K15052822	China	KY353802	KY353805
	Lactifluus sepiaceus	MEL2300727	AU	GU258293	GU265656
	Lactifluus sepiaceus	MEL1054958	AU_VIC	MW134808	n/a
	Lactifluus sepiaceus	P.Leonard 40509	NZ	GU258287	GU265650
	Lactifluus sinensis	K15060710 Holotype	China	KT900208	n/a
	Lactifluus sinensis	K15070203	China	KT900209	n/a
	Lactifluus sinensis	environmental sample HIB12	China	JX457047	n/a
	Lactifluus sp.	DS06-003 GENT	Malaysia	GU258231	GU265588
	Lactifluus sp.	AV05-283 GENT	USA	GU258259	n/a
	Lactifluus sp.	DPLewis6983	USA	GU258272	n/a
	Lactifluus sp.	Desjardin3564	n/a	GU258273	n/a
	Lactifluus sp. 10	P.Leonard 10409	AU	JF731001	JF731003
	Lactifluus sp. 10	MEL2305122	AU_QLD	MW134809	n/a
	Lactifluus sp. 10	MEL2332066	AU_QLD	MW134810	n/a
	Lactifluus sp. 11	PL26078	AU	n/a	MW128157
	Lactifluus sp. 12	R.E.Halling 6800	AU	JF731000	JF731002
	Lactifluus sp. 13	environmental sample RFLP61	AU	DQ388868	n/a
	Lactifluus sp. 13	environmental sample Toosoil16	AU	KC222796	n/a
	Lactifluus sp. 13	environmental sample Toosoil56	AU	KC222836	n/a
	Lactifluus subgerardii	AV05-389 GENT	USA	GU258271	n/a
	Lactifluus wirrabara	G.Gates_D.Ratkowsky 12-07-2003	AU	GU258306	GU265666
	Lactifluus wirrabara	G.Gates_D.Ratkowsky 17-01-2002	AU	GU258305	GU265665
	Lactifluus wirrabara	G.Gates_D.Ratkowsky 24-01-2004	AU	GU258307	n/a
	Lactifluus wirrabara	JET943 MEL	AU	GU258291	n/a
subg. Lactifluus sect. Lactifluus	Lactifluus acicularis	DS07-456 GENT	Thailand	HQ318224	HQ318125
	Lactifluus acicularis	KVP08-033 GENT	Thailand	HQ318242	HQ318150
	Lactifluus corrugis	JN2004-015 GENT	USA	JQ753820	JQ348262
	Lactifluus corrugis	AV05-291 GENT	USA	JQ753823	JQ348266
	Lactifluus crocatus	LTH268 GENT	Thailand	HQ318266	HQ318181
	Lactifluus crocatus	LTH202 GENT	Thailand	HQ318248	HQ318157
	Lactifluus dissitus	AV-KD-KVP09-082 GENT	India	n/a	JN389035
	Lactifluus distantifolius	DS07-461 GENT Isotype	Thailand	HQ318223	HQ318124
	Lactifluus distantifolius	LTH288 GENT	Thailand	HQ318274	HQ318193
	Lactifluus geoprofluens sp. nov.	MEL2145804 Holotype	AU_VIC	MW471123	MW471117
	Lactifluus jetiae	MEL2238281 Holotype	AU_VIC	MW134811	MW128158
	Lactifluus jetiae	MEL2341759	AU_VIC	MW134813	n/a
	Lactifluus leptomerus	AV-KD-KVP09-130 GENT	India	JN388971	JN389022
	Lactifluus leptomerus	AV-KD-KVP09-131 GENT Holotype	India	JN388972	JN389023
	Lactifluus longipilus	LTH273 GENT	Thailand	HQ318276	HQ318195
	Lactifluus longipilus	LTH168 GENT	Thailand	HQ318235	HQ318143
	Lactifluus maenamensis	KD 16-008	India	MF928075	n/a
	Lactifluus mexicanus	Montoya5276 Holotype	Mexico	MK211181	MK211190
	Lactifluus oedematopus	AV07-079 GENT	Belgium	JQ753835	JQ348270
	Lactifluus oedematopus	RW1228 GENT	France	HQ318216	HQ318116
	Lactifluus oedematopus	KVP12-001 GENT Neotype	Germany	KR364100	KR364232
	Lactifluus pagodicystidiatus	MEL2121979	AU_VIC	MW134815	MW128161
	Lactifluus pagodicystidiatus	MEL2150777 Holotype	AU_VIC	MW134816	MW128162
	Lactifluus pallidilamellatus	Leticia Montoya 4716	Mexico	JQ753824	JQ348268
	Lactifluus pinguis	LTH117 GENT Holotype	Thailand	HQ318211	HQ318111
	Lactifluus pinguis	LTH169 GENT	Thailand	HQ318221	HQ318121
	Lactifluus rugulostipitatus	MEL2329677 Holotype	AU_NT	MW134817	MW128163
	Lactifluus rugulostipitatus	MEL2329673	AU_NT	MW134819	n/a
	Lactifluus sp.	KIINA158 GENT	China	HQ318225	HQ318126
	Lactifluus sp.	AV-KD-KVP09-134 GENT	India	JN388978	JN389026
	Lactifluus sp.	AV-KD-KVP09-128	India	n/a	JN389020
	Lactifluus sp.	AV-KD-KVP09-137	India	n/a	JN389027
	Lactifluus sp.	AV-KD-KVP09-129	India	n/a	JN389021
	Lactifluus sp.	OSA-My-3993	Japan	n/a	AB238645
	Lactifluus sp.	OSA-My-3998	Japan	n/a	AB238650
	Lactifluus sp.	OSA-My-4003	Japan	n/a	AB238655
	Lactifluus sp.	OSA-My-3994	Japan	n/a	AB238646
	Lactifluus sp.	OSA-My-4014	Japan	n/a	AB238666
	Lactifluus sp.	OSA-My-4015	Japan	n/a	AB238667
	Lactifluus sp.	OSA-My-3999	Japan	n/a	AB238651
	Lactifluus sp.	LTH313 GENT	Thailand	HQ318272	HQ318190
	Lactifluus sp.	LTH133 GENT	Thailand	HQ318212	HQ318112
	Lactifluus sp.	KVP08-006 GENT	Thailand	HQ318229	HQ318136
	Lactifluus sp.	LTH231 GENT	Thailand	HQ318278	HQ318197
	Lactifluus sp.	LTH123 GENT	Thailand	HQ318222	HQ318122
	Lactifluus sp.	LTH294 GENT	Thailand	HQ318273	HQ318191
	Lactifluus sp.	KVP08-021 GENT	Thailand	HQ318233	HQ318140
	Lactifluus sp.	LTH170 GENT	Thailand	HQ318252	HQ318165
	Lactifluus sp.	LTH264 GENT	Thailand	HQ318264	HQ318179
	Lactifluus sp.	KVP08-008 GENT	Thailand	HQ318231	HQ318138
	Lactifluus sp.	LTH249 GENT	Thailand	n/a	HQ318176
	Lactifluus sp.	LTH284 GENT	Thailand	HQ318253	HQ318166
