Research Article |
Corresponding author: Eske De Crop ( eske.decrop@ugent.be ) Academic editor: Bryn Dentinger
© 2019 Eske De Crop, Jonas Lescroart, André-Ledoux Njouonkou, Ruben De Lange, Kobeke Van de Putte, Annemieke Verbeken.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
De Crop E, Lescroart J, Njouonkou A-L, De Lange R, Van de Putte K, Verbeken A (2019) Lactifluus bicapillus (Russulales, Russulaceae), a new species from the Guineo-Congolian rainforest. MycoKeys 45: 25-39. https://doi.org/10.3897/mycokeys.45.29964
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The milkcap genus Lactifluus is one of the most common ectomycorrhizal genera within Central African rainforests. During a field trip to the Dja Biosphere Reserve in Cameroon, a new Lactifluus species was found. Molecular and morphological analyses indicate that the species belongs to Lactifluus section Xerampelini and we formally describe it here as Lactifluus bicapillus sp. nov.
Ectomycorrhizal fungi, Gilbertiodendron, Lactarius , phylogeny, taxonomy, tropical Africa, Uapaca
Rainforests occur in Central Africa and form the main vegetation type in the Guineo-Congolian region (
Within Central African rainforests, the ECMRussulaceae genera Russula Pers. and Lactifluus (Pers.) Roussel are abundant (
About 20 Lactifluus species are known from the rainforests of Central Africa (
During fieldwork in Cameroon in 2012 and 2014, several Lactifluus specimens were found morphologically resembling yet different from the described species within L. subg. Pseudogymnocarpi (Pacioni & Lalli) De Crop. The phylogenetic results of
Sampling expeditions in Cameroon were carried out in May 2012 and May 2014, in the Guineo-Congolian rainforest of the Dja Biosphere Reserve (East Region of Cameroon), mainly in the vicinity of Somalomo and Lomié. During each expedition, four collections were made of an unknown and putative new milkcap species with characteristics of L. subg. Pseudogymnocarpi. The collections were found in either monodominant stands of Gilbertiodendron dewevrei, or mixed stands with Uapaca guineensis Müll. Arg., U. acuminata (Hutch.) Pax & K. Hoffm., and U. paludosa Aubrév. & Leandri as the main ECM hosts. Specimens were dried using a field drier and candles. The studied collections were deposited in the fungal herbarium of Ghent University (GENT).
Macroscopic features were all based on fresh material described in the field. Colour codes refer to
DNA was extracted using the CTAB extraction protocol described in
PCR products were sequenced using an automated ABI 3730 XL capillary sequencer (Life Technology) at Macrogen. Forward and reverse reads were assembled into contigs and edited where needed with the SEQUENCHER v. 5.0 software (Gene Codes Corporation, Ann Arbor, MI, USA).
A dataset was constructed, containing sequences of these recent collections, together with sequences of L. subg. Pseudogymnocarpi extracted from the dataset of
Specimens and GenBank accession numbers of DNA sequences used in the molecular analyses. The arrangement of the subgenera and sections in the table follows their position in the concatenated phylogeny of the genus Lactifluus (Fig.
