Revealing hidden species diversity in closely related species using nuclear SNPs, SSRs and DNA sequences - a case study in the tree genus Milicia

Background: 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. Results: 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. Conclusions: 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

Development of nuclear SNP markers for the timber tracking of the African tree species Sapelli, Entandrophragma cylindricum

We describe the development of new nuclear SNP markers for the genetic timber tracking of the geographical origin of Sapelli, Entandrophragma cylindricum (Meliaceae). Restriction associated DNA sequencing (RADseq) of two reference individuals yielded 1131 putative SNPs. Among those, 131 were selected to design four MassARRAY multiplexes and screened at 178 individuals. Seventy-two loci were selected for further use in genetic tracking

A set of nuclear SNP loci derived from single sample double digest RAD and from pool sequencing for large-scale genetic studies in Fagus sylvatica

Putative SNP loci derived from ddRADseq of 54 samples of European beech Fagus sylvatic

SNP_and_SSR_Milicia_genotypes

This file presents the genotypes (at 7 nuclear SSR and 62 nuclear SNP) of Milicia samples collected in West Africa

Chromatograms of length-variant heterozygotes in Milicia at At103

This Sequencher file contains, for each length-variant heterozygote, (i) the forward and reverse At103 chromatograms which were assembled into contigs: the double peaks were called either using the "Call secondary function" or manually; (ii) the two haplotypes (a and b) reconstructed by combining the information carried by the forward and reverse chromatograms using the program Champuru (http://jfflot.mnhn.fr/champuru/). These samples of the genus Milicia were sequenced at the nuclear region At103

DNA taxonomy in the timber genus Milicia: evidence of unidirectional introgression in the West African contact zone

DNA-based techniques are helpful in characterising hybridisation patterns in plant species. To be efficient in disentangling species boundaries and interspecific gene flow, it is recommended to combine various methodologies and types of markers. Here, we used different analytical tools (algorithms implemented in Structure, Tess, NewHybrids and HIest, and the haploweb approach) and three nuclear genetic markers (7 nuclear simple sequence repeat loci (SSRs), 62 single-nucleotide polymorphism loci (SNPs) and a single-copy gene region, At103) to revisit hybridisation patterns in the commercially important African tree genus Milicia. Samples were collected in the natural ranges of Milicia regia and Milicia excelsa in West Africa. Using real data sets, simulated purebreds and hybrid genotypes, we found that SNPs yielded results more consistent than SSRs; outputs from the Bayesian and maximum-likelihood analyses differed significantly using the SSRs, whereas they were perfectly congruent using SNPs. A proportion of 12.4% hybrids were detected amongst the SNP genotype samples. A haploweb analysis of At103 gene sequences confirmed the existence of interspecific hybrids. There was also a clear evidence of advanced generations of hybrids (backcrossed individuals) but only towards M. regia. Although more investigation is required for understanding the mechanisms responsible for this asymmetric introgression, we suggest that it may be due to the differences in flowering time between species and between sexes, combined with a maternal inheritance of flowering time.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Data from: DNA taxonomy in the timber genus Milicia: evidence of unidirectional introgression in the West African contact zone

DNA-based techniques are helpful in characterising hybridisation patterns in plant species. To be efficient in disentangling species boundaries and interspecific gene flow, it is recommended to combine various methodologies and types of markers. Here, we used different analytical tools (algorithms implemented in Structure, Tess, NewHybrids and HIest, and the haploweb approach) and three nuclear genetic markers (7 nuclear simple sequence repeat loci (SSRs), 62 single-nucleotide polymorphism loci (SNPs) and a single-copy gene region, At103) to revisit hybridisation patterns in the commercially important African tree genus Milicia. Samples were collected in the natural ranges of Milicia regia and Milicia excelsa in West Africa. Using real data sets, simulated purebreds and hybrid genotypes, we found that SNPs yielded results more consistent than SSRs; outputs from the Bayesian and maximum-likelihood analyses differed significantly using the SSRs, whereas they were perfectly congruent using SNPs. A proportion of 12.4% hybrids were detected amongst the SNP genotype samples. A haploweb analysis of At103 gene sequences confirmed the existence of interspecific hybrids. There was also a clear evidence of advanced generations of hybrids (backcrossed individuals) but only towards M. regia. Although more investigation is required for understanding the mechanisms responsible for this asymmetric introgression, we suggest that it may be due to the differences in flowering time between species and between sexes, combined with a maternal inheritance of flowering time

Development of new SNP and INDEL loci for the valuable African timber species Lophira alata

The timber of the species Lophira alata (azobe) is very popular for outdoor constructions, which favours its overexploitation and illegal logging. We sampled individuals from Liberia, Ivory Coast, Ghana, Nigeria, Cameroon, Gabon, Congo Brazzaville and Republic Democratic of Congo to discover new nuclear and plastidial SNP and INDEL loci through restriction associated DNA sequencing (RADSeq) and low coverage MiSeq genome sequencing. From an initial set of 397 loci, a final set of 126 loci was selected for timber tracking purposes.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Complete Chloroplast Genome Sequences of Four Meliaceae Species and Comparative Analyses

The Meliaceae family mainly consists of trees and shrubs with a pantropical distribution. In this study, the complete chloroplast genomes of four Meliaceae species were sequenced and compared with each other and with the previously published Azadirachta indica plastome. The five plastomes are circular and exhibit a quadripartite structure with high conservation of gene content and order. They include 130 genes encoding 85 proteins, 37 tRNAs and 8 rRNAs. Inverted repeat expansion resulted in a duplication of rps19 in the five Meliaceae species, which is consistent with that in many other Sapindales, but different from many other rosids. Compared to Azadirachta indica, the four newly sequenced Meliaceae individuals share several large deletions, which mainly contribute to the decreased genome sizes. A whole-plastome phylogeny supports previous findings that the four species form a monophyletic sister clade to Azadirachta indica within the Meliaceae. SNPs and indels identified in all complete Meliaceae plastomes might be suitable targets for the future development of genetic markers at different taxonomic levels. The extended analysis of SNPs in the matK gene led to the identification of four potential Meliaceae-specific SNPs as a basis for future validation and marker development