	Lactifluus sp.	KVP08-026 GENT	Thailand	HQ318238	HQ318146
	Lactifluus sp.	DED7577	USA	n/a	HQ318188
	Lactifluus sp.	AV05-394 GENT	USA	GU258300	GU265660
	Lactifluus sp.	EIU-ASM10990	USA	JQ358921	JN940236
	Lactifluus sp.	AV04-209 GENT	USA	JN388977	JN388998
	Lactifluus sp.	EIU-ASM11130	USA	JQ358938	JN940223
	Lactifluus sp.	SAM310809-02 TENN	USA	MF773609	n/a
	Lactifluus sp.	MycoMap10398	USA	MH975019	n/a
	Lactifluus sp.	AV05-337 GENT	USA	JQ753821	n/a
	Lactifluus sp.	AV04-167 GENT	n/a	JQ753827	JQ348273
	Lactifluus sp. 8	environmental sample RFLP38	AU_QLD	DQ388845	n/a
	Lactifluus sp. 8	environmental sample RFLP39	AU_QLD	DQ388846	n/a
Lactifluus sp. 8	environmental sample RFLP5	AU_QLD	DQ388812	n/a
Lactifluus sp. 9	REH9320 NY	AU	KR364096	KR364228
Lactifluus sp. 9	environmental sample Toosoil58	AU_QLD	KC222838	n/a
Lactifluus subvolemus	KVP08-048 GENT	Slovenia	JQ753927	JQ348379
Lactifluus subvolemus	LAS75_092-A	Sweden	n/a	JQ348348
Lactifluus versiformis	AV-KD-KVP09-047 GENT	India	JN388964	JN389032
Lactifluus versiformis	AV-KD-KVP09-014 GENT Holotype	India	JN388963	JN389029
Lactifluus vitellinus	LTH348 GENT	Thailand	HQ318251	HQ318164
Lactifluus vitellinus	KVP08-024 GENT Holotype	Thailand	HQ318236	HQ318144
Lactifluus volemus	UE09.08.2004-5 UPS	n/a	DQ422008	DQ422008
subg. Lactifluus sect. Piperati	Lactifluus albopicri	MDB_F12_18	AU_NT	MN598888	MN598864
	Lactifluus albopicrus	MEL2297391 Type	AU_VIC	MN598874	MN598855
	Lactifluus austropiperatus	PERTH07550324 Type	AU_QLD	MN614115	MN614111
	Lactifluus austropiperatus	MEL2150778	AU_VIC	MN614116	MN614112
	Lactifluus dwaliensis	LTH67 GENT	Thailand	KF220108	KF220203
	Lactifluus glaucescens	M.Lecomte_2002-20-9-3	France	KF220031	KF220134
	Lactifluus leucophaeus	AV97-382 GENT	Papua New Guinea	GU258299	GU265640
	Lactifluus piperatus	M.Lecomte_2001-8-19-23	France	KF220120	KF220212
subg. Lactifluus sect. Tenuicystidiati	Lactifluus subpruinosus	KUN_F76034	China	KC154110	KC154136
	Lactifluus tropicosinicus	KUN_F59626	China	KC154120	KC154146
subg. Pseudogymnocarpi	Lactifluus armeniacus	EDC14-501 GENT Holotype	Thailand	KR364127	n/a
	Lactifluus sp.	TENN065929	USA	KR364102	KR364233
	Lactifluus sp.	JN2011-012 GENT	Vietnam	KR364045	KR364171
	Lactifluus sp. 7	AQ797939	AU_QLD	n/a	MW128164
	Lactifluus sp. 7	FG2018031	AU_QLD	MW134820	MW128165
	Lactifluus sp. 7	AQ794627	AU_QLD	MW134821	MW128166
	Lactifluus volemoides	TS0705 Holotype	Tanzania	KR364038	KR364165
subg. Pseudogymnocarpi sect. Pseudogymnocarpi	Lactifluus cf. pseudogymnocarpus	AV05-085 GENT	Malawi	KR364012	n/a
	Lactifluus cf. pumilus	EDC12-066 GENT	Cameroon	KR364067	n/a
	Lactifluus flavellus	MD393 Holotype	Togo	LK392594	n/a
	Lactifluus gymnocarpoides	JD885	DR Congo	KR364074	n/a
	Lactifluus holophyllus	ASIS19960	South Korea	MF611684	MF611659
	Lactifluus holophyllus	ASIS22632	South Korea	MF611685	n/a
	Lactifluus holophyllus	SFC20150812-63 Holotype	South Korea	MF611683	n/a
	Lactifluus hygrophoroides	AV05-251 GENT	USA	HQ318285	HQ318208
	Lactifluus hygrophoroides	EIU-ASM10004 clone c4	USA	JQ358911	n/a
	Lactifluus longisporus	AV94-557 GENT	Burundi	KR364118	KR364244
	Lactifluus longisporus	AV11-025 GENT	Tanzania	KR364054	n/a
	Lactifluus luteolamellatus	MHHNU8297	China	MK167429	n/a
	Lactifluus luteolamellatus	SFC20150818-39	South Korea	MF611680	n/a
	Lactifluus luteopus	AV94-463 GENT Type	Burundi	KR364119	n/a
	Lactifluus luteopus	EDC11-087 GENT	Tanzania	KR364049	KR364176
	Lactifluus medusae	EDC12-152 GENT	Cameroon	KR364069	n/a
	Lactifluus pseudohygrophoroides	SFC20140821-45 Holotype	South Korea	MF611682	MF611657
	Lactifluus pseudohygrophoroides	SFC20150813-71	South Korea	MF611681	n/a
	Lactifluus pseudoluteopus	LTH155 GENT	Thailand	HQ318286	HQ318210
	Lactifluus pseudoluteopus	FH12-026 GENT	Thailand	KR364084	n/a
	Lactifluus pseudoluteopus	environmental sample CD15	Thailand	FJ644702	n/a
	Lactifluus rugatus	EP 1212_7 LGAM-AUA	Greece	KR364104	KR364235
	Lactifluus rugatus	PA2010R	Greece	MH125243	n/a
	Lactifluus rugatus	4_01_2015	Italy	KU885436	n/a
	Lactifluus sp.	environmental sample L7524_Russ MAD37	Madagascar	FR731264	n/a
	Lactifluus sp.	environmental sample T071b	Thailand	JN969388	n/a
	Lactifluus sp.	MycoMap6251	USA	MK560130	n/a
	Lactifluus sp.	MycoMap6284	USA	MK560131	n/a
	Lactifluus sp.	FLAS-F-61011	USA	MH016945	n/a
	Lactifluus sp.	KUNF58696	n/a	KC154100	n/a
	Lactifluus sp.	KV181	AU_NSW	MW471124	n/a
	Lactifluus sudanicus	AV11-174 MD105	Togo	HG426469	KR364186
	Lactifluus sudanicus	MD148	Togo	HG426476	n/a