Species | Voucher collection (herbarium) | Country | ITS accession no. | LSU accession no. |
---|---|---|---|---|
Genus Lactifluus | ||||
Lactifluus subg. Pseudogymnocarpi | ||||
Lactifluus sect. Pseudogymnocarpi | ||||
L. cf. longisporus | AV 11-025 (GENT) | Tanzania | KR364054 | KR364181 |
L. cf. pseudogymnocarpus | AV 05-085 (GENT) | Malawi | KR364012 | KR364139 |
L. cf. pumilus | EDC 12-066 (GENT) | Cameroon | KR364067 | KR364196 |
L. gymnocarpoides | JD 885 (BR) | Congo | KR364074 | KR364203 |
AV 05-184 (GENT) | Malawi | KR364024 | KR364151 | |
L. hygrophoroides | AV 05-251 (GENT) | North America | HQ318285 | HQ318208 |
L. longisporus | AV 94-557 (Isotype, GENT) | Burundi | KR364118 | KR364244 |
L. luteopus | AV 94-463 (Isotype, GENT) | Burundi | KR364119 | None |
L. medusae | EDC 12-152 (GENT) | Cameroon | KR364069 | KR364198 |
L. pseudoluteopus | FH 12-026 (GENT) | Thailand | KR364084 | KR364214 |
L. rugatus | EP 1212/7 (LGAM-AUA) | Greece | KR364104 | KR364235 |
L. sudanicus | AV 11-174 (Isotype, GENT) | Togo | HG426469 | KR364186 |
Lactifluus sect. Xerampelini | ||||
L. bicapillus sp. nov. | EDC 12-176 (GENT) | Cameroon | KR364070 | KR364199 |
EDC 12-174 (GENT) | Cameroon | MH549201 | MH549201 | |
EDC 14-245 (GENT) | Cameroon | MH549204 | MH549204 | |
EDC 12-169 (GENT) | Cameroon | MH549200 | MH549200 | |
EDC 14-249 (Holotype, GENT) | Cameroon | MH549203 | MH549203 | |
EDC 14-284 (GENT) | Cameroon | KX499395 | None | |
EDC 14-238 (GENT) | Cameroon | MH549202 | MH549202 | |
EDC 12-071 (GENT) | Cameroon | KX499396 | KX622762 | |
L6470/Gab40 (env. seq.) | Gabon | FR731875 | None | |
L. cf. pseudovolemus | ADK 2927 (GENT) | Benin | KR364113 | KR364243 |
L. goossensiae | AB 320 (GENT) | Guinea | KR364132 | KR364252 |
L. kivuensis | JR Z 310 (Holotype, GENT) | Congo | KR364027 | KR364154 |
L. rubiginosus | JD 959 (BR) | Congo | KR364081 | KR364210 |
BB 3466 (Holotype, BR) | Zambia | KR364014 | KR364250 | |
L. persicinus | EDC 12-001 (Holotype, GENT) | Cameroon | KR364061 | KR364190 |
L. xerampelinus | TS 1116 (Isotype, GENT) | Tanzania | KR364039 | KR364166 |
Clade 8 | ||||
L. sp. | JN 2011-012 (GENT) | Vietnam | KR364045 | KR364171 |
TENN 065929 (TENN) | North America | KR364102 | KR364233 | |
L. armeniacus | EDC 14-501 (Isotype, GENT) | Thailand | KR364127 | None |
L. volemoides | TS 0705 (Holotype, H) | Tanzania | KR364038 | KR364165 |
Lactifluus sect. Aurantiifolii | ||||
L. aurantiifolius | AV 94-063 (Isotype, GENT) | Burundi | KR364017 | KR364144 |
Lactifluus sect. Rubroviolascentini | ||||
L. aff. rubroviolascens | EDC 12-051 (GENT) | Cameroon | KR364066 | KR364195 |
L. carmineus | AV 99-099 (Holotype, GENT) | Zimbabwe | KR364131 | KR364251 |
L. denigricans | EDC 11-218 (GENT) | Tanzania | KR364051 | KR364178 |
L. kigomaensis | AV 11-006 (Holotype, GENT) | Tanzania | KR364052 | KR364179 |
L. subkigomaensis | EDC 11-159 (GENT) | Tanzania | KR364050 | KR364177 |
Lactifluus sect. Polysphaerophori | ||||
L. pegleri | PAM/Mart 12-091 (LIP) | Martinique | KP691416 | KP691425 |
L. sp. | RC/Guy 09-036 (LIP) | French Guiana | KJ786645 | KJ786550 |
MR/Guy 13-145 | French Guiana | KJ786691 | KJ786595 | |
MCA 3937 (GENT) | Guyana | KR364109 | KR364240 | |
L. veraecrucis | M 8025 (Holotype, ENCB) | Mexico | KR364112 | KR364241 |
Lactifluus subg. Lactifluus | ||||
Lactifluus sect. Lactifluus | ||||
L. corrugis s.l. | AV 05-392 (GENT) | North America | JQ753822 | KR364143 |
L. versiformis | AV-KD-KVP 09-045 (Holotype, GENT) | India | JN388967 | JN389031 |
L. vitellinus | KVP 08-024 (GENT) | Thailand | HQ318236 | HQ318144 |
L. volemus | KVP 11-002 (GENT) | Belgium | JQ753948 | KR364175 |
Sequences were aligned using the online version of the multiple sequence alignment program MAFFT v. 7 (
Sequence data were divided into the following partitions: partial 18S, ITS1, 5.8S, ITS2 and partial 28S. Maximum likelihood (ML) analyses were conducted with RAxML v. 8.0.24 (
Our molecular results show that the recently collected specimens form a well-supported monophyletic clade within Lactifluus subg. Pseudogymnocarpi, L. sect. Xerampelini (Fig.
