title: Revealing hidden species diversity in closely related species using nuclear SNPs, SSRs and DNA sequences - a case study in the tree genus Milicia.
abstract: Species delimitation in closely related plant taxa can be challenging because (i) reproductive barriers are not always congruent with morphological differentiation, (ii) use of plastid sequences might lead to misinterpretation, (iii) rare species might not be sampled. We revisited molecular-based species delimitation in the African genus Milicia, currently divided into M. regia (West Africa) and M. excelsa (from West to East Africa). We used 435 samples collected in West, Central and East Africa. We genotyped SNP and SSR loci to identify genetic clusters, and sequenced two plastid regions (psbA-trnH, trnC-ycf6) and a nuclear gene (At103) to confirm species' divergence and compare species delimitation methods. We also examined whether ecological niche differentiation was congruent with sampled genetic structure.
  
  
  
  West African M. regia, West African and East African M. excelsa samples constituted three well distinct genetic clusters according to SNPs and SSRs. In Central Africa, two genetic clusters were consistently inferred by both types of markers, while a few scattered samples, sympatric with the preceding clusters but exhibiting leaf traits of M. regia, were grouped with the West African M. regia cluster based on SNPs or formed a distinct cluster based on SSRs. SSR results were confirmed by sequence data from the nuclear region At103 which revealed three distinct 'Fields For Recombination' corresponding to (i) West African M. regia, (ii) Central African samples with leaf traits of M. regia, and (iii) all M. excelsa samples. None of the plastid sequences provide indication of distinct clades of the three species-like units. Niche modelling techniques yielded a significant correlation between niche overlap and genetic distance.
  
  
  
  Our genetic data suggest that three species of Milicia could be recognized. It is surprising that the occurrence of two species in Central Africa was not reported for this well-known timber tree. Globally, our work highlights the importance of collecting samples in a systematic way and the need for combining different nuclear markers when dealing with species complexes. Recognizing cryptic species is particularly crucial for economically exploited species because some hidden taxa might actually be endangered as they are merged with more abundant species.
authors.affiliation: Nature + asbl / TERRA Research Centre, Central African Forests, Gembloux Agro-Bio Tech, University of Liege, Passage des Déportés 2, 5030, Gembloux, Belgium. k.dainou@natureplus.be.|Université d'Agriculture de Kétou, BP 43, Kétou, Benin. k.dainou@natureplus.be.
authors.forename: Kasso
authors.identifier: 0000-0003-2486-1895
authors.initials: K
authors.lastname: Daïnou
authors.affiliation: Thünen Institute of Forest Genetics, Sieker Landstrasse 2, 22927, Grosshansdorf, Germany.
authors.forename: Céline
authors.initials: C
authors.lastname: Blanc-Jolivet
authors.affiliation: Thünen Institute of Forest Genetics, Sieker Landstrasse 2, 22927, Grosshansdorf, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Degen
authors.affiliation: Kenya Forestry Research Institute, Biotechnology Section, P. O. Box 20412-00200, Nairobi, Kenya.
authors.forename: Priscilla
authors.initials: P
authors.lastname: Kimani
authors.affiliation: IRAF/CENAREST, BP 842 Gros-bouquet, Libreville, Gabon.
authors.forename: Dyana
authors.initials: D
authors.lastname: Ndiade-Bourobou
authors.affiliation: TERRA Research Centre, Central African Forests, Gembloux Agro-Bio Tech, University of Liege, Passage des Déportés 2, 5030, Gembloux, Belgium.
authors.forename: Armel S L
authors.initials: AS
authors.lastname: Donkpegan
authors.affiliation: TERRA Research Centre, Central African Forests, Gembloux Agro-Bio Tech, University of Liege, Passage des Déportés 2, 5030, Gembloux, Belgium.
authors.forename: Félicien
authors.initials: F
authors.lastname: Tosso
authors.affiliation: Evolutionary Biology and Ecology - CP 160⁄12, Faculté des Sciences, Université Libre de Bruxelles, Av. F. Roosevelt 50, 1050, Brussels, Belgium.
authors.forename: Esra
authors.initials: E
authors.lastname: Kaymak
authors.affiliation: Service of Wood Biology, Royal Museum for Central Africa, Tervuren, Belgium.
authors.forename: Nils
authors.initials: N
authors.lastname: Bourland
authors.affiliation: TERRA Research Centre, Central African Forests, Gembloux Agro-Bio Tech, University of Liege, Passage des Déportés 2, 5030, Gembloux, Belgium.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Doucet
authors.affiliation: Evolutionary Biology and Ecology - CP 160⁄12, Faculté des Sciences, Université Libre de Bruxelles, Av. F. Roosevelt 50, 1050, Brussels, Belgium.
authors.forename: Olivier J
authors.initials: OJ
authors.lastname: Hardy
mesh_terms: D000350:Africa, Central; D000351:Africa, Eastern; D000354:Africa, Western; D018744:DNA, Plant; D040342:Genetic Structures; D005838:Genotype; D018895:Microsatellite Repeats; D005810:Multigene Family; D010802:Phylogeny; D020641:Polymorphism, Single Nucleotide; D019685:Rosales / Q000145:classification / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D061350:Sympatry; D014197:Trees / Q000145:classification / Q000235:genetics*
chemical_list: D018744:DNA, Plant
keywords: Cryptic species; DNA sequences; Microsatellites; Milicia; SNP; Species delimitation; Tropical forests