Phylogenetic analyses of the concatenated ITS+LSU alignment was performed with Maximum Likelihood (ML) in RAxML v. 8.2.12 (Stamatakis ) using the CIPRES Science Gateway v. 3.3 (Miller ). The final alignment comprised 211 specimens (195 ITS and 146 LSU sequences), consisting of 2 087 bp including gaps. Gaps in alignments were treated as missing data. The tree was visualised in FigTree v. 1.4.2 (Rambaut 2009).

RESULTS

Sequestrate forms arose independently in at least three sections in two subgenera of Lactifluus: subg. Lactifluus sect. Gerardii (Lf. dendriticus comb. nov.) and sect. Lactifluus (Lf. geoprofluens sp. nov.), and subg. Pseudogymnocarpi sect. Pseudogymnocarpi (Lf. sp. prov. KV181). Lactifluus dendriticus comb. nov. appears to be a wide-spread species from eastern Australia, ectomycorrhizal with Eucalyptus and Acacia. It is in a strongly supported clade, sister to Lf. wirrabara, three provisional Australian species with agaricoid basidiomes (Lf. sp. prov. 10–12), an environmental sequence with unknown basidiome form Lf. sp. prov. 13 from Australia, Lf. limbatus from Malaysia, Lf. sinensis from China, Lf. coniculus from Sri Lanka, and Lf. midnapurensis from India in section Gerardii (Fig. 1). All of these Asian taxa are associated with lowland tropical rainforest dominated by Dipterocarp taxa (Stubbe , Song , Phookamsak ).

Fig. 1.

Maximum Likelihood tree based on ITS and LSU sequences for subgenus Lactifluus sections Gerardii, Piperati, Ambicystidiati, and Allardi; subgenus Gymnocarpi sections Tomentosi, Phlebonemi, Panuioidei, Nebulosi, Luteoli, Gymnocarpi, and Lf. vellereus from Lf. sect. Albati, subg. Lactariopsis, with outgroup Multifurca. Bold lines indicate ML support ≥ 90 %. Bold text sequences of sequestrate taxa generated for this study. Red text: Australian specimens or sequences, blue text: New Zealand specimens, green text: New Caledonia specimens or sequences.