Overview Maximum Likelihood tree of Lactifluus subg. Pseudogymnocarpi, based on concatenated ITS and LSU sequence data. Sequences of the here described species Lactifluus bicapillus are written in bold. Maximum Likelihood bootstrap values > 70 are shown. Numbers of undescribed sections refer to
Lactifluus bicapillus differs from L. xerampelinus by its yellowish-orange to dark red cap, fertile lamella edge, a lampropalisade with two types of terminal elements as pileipellis type, and a distribution in the Guineo-Congolian rainforest.
CAMEROON. East Region, Haut-Nyong division, Somalomo subdivision, Dja Biosphere Reserve, alt. ca 640 m, 3°21.83'N, 12°44.18'E, rainforest with Uapaca paludosa and U. guineensis, 14 May 2014, leg.: De Crop & Verbeken, EDC 14-249 (GENT!).
Basidiocarps medium-sized. Pileus 34–79 mm in diameter, firm, infundibuliform to deeply infundibuliform, planoconvex with central depression when younger; margin involute when juvenile, becoming inflexed up to reflexed when older; edge entire, sometimes eroded when older; surface felty to chamois leather-like, often slightly pruinose in the centre, often grooved, concentrically wrinkled, in young specimens completely velutinous and somewhat translucent; rubiginous (7D6–7) in centre, becoming paler and more orange towards the margin (6C5–6 to 5A5–6); young specimens dark reddish or burgundy in centre, to bright orange or yellow at the margin (8F6 to 7B6, to 6A5, 4AG); secondary velum absent. Stipe 16–39 × 6–12 mm, cylindrical to slightly tapering downwards, often laterally curved near the base, central to eccentric insertion to pileus, entire or bruised appearance, sometimes with white flocks near the base; surface smooth and felty, sometimes pruinose, yellowish orange (5AB5–6), becoming slightly paler and more yellow near the base and/or lamellae (5A4–5). Lamellae intervenose, transvenose, sparingly bifurcating; attachment adnate to decurrent with some lamellae forming a small tooth; juveniles not brittle, rather thin, older specimens brittle to very brittle, thick to very broad; edge entire and concolourous; distant, 3–5 + 6–9 L+l/cm, between 2 lamellae often 3 lamellulae, with regular short long-short pattern; creamy yellow (3A2) to yellowish orange (4A4). Context white, with a faint yellow tinge, colour not changing when cut, but in 1 collection (EDC 14-238) becoming brown when damaged, rather solid and full, smell sweet or not distinct, taste mild. Latex white, somewhat astringent, rather abundant, becomes less abundant and more watery with age, mild, colour rarely changing brownish when isolated. Chemical reactions unchanging with Fe2SO4; context faint blue after 5 sec. with guaiac.
