Mycol Progress (2016) 15:58 DOI 10.1007/s11557-016-1198-4 ORIGINAL ARTICLE Lactarius megalopterus,anewangiocarpousspecies from a tropical rainforest in Central Africa, shows adaptations to endozoochorous spore dispersal 1,2 3,4 5 Ludwig Beenken & Moses N. Sainge & Alexander Kocyan Received: 22 December 2015 /Revised: 3 May 2016 /Accepted: 13 May 2016 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2016 Abstract A new sequestrate Lactarius species was found in a with the protection of the delicate hymenium against desicca- humid evergreen tropical rainforest dominated by Fabaceae of tion in arid habitats or against frost in cold habitats. However, the subfamily Caesalpinioideae in Cameroon, Central Africa. these cannot be the selective factors in warm and humid areas It is described here as new to science and is named Lactarius like the tropics. This controversy is exemplarily studied and megalopterus, referring to its spore ornamentation of extraor- discussed in the family of Russulaceae, especially in the genus dinarily high wings. Anatomical characters and molecular sys- Lactarius. Characters shown by the angiocarpous sporocarp tematic analyses confirm its relationship to Lactarius subge- of the new Lactarius, such as thick-walled statismospores, an nus Plinthogali. Phylogenetic analyses based on two nuclear aromatic smell and mild taste, can be interpreted as adapta- DNA regions revealed its close relationship to Lactarius tions to endozoochorous spore dispersal by mammals. angiocarpus, which is also an angiocarpous species from Therefore, here we prefer the alternative hypothesis that se- Zambia in Africa. Molecular studies have shown that tuber- questrate sporocarps are the result of adaptation to like, sequestrate sporocarps evolved independently in several endozoochorous spore dispersal. lineages of Basidiomycota. The findings of sequestrate fungi in tropical rainforests raise questions regarding the evolution- Keywords Russulaceae . Lactarius subgenus Plinthogali . ary benefit of enclosing the spore-producing hymenium. The Mycophagy . Endozoochory syndrome . Cameroon enclosure of spore-producing tissue has often been associated Introduction Section Editor: Zhu-Liang Yang The milk-cap genera Lactarius Pers. and Lactifluus (Pers.) * Ludwig Beenken Roussel show high diversity in tropical regions, especially in [email protected] Africa, where about 100 species are identified up to now (Van Rooij et al. 2003; Douanla-Meli and Langer 2009; Van de 1 ETH Zurich, Institute of Integrative Biology, Universitaetstrasse 16, Putte et al. 2009; Verbeken and Walleyn 2010; Maba et al. 8092 Zurich, Switzerland 2014) and even more species are to be expected (Verbeken 2 Present address: Swiss Federal Institute for Forest, Snow and and Buyck 2002). They are among the most important and Landscape Research WSL, Zürcherstrasse 111, diverse genera of ectomycorrhizal fungi in this region 8903 Birmensdorf, Switzerland (Verbeken and Buyck 2002; Verbeken and Walleyn 2010). 3 Tropical Plant Exploration Group (TroPEG), P.O. Box 18, Thirteen Lactarius and Lactifluus species have been reported Mundemba, Ndian, South West Region, Cameroon from Cameroon, and four of these (Lactarius desideratus 4 Department of Environmental and Occupational Studies, Faculty of Verbeken & Stubbe, L. dewevrei Douanla-Meli, L. uapacae Applied Science, Cape Peninsula University of Technology, Cape Town Campus, Keizersgracht, P.O. Box 652, Cape Town 8000, Verbeken & Stubbe, L. undulatus Verbeken) have recently South Africa been described as new to science (Verbeken et al. 2008; 5 Institute of Biochemistry and Biology, Biodiversity Research/ Douanla-Meli and Langer 2009). Until now, only two seques- Systematic Botany, University of Potsdam, Maulbeerallee 2a, trate Lactarius species were known from sub-Saharan tropical D-14469 Potsdam, Germany Africa: the secotioid Lactarius dolichocaulis (Pegler) 58 Page 2 of 10 Mycol Progress (2016) 15:58 Verbeken & Eberhardt and the angiocarpous L. angiocarpus on dried material. Spores were described and drawn after treat- Verbeken & Eberhardt (Verbeken and Walleyn 2010). Both ment with Melzer’s reagent. The measurements (without or- were collected in the relativelydryZambezianmiombowood- nament) are based on 25 observed spores. Length, width, lands (Eberhardt and Verbeken 2004;Pegler1982). In the pres- length/width ratio (Q) and volume (V) are given as mini- ent study, a third, new sequestrate Lactarius species is described mum–mean–maximum values. The volumes are calculated from a very humid tropical rainforest in Southwest Cameroon. using the formula of the rotational ellipsoid. Sequestrate sporocarps are well known from truffles and oth- Basidia were stained with ammoniacal Congo red follow- er Ascomycota, but