Lactifluus geoprofluens sp. nov. is in a weakly supported clade with two agaricoid species Lf. jetiae nom. prov. (Fig. 3C) and Lf. rugulostipitatus nom. prov. (in press; these provisional species will be published elsewhere), a third provisional species known only from environmental sequences (Lf. sp. prov. 8), several undescribed taxa with agaricoid basidiomes from Thailand and Japan, and Lf. distantifolius and Lf. Longipilus (Fig. 2). More data are required to reliably recognise sister relationships as branch support values are low in this mixed clade of mostly undescribed taxa.

Fig. 3.

Basidiomata of new sequestrate species and representative agaricoid species. A. Lactifluus wirrabara (JET 943; photo J.E. Tonkin). B. Lf. dendriticus (photo T. Lebel). C. Lf. jetiae (photo J.E. Tonkin). D. Lf. geoprofluens (photo L. Vaughan). E. Lf. rugatus (photo U. Pero). F. Lf. sp. prov. KV181 (photo T. Lebel). Scale bars = 10 mm

Fig. 2.

Maximum Likelihood tree based on ITS and LSU sequences for subgenus Lactifluus sections Lactifluus and Tenuicystidiati; and subgenus Pseudogymnocarpi section Pseudogymnocarpi and an unnamed clade, and exemplars from sections Lactariopsis, Neotropicus and Edules. Bold lines ML ≥ 90 %. Bold text sequences generated for this study. Red text: Australian specimens or sequences.

A single sequence of the sequestrate Lactifluus sp. prov. KV181 is in a clade with a mix of species from diverse geographic locations, Lf. pseudoluteopus from Thailand, Lf. hygrophoroides from North America, Lf. rugatus from Southern Europe, and Lf. holophyllus and Lf. luteolamellatus from South Korea in sect. Pseudogymnocarpi. While we did take a small sample for DNA in the field, unfortunately, the single collection of Lf. sp. prov. KV181 was destroyed by mould contamination after drying and discarded, and no further collections of this sequestrate taxon were found while looking through abundant material of sequestrate lactarioid taxa in herbaria. Thus while a full description cannot be provided at this time, we do provide a photo of basidiomes, and a partial description, in the hope that further material will be found.

Taxonomy

Section Gerardii

(T. Lebel) T. Lebel, J. Cooper & Nuytinck, comb. nov. MycoBank MB 838127. Figs 3B, 4A–F.

Fig. 4.

Lactifluus dendriticus. A. Cross-section of pileipellis with dendritic terminal elements. B. Scalp section showing dendritic terminal elements. C. Dendritic terminal elements. D. Basidiospores. E. Hymenial trama with pseudocystidia. F. Scanning Electron Microscopy photo micrograph of basidiospores. Scale bars: A–D, F = 10 μm, E = 50 μm.

Basionym: Zelleromyces dendriticus T. Lebel, Australasian Mycologist 21: 4. 2002. figs 1–4. MB 373383.

Etymology: The specific epithet, “dendriticus” (L), refers to the dendritic terminal element of the pileipellis.

Diagnosis: This species can be distinguished by the pale sequestrate basidiomes, unique dendritic terminal elements forming a short turf in the pileipellis, and globose spores.

Typus: Australia, Victoria, Errinundra National Park, The Gap Scenic Reserve, Gunmark Road, 1.3 km southeast of Survey Road, Claridge site 124, 28 May 1996, A. Jumpponen & J. Trappe 18478 (holotype MEL 2063482, isotype OSC 80519).

Basidiomes hypogeal, sequestrate, 5–25 × 5–20 mm, globose to subglobose, slightly irregular or furrowed near point of attachment; locules exposed at point of attachment in some basidiomata as pileus is incomplete, surface dry, smooth to minutely fibrillose or verrucose, white with a faint yellowish tinge and often with small darker yellow-brown patches, pileipellis thin, off-white in cross-section. Hymenophore loculate, locules large 0.5–2 mm diam, somewhat irregular, white when young, becoming pale yellow to pale brown-yellow with maturity, pale tan when dried. Stipe-columella absent. Latex scant to abundant, white, unchanging. Taste mild. Odour mildly sweet, pleasant. Basidiospores 9.0–10.8 × 8.5–10.6 μm (x̄ =9.8 ± 0.53 × 9.69 ± 0.64, n = 50), globose [Q=1.0–1.05 (x̄ = 1.01 ± 0.02, n = 50)], walls weakly amyloid, ornamentation a fine, complete to almost complete regular mesh reticulum ± 0.5–0.7 μm high, with few scattered, isolated warts also present; hilar appendix small, central, plage absent. Basidia 33–65 × 7–13 μm, elongated clavate to cylindrical or rarely narrowly ventricose, hyaline, thin-walled, with 2 or 4 robust, slightly curved sterigmata 4–10 × 2–3.5 μm. Hymenophoral trama 14–22 μm wide, a narrow central strand of crowded, hyaline hyphae 2–4 μm diam, with slightly thickened walls intermixed with scattered sinuous laticiferous hyphae 3–7.5 μm diam, sphaerocytes absent; subhymenium 15–35 μm wide, with two or three tiers of ± isodiametric cells 8–15 μm diam. Pseudocystidia absent or when present, 5–11 μm diam, thin-walled, cylindrical to narrowly clavate, scattered, with oily refractive contents; arising in trama from laticiferous hyphae extending up through the subhymenium. Pileipellis 18–45 μm wide, composed of a very narrow cutis with an almost complete turf of thick-walled (throughout length) dendritic terminal elements and scattered rare laticiferous hyphae when young, this turf becoming patchy as basidiomata expands; no gelatinous matter or very little in some basidiomes apparent amongst terminal elements dendritic terminal elements less branched and intricate when young, variously and often elaborately branched when older, coralloid to irregularly repeatedly branched with coralloid tips, 18–32 μm high, 4–25 μm wide, 3–6 μm at base, walls thick. Pileus trama 32–60 μm wide, of tightly interwoven, subgelatinous, hyaline hyphae 2–4 μm diam, more crowded towards the surface than within, with scattered to common, sinuous laticiferous hyphae 3–8 μm diam, refractive in KOH; sphaerocytes absent.