Basidiospores [6.2]–7.3–7.9–[9.6](10.3) × [4.6]–5.5–5.9–[6.8] μm; ellipsoid, with Q = (1.22)1.31–1.39(1.51); ornamentation amyloid, composed of low ridges and warts, up to 0.2 μm high, forming an incomplete to complete reticulum; plage inamyloid or centrally amyloid. Basidia 43–62 × 8–12 μm, rather long, narrowly subclavate, 1-, 2- or 4-spored; content oleiferic. Sterile elements abundant, 19.5–40 × 3.5–5.5 μm, not emergent, cylindrical, septate with clamp-like bulges under the septum, with rounded apex. Pleurocystidia absent. Pleuropseudocystidia very scarce in mature specimens, abundant in young specimens, narrowly and irregular cylindrical to flexuose, 3.3–4.6 μm diam., not emerging, apex obtuse, oleiferic content. Lamellae-edge fertile, consisting of basidioles with some basidia. Marginal cells absent. Hymenophoral trama cellular, with sphaerocytes and abundant lactifers. Pileipellis a lampropalisade, up to 275 μm thick; terminal elements of two types, without transitional forms: the first type long and slender, thick-walled and often septate, with a wide base, up to 7 μm, and growing thinner towards the apex, down to 1–2 μm, length 52–92 μm, often narrowing rather abruptly, and twisted; the second type short and broad, also thick-walled and often septate, not specifically narrower towards the apex, often twisted, 20–44 × 5–7 μm; subpellis composed of mostly rounded cells. Stipitipellis similar to pileipellis but not as thick; terminal elements of the long type 52–75 × 5–7 μm; terminal elements of the short type 22–29 × 5–7 μm. Clamp-connections absent.
Basidiomata of Lactifluus bicapillus. a–c Basidiomata of Lactifluus bicapillus (EDC 12-176, EDC 12-174, holotype EDC 14-249 resp.) d Detail of lamellae (EDC 14-176), e) young specimen (EDC 12-169) f Detail of latex (EDC 12-169) g Detail of brown colour change of the latex (EDC 14-238) (photographs a–f by E. De Crop, g by A. Verbeken).
Microscopic features of Lactifluus bicapillus a Basidiocarps (from EDC 12-071, EDC 12-169, EDC 12-174, EDC 12-176, and EDC 14-249) b Basidia (from EDC 12-071, and EDC 14-249) c Sterile elements from the hymenium (from EDC 12-169) d Pleuropseudocystidia (from EDC 12-169) e Basidiospores (from EDC 14-249). Illustrations by E. De Crop, J. Lescroart and A. Verbeken. Scale bar: 10 μm.
Microscopic features of Lactifluus bicapillus (continued) a Terminal elements of the pileipellis (from EDC 12-071) b Terminal elements of the stipitipellis (from EDC 12-176) c Section through the pileipellis (from holotype EDC 14-249). Illustrations by E. De Crop and J. Lescroart. Scale bar: 10 μm.
Known from Cameroon and Gabon.
Guineo-Congolian rainforest, scattered on forest floor under Gilbertiodendron dewevrei, Uapaca guineensis, U. acuminata, and U. paludosa.
A combination of ‘bi’ and ‘capillus’, referring to the two types of terminal elements in the pileipellis and stipitipellis.
Unknown.
Cameroon. East Region, Haut-Nyong division, Somalomo subdivision, Koulou village, alt. ca 650 m, 3°23.61'N, 12°44.50'E, rainforest, Gilbertiodendron dewevrei, Uapaca guineensis, U. acuminata, 15 May 2012, De Crop, EDC 12-071 (GENT); East Region, Haut-Nyong division, Lomié subdivision, Bosquet village, alt. ca 610 m, 3°07.82'N, 13°53.36'E, rainforest with many Uapaca trees, on a riverbank, Uapaca guineensis, 24 May 2012, De Crop, EDC 12-169 (GENT); Ibidem, Gilbertiodendron dewevrei, De Crop, EDC 12-174 (GENT); Ibidem, Uapaca guineensis, EDC 12-176 (GENT); East Region, Haut-Nyong division, Somalomo subdivision, Dja Biosphere Reserve, alt. ca 650 m, 3°21.90'N, 12°44.15'E, rainforest, Uapaca paludosa, U. guineensis, 14 May 2014, De Crop & Verbeken, EDC 14-238 (GENT); Ibidem, alt. ca 640 m, 3°21.83'N, 12°44.18'E, De Crop & Verbeken, EDC 14-249 (GENT); Ibidem, alt. ca 650 m, 3°19.87'N, 12°45.42'E, rainforest, near the river, Uapaca sp., 17/05/2014, De Crop & Verbeken, EDC 14-284 (GENT).