have evolved fairly often in several clades ing brief aqueous potassium hydroxide (KOH) preparation. within the Basidiomycota as well (e.g. Bruns et al. 1989; Basidia lengths exclude sterigmata. Peridiopellis and Læssøe and Hansen 2007; Wilson et al. 2011; Smith et al. subperidial and hymenophoral tramas were studied from radi- 2015). Among Basidiomycota, sequestrate sporocarps have al hand sections in 2 % KOH. In addition, cryosections were been detected in the family Russulaceae (e.g. Miller et al. obtained from the dried basidiocarp and the adherent 2001;LebelandTonkin2007), especially in the genus rhizomorph, which had been previously macerated in aqueous Lactarius (e.g. Verbeken et al. 2014). Such an evolutionary KOH and incubated in glycerol water. The sections, 30– transition to sequestrate sporocarps enclosing the delicate hyme- 60 μm thick, were stained with cotton blue/lactic acid. The nium can be interpreted as a mechanism of protection against terminology for peridiopellis (as pileipellis) structures and desiccation in arid habitats (e.g. Thiers 1984;Brunsetal.1989) hymenial elements is according to Heilmann-Clausen et al. or against frost in cold areas (Trappe 1988;Maseretal.2008). (1998) and Verbeken and Walleyn (2010). Scanning electron However, recent records of angiocarpous fungi from humid microscopy (SEM) images were obtained for air-dried mate- tropical rainforests contradict these climate-based explanations rial after sputtering with gold. (e.g. Verbeken et al. 2014;Smithetal.2015). Hence, in tropical regions and in humid temperate climates, other factors may Molecular analysis trigger the evolution to angiocarpous sporocarps (e.g. Bougher and Lebel 2001;TrappeandClaridge2005). DNA was isolated from a small piece of gleba from the dried Aspecialkindofsequestratefungiarethosethatare fruit body using the Dynabeads DNA DIRECT Universal kit adapted to mycophagous animals that feed from the sporo- (Dynal Biotech), according to the instructions for fungal tis- carps and transport the spores within their intestines. This sue. The ITS1, 5.8S and ITS2 regions of the nuclear rDNA endozoochorous spore dispersal (endozoochory) requires the were amplified by polymerase chain reaction (PCR) using the evolution of several characters that attract feeding animals, primers ITS1F and ITS4B (Gardes and Bruns 1993). Cycle and at the same time requires that fungi develop protective sequencing reactions were carried out with primers ITS1 and features against destructive digestion of the spores (Fogel ITS4 (White et al. 1990) using the BigDye Terminator kit v3.1 and Trappe 1978; Johnson 1996; Maser et al. 2008). The (Applied Biosystems, Foster City, CA, USA). The LSU was angiocarpous sporocarp of the newly described Lactarius amplified and sequenced using the primers LR0R and LR3 has such features and may be suited to endozoochorous spore (Vilgalys and Hester 1990). Processed cycle sequencing prod- dispersal. ucts were run on an ABI PRISM 3100-Avant Genetic To elucidate diagnostic characters of this new species, we Analyzer capillary sequencer (Applied Biosystems). conducted a detailed descriptive anatomical study along with Sequences were edited with Sequencher 4.10 software a molecular systematic study of two nuclear DNA sequence (Gene Codes, Ann Arbor, MI, USA) and deposited at regions to obtain phylogenetic information in a wider GenBank. The ITS and LSU sequences were compared with Lactarius context. We also reviewed the available literature accessions deposited at GenBank by applying the Basic Local on endozoochorous spore dispersal and examined established Alignment Search Tool (BLAST) with the nucleotide search views on the evolution of this feature. option (blastn) to classify the Lactarius species. Subsequently, ITS and LSU Lactarius sequences were obtained from GenBank in order to compile independent alignments to test Materials and methods for the phylogenetic position of the new Lactarius species (for GenBank accession numbers see Fig. 1). Alignments were Morphological analysis performed using MUSCLE 3.8.31 (Edgar 2004). All of the ingroup taxa belong to Lactarius subg. Plinthogali. The non- The description of the macroscopic features is based on field Plinthogali L. hispidulus was used as outgroup (derived from observations of a fresh fruit body discovered during an expe- Verbeken et al. 2014 and Stubbe and Verbeken 2012). dition to Cameroon in 2002. The new species was collected Ambiguously aligned regions were delimited and ex- only once at the type locality, and thus all data presented here cluded from phylogenetic analyses with Gblocks version are from the type specimen. Microscopic characters are based 0.91b (Castresana 2000). Mycol Progress (2016) 15:58 Page 3 of 10 58 Lactarius