Ecology and Distribution: Sporulating in April–July in small to large groups, at mid to higher elevations in the mountains of south eastern Australia in Victoria, Tasmania, and New South Wales. Hypogeal in mixed forests of Eucalyptus fastigata, E. cypellocarpa, E. radiata, E. dalrympleana, E. globoidea, E. obliqua, E. pauciflora, E. stellulata, E. regnans, Acacia dealbata, A. melanoxylon, A. aculeatissima, A. cognata, or A. mearnsii.

Additional specimens examined: Australia, Victoria, Kinglake National Park, 120 m down Mountain Creek Track, 8 Jul. 1993, M.A. Castellano OSC 80520 (MEL 2063479); East Gippsland, Errinundra National Park, The Gap Scenic Reserve, Gunmark Road, 1.3 km SE of Survey Road, Claridge Site 124, 30 May 2003, A.W. Claridge, W. Colgan III, A. Jumpponen, I. Kratzer AWC5079 (CANB 748631); ibid., Gap Road, 50 m E of Bonang River Bridge, Claridge Site 117, 9 May 2003, A.W. Claridge AWC4731 (CANB 65451); ibid. Claridge Site 117, 27 May 1999, J.M. Trappe AWC2589 (MEL 2105044); ibid., 27 May 1999, J.M. Trappe AWC2588 (MEL 2105043); ibid., Gap Road, 3.9 km E of Bonang Highway, Claridge site 118, 28 May 1996, J.M. Trappe 18434 (MEL 2063477); ibid., Gap Road, 2.1 km west of track to Result Creek Falls, Claridge Site 138, 11 Jun. 1996, J.M. Trappe 19022 (MEL 2063472); ibid., Gap Road, 5.2 km E of Junction with Bonang Highway, Claridge Site 119, 9 May 2003, J.M. Trappe & A.W. Claridge AWC4741 (DAR 76661, CANB 748620, MEL 2292167) (also in K, NY, BPI, FH); ibid., Claridge Site 119, 26 May 2001, T. Lebel AWC3994 (CANB 736252); ibid., Gap Road, 2.1 km west of track to Result Creek Falls, Claridge Site 138, 11 Jun. 1996, A.M. Jumpponen JMT19021 (MEL 2063473); Rich Forest Management Block, Jack Road, 0.7 km N of Winter Road, Claridge Retrospective Study Site R19, 27 Apr. 1996, A.W. Claridge AWC352 (CANB 669623); Lind National Park, Euchre Valley Road, 0.2 km W of Junction with Lind Park Road, Claridge Site 74, 29 May 1999, A.W. Claridge AWC2746B (MEL 210507); Murrungowar Forest Management Block, Princes Highway, 0.4 km E of Junction with Bendoc Ridge Road, Claridge Site 64, 26 May 1999, T. Lebel AWC2556 (MEL 2105041); 2.4 km N of Baw Baw National Park,16 Jun. 1994, J.M. Trappe H6792 (PERTH 7593031); 20 km west of Mansfield near Mt Buller, 9 Jul. 1993, T. Lebel JMT14063 (MEL 2063478); Alpine National Park, Black Mountain Road, Rams Horn Track. Claridge Site No. 78, 11 Nov. 2008, A.W. Claridge JMT18089 (MEL 2326248); Noojee State Park, ca. 1 km from Tooronga Road, along Link Road in small carpark area, 25 Feb. 2002, S.H. Lewis 791 (MEL 2061947); North east of Marysville on Lady Talbot Drive, wishing Well Track, 19 Apr. 1999, T. Lebel TL5 (MEL 2063377); New South Wales, Bombala, Bondi Gulf Nature Reserve, Bondi Gulf Road, 4 km E of Cann Valley Highway, Claridge Site 53, 15 Jun. 1999, J.M. Trappe AWC2972 (CANB 748185); Tasmania, Break O’Day, Elephant Pass, 16 kms southeast of St Marys, 2 May 1990, N. Malajczuk MEL 2063481; Elephant Pass, 2 May 1990, J.M. Trappe H1373 (PERTH 7673167).