Lactifluus bicapillus is recognized in the field by its yellowish-orange to dark-red cap, a concolourous or somewhat paler stipe, yellow lamellae, and unchanging white latex. L. bicapillus is placed in L. subg. Pseudogymnocarpi, L. sect. Xerampelini. Species in this section are characterized by yellowish-orange to reddish-brown caps, a palisade-like structure as pileipellis, the absence of true pleurocystidia, and generally low ornamented basidiospores (not higher than 0.2 μm) ranging from verrucose to almost completely reticulate (
Lactifluus sect. Xerampelini is exclusively known from Africa and contains six described species (Fig.
Basidiomata of described species of Lactifluus sect. Xerampelinia L. xerampelinus (EDC 11-113) bL.kivuensis (JR Z 233) cL.cf. pseudovolemus (ADK 2968) d L. rubiginosus (EDC 11-120) e L. persicinus (EDC 12-001, holotypus) f L. bicapillus (EDC 14-249, holotypus) (photographs a, d–f by E. De Crop, b by J. Rammeloo and c by A. De Kesel).
Lactifluus bicapillus differs in ecology from all but one species of L. sect. Xerampelini. Species from this section occur in woodlands, gallery forests and rainforests (
Macroscopically, L. bicapillus differs from the other species of this section by a combination of bright cap colours, which vary from dark red to bright orange near the edge, cream white lamellae and pale yellow-orange stipe colours in adult basidiocarps (Fig.
All species from L. sect. Xerampelini have ellipsoid to elongate basidiospores, with amyloid ornamentation composed of very low warts and ridges (up to 0.2 µm high) that are isolated, aligned or forming an incomplete reticulum. All seven species have long and slender basidia, mostly cylindrical and 4-spored. However, 1- and 2-spored basidia are present in L. bicapillus, L. persicinus, and L. pseudovolemus. True cystidia are absent in all species. Pleuropseudocystidia are scarce in L. bicapillus, L. persicinus, and L. kivuensis, abundant in the other species. These pleuropseudocystidia are occasionally emergent in all species; however, emergent pleuropseudocystidia were not observed in L. bicapillus. Lactifluus persicinus and L. bicapillus have a fertile lamellar edge, whilst the others have a sterile lamellar edge (or unknown in L. pseudovolemus and L. goossensiae).
All species of this section have palisade-like structures as pileipellis. Lactifluus bicapillus, L. persicinus, and L. goossensiae have a lampropalisade with thick-walled terminal elements. Lactifluus pseudovolemus has a palisade in which the elements of the pileipellis are slightly thickened. Lactifluus kivuensis, L. xerampelinus, and L. rubiginosus have a palisade to trichopalisade, with only thin-walled elements of the pileipellis. Only Lactifluus bicapillus, L. persicinus, and L. goossensiae have terminal elements that are narrow near the apex. Furthermore, L. bicapillus is the only species within this section with two types of terminal elements in the pilei- and stipitipellis.
With the finding of Lactifluus bicapillus, L. sect. Xerampelini now contains seven described species, all from sub-Saharan Africa. Together with the recently described L. persicinus (
The first author is supported by the “Special Research Fund Ghent University” (BOF, grants B/13485/01 and BOF-PDO-2017-001201). The 2012 survey in Cameroon was financially supported by the Faculty Committee Scientific Research (FCWO) of Ghent University. The 2014 survey in Cameroon was financially supported by the Research Foundation Flanders (FWO, grant V416214N) and by the King Leopold III Fund for Nature Exploration and Conservation. We express our gratitude to all who helped during fieldwork, especially to the conservators and Ecogards in post in the Dja Biosphere Reserve (from 2012 to 2014) and Mr Tchana Tchonkui Merlin. We would like to thank Viki Vandomme for conducting lab work. We thank André De Kesel and Jan Rammeloo for providing pictures of Lactifluus species. We thank the reviewers and the editor for their constructive suggestions and detailed comments on the manuscript.