Lactifluusbicapillus (Russulales, Russulaceae), a new species from the Guineo-Congolian rainforest

Abstract 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 LactifluussectionXerampelini and we formally describe it here as Lactifluusbicapillussp. nov.

Within Central African rainforests, the ECM Russulaceae genera Russula Pers. and Lactifluus (Pers.) Roussel are abundant (Douanla-Meli and Langer 2009;Verbeken and Buyck 2002;Verbeken et al. 2008;Verbeken and Walleyn 2010). The milkcap genus Lactifluus is mainly distributed in the tropics ). It is a species-rich genus with about 160 species distributed worldwide, of which the majority is found in tropical Asia (Le et al. 2007b;Stubbe et al. 2010; Van de Putte et al. 2010), tropical Africa ( Van de Putte et al. 2009;Verbeken and Walleyn 2010;De Crop et al. 2012, 2016Maba et al. 2014Maba et al. , 2015aDelgat et al. 2017;De Lange et al. 2018) and the Neotropics (Henkel et al. 2000;Miller et al. 2002;Smith et al. 2011;. The genus is relatively understudied and many species remain undescribed due to this mainly tropical distribution. Furthermore, the genus is known for its many species complexes with morphologically cryptic species ; Van de Putte et al. 2010De Crop et al. 2014;. About 20 Lactifluus species are known from the rainforests of Central Africa (Verbeken and Walleyn 2010). The actual diversity is expected to be higher for several reasons: (i) the ECM flora is present in most parts of the tropical African rainforest, (ii) most countries in the region are understudied due to difficult political situations or challenging sampling conditions, (iii) seasonality in the rainforest is less pronounced, which makes it difficult to assess the exact fruiting period of these fungi and the fruiting of fungi can be missed during short sampling periods, and (iv) Lactifluus is known for its morphologically cryptic diversity with several species complexes occurring. Traditional species descriptions were often based on morphology and this morphologically cryptic diversity makes it difficult to correctly assess the number of species based on morphology alone.
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 De , based on four nuclear genes, revealed that this species is new to science. A preliminary microscopic study confirmed the deviating morphology of the Cameroonian collections and a more detailed study of all available material was initiated. In this study, molecular and morphological examinations were performed, the collections were compared with closely related species, and a new species, Lactifluus bicapillus, was described based on these results.

Sampling
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).

Morphology
Macroscopic features were all based on fresh material described in the field. Colour codes refer to Kornerup and Wanscher (1978). Microscopic features were studied from dried material. Morphological terminology followed Verbeken and Walleyn (2010). Elements of the pileipellis and hymenium were mounted in Congo Red in L4. Sections of the pileipellis and stipitipellis were first mounted in 10% KOH to enhance cell expansion and then mounted in Congo Red dissolved in water. Basidium length excludes sterigmata length. Measurements are given as MIN-MAX, except for basidiospores. Basidiospores were measured in side view in Melzer's reagent, excluding the ornamentation, and measurements are given as described in Nuytinck and Verbeken (2005): (MIN) [Ava −2 × SDa] -Ava -Avb -[Avb + 2 × SDb] (MAX), in which Ava/b = lowest/highest mean value for the measured collections, SDa/b = standard deviation of the lowest/highest mean value. MIN/MAX = lowest/highest value measured and only given when they exceed [Ava −2 × SDa] or [Avb + 2 × SDb] respectively. Q stands for 'quotient length/width' and is given as MINQ -Qa -Qb -MAXQ, in which Qa/b = lowest/highest mean quotient for the measured specimens, MIN/MAXQ = minimum/maximum value over the quotients of all available measured basidiospores. Line drawings were made with the aid of a drawing tube at the original magnifications: 6000 × for basidiospores (Zeiss axioscop 2 microscope), 1000 × for individual elements and sections (Olympus CX31 microscope).

Phylogenetic analysis
DNA was extracted using the CTAB extraction protocol described in Nuytinck and Verbeken (2003). Protocols for PCR amplification follow Le et al. (2007a). Two nuclear markers that were previously shown to be informative within this subgenus (De Table 1. 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. 1).

Species
Voucher collection (herbarium) Country ITS accession no.

Species
Voucher collection ( Crop et al. 2017) were used: (1) the internal transcribed spacer region of ribosomal DNA (ITS), comprising the ITS1 and ITS2 spacer regions and the ribosomal gene 5.8S, using primers ITS-1F and ITS4 (Gardes and Bruns 1993;White et al. 1990) and (2) a part of the ribosomal large subunit 28S region (LSU), using primers LR0R and LR5 (Moncalvo et al. 2000). 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 De . Furthermore, sequences were compared to sequences in the Unite database using Blastn (Abarenkov et al. 2010). One environmental sequence was found within the same Species Hypothesis and was added to the dataset. The outgroup consisted of four species of L. subg. Lactifluus (Table 1).
Sequences were aligned using the online version of the multiple sequence alignment program MAFFT v. 7 (Katoh and Standley 2013), using the E-INS-I strategy.
Trailing ends of the alignment were trimmed and sequences were manually edited when necessary in MEGA 6 (Tamura et al. 2013). The alignment can be acquired from the first author and TreeBASE (S22916, http://purl.org/phylo/treebase/phylows/study/TB2:S22916).
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 (Stamatakis 2014), where a ML analysis was combined with the Rapid Bootstrapping algorithm with 1000 replicates under the GTRCAT option (Stamatakis et al. 2008). All analyses were performed on the CIPRES Science Gateway (Miller et al. 2010).

Discussion
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 palisadelike 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 bicapillus perfectly concurs with these morphological characteristics, providing additional support for its placement in L. sect. Xerampelini.
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 (Verbeken and Walleyn 2010). Lactifluus xerampelinus and L. rubiginosus are found in miombo woodland in East Africa, while L. goossensiae is known from both Sudanian woodland and Central African gallery forests. Lactifluus persicinus and L. pseudovolemus occur in West African gallery forests. Both L. kivuensis and L. bicapillus are found in the Guineo-Congolian rainforest, associated with Gilbertiodendron dewevrei and Uapaca species.
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. 5).
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 (Delgat et al. 2017), L. bicapillus was found during two sampling expeditions in Cameroon. Even though those expeditions only covered a small area of the Guineo-Congolian rainforest and gallery forests, we collected at least five species new to science ). This highlights the large Lactifluus diversity in Africa, with many areas still unexplored and probably many new species still to be found.