Notes: A revised description of Lactifluus dendriticus is provided here to enable better comparisons to other taxa, and to incorporate more data from fresh collections. Detailed illustrations of spores, pileipellis and hymenophore structure and constituent elements were provided as part of the original description (Lebel 2002). Lactifluus dendriticus is widespread in eucalypt forests occurring in regions with relatively higher rainfall. Its basidiomata do not always produce a latex when cut or bruised. Microscopically, Lactifluus dendriticus is unique, with distinct highly branched pileal terminal elements (Fig. 4A–C) that have not been observed in any other species of sequestrate or currently known agaricoid Russulales taxa.

Taxa in the larger clade, Lactifluus wirrabara, Lf. limbatus, Lf. coniculus, Lf. sinensis, and Lf. midnapurensis all have brownish agaricoid basidiocarps (Fig. 3A), and spores with a low almost complete to complete reticulum; however these species have smaller, subglobose to broadly ellipsoid spores, and very different pileicystidia or pileal terminal elements.

Section Lactifluus

T. Lebel, Castellano, Claridge & Trappe, sp. nov. MycoBank MB 838126. Figs 3D, 5A–F.

Fig. 5.

Lactifluus geoprofluens A. Lampropallisade pileipellis terminal elements (te), subpellis (sp) and context (c). B. Pileipellis terminal elements (te) and subpellis (sp). C. Hymenial trama. D–E. Basidiospores. F. Scanning Electron Microscopy photo micrograph of basidiospores. Scale bars: A, C = 50 μm; B = 20 μm; D–F =10 μm.

Etymology: The species epithet, “profluens”, from Latin, meaning “freely flowing” referring to the abundantly flowing latex in cut basidiomes of this species, and “geo” in reference to the hypogeal basidiomes.

Diagnosis: Differs from other currently known lactarioid sequestrate taxa by the faint fishy odour, abundant white latex, globose to occasionally barely subglobose basidiospores, up to 10.5 × 10.0 μm, ornamented with a robust amyloid reticulum, and presence of dendritic pileal terminal elements

Typus: Australia, Victoria, East Gippsland, Rich Forest Management Block, near Jack Road 0.7 km N of junction with Winter Road, Claridge site R19, 29 Feb. 1996, A.W. Claridge AWC201 (holotype MEL 2145804).

Basidiomes hypogeal, sequestrate, 5–15 mm broad, globose to subglobose or irregular, surface dry, smooth to rugulose and irregularly folded radially particularly around the point of attachment to substrate, minutely pruinose and velutinous, pale orange-ochre to almost reddish-brown in folds; pileus in cross-section thin (≤ 0.5 mm), pale orange becoming orange-brown in dried specimens. Hymenophore loculate, locules 0.5–2 mm, labyrinthine and vaguely radial from columella attachment, pale orange-ochre to reddish orange-brown. Stipe-columella present or absent, much reduced if present and variable, 0.5–3 mm long × 0.5–2 mm wide, irregularly cylindrical, slightly tapering towards the base, orange-ochre to reddish-brown in patches, minutely pruinose and finely rugulose; context golden to reddish-brown. Latex white, unchanging, abundant along the pileipellis edges and the interior of the hymenophore. Taste mild. Odour not obvious or slightly fishy when fresh. Basidiospores 8.0–10.5 × 8.0–10.0 μm (x̄ = 9.85 ± 0.43 × 9.79 ± 0.35, n = 40), globose to occasionally barely subglobose [Q = 1.00–1.05 (x̄ = 1.02 ± 0.02, n = 40)], walls amyloid between ridges and plage distally to completely amyloid, ornamentation reticulate with ridge apices to 1 μm tall. Basidia 31–52 × 8–14 μm (x̄ = 44.18 ± 5.33 × 11.25 ± 1.08, n = 19), 3–6 μm wide at base (x̄ = 3.87 ± 0.54, n = 16), clavate, mostly 4-spored but occasionally 3-spored; sterigmata 3–10 × 1–2.5 μm, (x̄ = 7.18 ± 2.20 × 2.00 ± 0.51, n = 12); basidioles 30–51 × 6–11 μm (x̄ = 39.89 ± 4.46 × 9.14 ± 1.41, n = 14), 2–5 μm wide at base (x̄ = 3.36 ± 0.74, n = 14), cylindrical to barely clavate. Hymenophoral trama comprised of interwoven and parallel tightly packed hyphae 2–3 μm diam, interspersed with sinuous laticiferous hyphae 5–7 μm diam (x̄ = 5.64 ± 0.31, n = 8) and occasional sphaerocytes 30–45 × 17–38 μm; subhymenium composed of chains of 3–4 inflated cells 8–21 × 6–17 μm (x̄ = 11.30 ± 6.70 × 10.40 ± 5.72, n = 18), laticiferous hyphae present and occasionally extending into pseudocystidia 3–8 μm diam., thin-walled, cylindrical to slightly tortuous, with oily refractive contents. Hymenial cystidia scarce, when present not well differentiated, 31–52 × 7–11 μm (x̄ = 46.34 ± 4.78 × 9.56 ± 1.16, n = 12), subcylindrical tapering to apex, apex mucronate or obtuse, rarely emergent above hymenium, hyaline, thin-walled, contents scattered when present. Pileipellis a lampropalisade; subpellis composed of 3–6 layers of thin-walled polygonal cells 8–19 × 7–17 (x̄ = 15.02 ± 3.28 × 13.20 ± 2.06, n = 12) μm; lamprocystidia 18–27 × 2–5 μm (x̄ = 24.32 ± 4.12 × 3.73 ± 1.02, n = 15), elongate cylindrical and slightly sinuate, tapering toward apex, apex mucronate or capitate or obtuse, thick-walled, basal width 2–5 μm (x¯ = 3.73 ± 1.02, n = 15), and apical width 1–3 μm (x̄ = 1.59 ± 0.35, n = 15); pileus trama similar to hymenophoral trama, heteromerous, hyphae tightly compressed and interwoven, sphaerocytes not abundant.

Ecology and distribution: Sporulating in February–June in cool temperate forests in NE Victoria. Hypogeal in mixed forests of Eucalyptus maculata, E. muellerana, E. paniculata, E. obliqua, E. cypellocarpa, E. delegatensis, Angophora floribunda, E. longifolia, E. muellerana, E. sieberi, E. baxteri, E. botryoides, with an Acacia cognata, A. verticillata, A. melanoxylon, A. mucronata, A. longifolia, A. mearnsii, A. implexa understorey.

Additional specimens examined: Australia, Victoria, East Gippsland, Mimosa Rocks National Park, Tanja-Tathra Road, 0.5 km N of junction with track Leading to ‘Gillards’, Claridge Site 40, 7 Jun. 2003, J.M. Trappe, A.W. Claridge, A. Jumpponen AWC5539 (OSC 148637); ibid., Murrangowar Forest Management Block, Bendoc Ridge Road, 0.4 km from Princes Hwy, Claridge Site 66, 2 Jun. 2003, M. Vavrek AWC5306 (OSC 148635); ibid., Cape Conran-Sydenham Inlet Coastal Park, Swampy Creek trail, Crossing of Swampy Creek, Claridge site 128, 31 May 2003, A. Jumpponen AWC5204 (OSC 148634); ibid., East Gippsland, Rich Forest Management Block, junction of Mills Road and Jack Rich Divide Track, Claridge Retrospective study site R8, 18 Jul. 1996, B. Gunn JMT19613 and AWC452 (OSC 159173); ibid., Nunniong Forest Management Block, off Nunniong Road, Claridge site 106 (relocated 1999), 18 May 2001, T. Lebel AWC3614 (OSC 148633); Ben Boyd National Park, Bittangabee Picnic Area, Bittangabee Creek, Crossing of Walking Track leading N from Picnic Area, Claridge Site 52, 4 Jun. 2003, A. Jumpponen AWC5415 (OSC 148636); Lind National Park, Euchre Valley Road, 0.2 km W of Junction with Lind Park Road, Claridge site 13 (1999 relocation), 29 May 2001, W. Colgan III AWC4223 (OSC 148632).

Notes: This hypogeous, sequestrate fungus is known from cool-temperate mixed species Eucalyptus forest in East Gippsland, Victoria. It differs from the other two sequestrate taxa in often having a distinct columella, radial-locular hymenophore and abundant white latex in all tissues. Lactifluus geoprofluens shares with other species in Lactifluus section Lactifluus the fishy odour, velutinous orange-brown pileus and a lampropalisade pileipellis structure. The basidiospores in Lf. geoprofluens are large (up to 10.5 × 10 μm) and globose (Q = 1–1.05), unique among the Australian section Lactifluus species.

Section Pseudogymnocarpi

KV181 Fig. 3F.

Basidiomes sequestrate, up to 19 mm broad, globose to subglobose, surface dry, smooth to minutely pruinose and velutinous, pale creamy yellow-tan with some minor slightly darker bruising; pileipellis in section thin (≤ 0.5 mm), pale tan. Hymenophore loculate, empty locules 0.5–2 mm, labyrinthine, orange-ochre. Stipe absent; columella present, percurrent central strand, 0.5–2 mm wide, white to pale cream. Latex white turning slightly yellowish, abundant, particularly along the pileipellis edges and the interior of the hymenophore near columella. Taste mild. Odour faint, fungal.

Distribution and habitat: Sporulating in June in cool temperate open woodland in the eastern tablelands of New South Wales. Hypogeal in mixed Eucalyptus forest.

Sequence data: Australia, New South Wales, Gibraltar Range National Park, Mulligans Dr., 16 Jun. 2006, K. Vernes, T. Lebel & A. O’Malley KV181.

Notes: We provide the image of the basidiome and general notes in the hope that further material will be found. Our analyses show that this taxon is in a strongly supported section Pseudogymnocarpi, however support values within the clade are low (Fig. 2). The majority of agaricoid species currently known within this clade (e.g. Lf. rugatus, Lf. pseudoluteopus, Lf. hygrophoroides) have pale orange to orange-red pilei and stipe, pale coloured lamellae, abundant white latex (Fig. 3E), and an association with Fagaceae or Pinaceae. The lack of microscopic detail, and additional data for Australasian agaricoid taxa or environmental sequences, precludes any statements regarding how well this sequestrate taxon fits in this clade generally.

DISCUSSION

Both agaricoid and pleurotoid basidiome forms have been found in Lactifluus. In this predominantly agaricoid genus, the pleurotoid habit has at least seven different origins in three subgenera: subg. Lactifluus section Gerardii, subg. Lactariopsis and subg. Gymnocarpi (De Crop ). No pleurotoid forms are thus far known in Lactarius, but they have been found in Russula (Buyck & Horak 1999, Henkel ). Before the present study, sequestrate forms were not known from Lactifluus, but have evolved many times over in Lactarius; the evolution of a sequestrate basidiome form thus appears to be a rare event in Lactifluus.

All three sequestrate Lactifluus species have a phylogenetic affinity with Southeast Asian taxa. Some, like Lf. geoprofluens and Lf. dendriticus, probably evolved in Australia from local ancestors (that have a SE Asian descent - or other way around; cannot be determined at this point). However, the isolated position of Lf. sp. prov. KV181 is intriguing, as apart from Asian relatives, a European and a North American species are also closely related. This could be due to under sampling of the Australian and Malesian taxa in particular. However, placement within section Pseudogymnocarpi is well supported (BS 98 %).

While the number of species in Lactarius with sequestrate basidiome forms is much greater (in the order of 25–30 undescribed and nine named species known for Australasia; and a further 22 spp. worldwide) than in Lactifluus, all sequestrate taxa also appear to have evolved as independent and isolated incidents within the genus (Trappe , Desjardin 2003, Verbeken , Sheedy ). None of the sequestrate taxa have evolved into a clade containing a large diversity of species with similar basidiome forms, all indicative of relatively recent origin of this basidiome form in Lactarius (Verbeken ) and Lactifluus. The recent description of several sequestrate Lactarius from tropical forests (Verbeken , b, Buyck ), and discovery of hidden diversity of Australasian Lactifluus species lend support to the theory that sequestrate basidiome forms are essentially an excellent way to maintain a fairly constant relative humidity in the enclosed hymenophore, allowing full development of spores to ensue, regardless of arid, seasonally dry or tropical, seasonally waterlogged conditions (Beever & Lebel 2014).

Unsurprisingly, given the trouble with morphological differentiation of Lactifluus and Lactarius agaricoid species, it is also difficult to distinguish taxa with sequestrate basidiomes. As a broad generalisation, in Australia, the majority of sequestrate Lactarius have yellow to orange to red tinged basidiomes, whereas two of the three Lactifluus taxa described in this study have white to pale cream basidiomes. Pale basidiome colouring is more common in Australasian sequestrate russuloid taxa (Beaton , Lebel & Trappe 2000, Lebel & Castellano 2002, Lebel 2002a, b, 2003a, b, Lebel & Tonkin 2007). General trends in microscopic differentiation between Lactarius and Lactifluus were formulated by Verbeken & Nuytinck (2013) and can be summarised as: (i) thick-walled elements in the pileipellis and stipitipellis, as well as lamprocystidia, are generally present in Lactifluus and very rarely observed in Lactarius, and (ii) a hymenophoral trama composed of sphaerocytes (as in Russula) is common in Lactifluus but is rarely observed in Lactarius. Both trends don’t seem to hold up for the sequestrate Lactifluus species. The loss of thick-walled elements in pileus and hymenium in sequestrate forms could be due to increased folding and loculisation of the hymenophoral trama in combination with loss of ballistospory (i.e. no longer require spacing to allow for spore drop). As the basidiomes are submersed in the soil, dispersal of spores by vectors such as marsupial mammals or insects, is highly likely; by having thin walls the ripe basidiomes would be easier to break open or eat. The highly branched, angular pileal dendritic terminal elements of Lf. dendriticus are unique. The presence of distinctively shaped pileicystidia or terminal elements is more typical of species of Russula but rarely seen in milkcaps (Verbeken 1996a, Verbeken & Walleyn 2010). Initially thought to be a contaminant, perhaps a mycenoid mycoparasite or virus, but these terminal elements are found consistently in multiple basidiomes from geographically distant collections, and no DNA sequences for mycenoid taxa were ever recovered as contaminants.

In Lactifluus section Gerardii the majority of species have a brown stipe and pileus contrasting with white mostly distant lamellae, reticulate spore ornamentation < 2 μm high, a palisade pileipellis structure, and are generally lacking in macrocystidia (Stubbe , De Crop , 2018). The basidiomes can also be small, white and pleurotoid, with thick-walled terminal elements in the pileipellis (Stubbe , Latha , De Crop ). Several of the agaricoid and pleurotoid taxa have latex that changes colour on exposure, however this is not the case for Lf. dendriticus. While the pale sequestrate basidiomes of Lf. dendriticus can be easily mistaken for sequestrate russuloid taxa as latex production is variable, the more open form of locules in the hymenophoral tissue tend to suggest a lactarioid taxon. However, it is the unique dendritic pileal terminal elements of Lf. dendriticus that are intriguing. How does a cystidium evolve from a simple filamentous, perhaps irregularly capitate form, to a highly branched, sharp angled form?

Lactifluus geoprofluens is readily distinguished from Australian and international species in Lf. section Lactifluus by lacking lamprocystidia, having large globose basidiospores and a sequestrate basidiome form. It is the first known sequestrate member of Lf. section Lactifluus (De Crop ). Unfortunately, we do not have an image of the fresh basidiomes of Lf. geoprofluens, however the field notes all state pale orange-ochre to reddish brown in colour, with slightly paler hymenophoral tissue.

We currently lack data to describe Lf. sp. prov. KV181 as a new species, but we do provide a picture and a description of field characteristics. It is the first time a non-agaricoid representative of Lf. subg. Pseudogymnocarpi is documented, and the first Australasian representative in section Pseudogymnocarpi. We look forward to obtaining new material for further examination.