Small to medium-sized central African forest artiodactyls constitute a diverse yet heavily hunted group composed primarily of species within the genera Cephalophus, Neotragus, Tragelaphus and Hyemoschus. Of these genera, Cephalophus is the richest with as many as seven sympatric species known to occur in central African forests. However, differentiating species from their faeces or from tissue where the whole carcass is unavailable is very difficult. In order to develop a robust molecular diagnostic for species identification, a database of mitochondrial cytochrome b (553 bp) and control region ($675 bp) sequences was compiled from all forest Cephalophus species and other similarly sized, sympatric Tragelaphus, Neotragus and Hyemoschus species. Reference phylogenies from each marker were then used to recover the identity of sequences obtained from unknown faecal samples collected in the field. Results were then compared to determine which region best recovered species identity with the highest statistical support. Restriction fragment length polymorphisms (RFLPs) were also assessed as an alternative method for rapid species identification. Of the methods examined, tree-based analyses built on a geographically comprehensive database of control region sequences was the best means of reliably recovering species identity from central African duikers. However, three sister taxa appear indistinguishable (Cephalophus callipygus, Cephalophus ogilbyi and Cephalophus weynsi) and not all species were monophyletic. This lack of monophyly may be due to incomplete lineage sorting commonly observed in recently derived taxa, hybridization or the presence of nuclear translocated copies of mitochondrial DNA. The high level of intra-specific variation and lack of robust species-specific diagnostic sites made an RFLP-based approach to duiker species identification difficult to implement. The tree-based control region diagnostic presented here has many important applications including fine-scale mapping of species distributions, identification of confiscated tissue and environmental impact assessments.

BackgroundDuikers in the subfamily Cephalophinae are a group of tropical forest mammals believed to have first originated during the late Miocene. However, knowledge of phylogenetic relationships, pattern and timing of their subsequent radiation is poorly understood. Here we present the first multi-locus phylogeny of this threatened group of tropical artiodactyls and use a Bayesian uncorrelated molecular clock to estimate divergence times.ResultsA total of 4152 bp of sequence data was obtained from two mitochondrial genes and four nuclear introns. Phylogenies were estimated using maximum parsimony, maximum likelihood, and Bayesian analysis of concatenated mitochondrial, nuclear and combined datasets. A relaxed molecular clock with two fossil calibration points was used to estimate divergence times. The first was based on the age of the split between the two oldest subfamilies within the Bovidae whereas the second was based on the earliest known fossil appearance of the Cephalophinae and molecular divergence time estimates for the oldest lineages within this group. Findings indicate strong support for four major lineages within the subfamily, all of which date to the late Miocene/early Pliocene. The first of these to diverge was the dwarf duiker genus Philantomba, followed by the giant, eastern and western red duiker lineages, all within the genus Cephalophus. While these results uphold the recognition of Philantomba, they do not support the monotypic savanna-specialist genus Sylvicapra, which as sister to the giant duikers leaves Cephalophus paraphyletic. BEAST analyses indicate that most sister species pairs originated during the Pleistocene, suggesting that repeated glacial cycling may have played an important role in the recent diversification of this group. Furthermore, several red duiker sister species pairs appear to be either paraphyletic (C.callipygus/C. ogilbyi and C. harveyi/C. natalensis) or exhibit evidence of mitochondrial admixture (C. nigrifrons and C. rufilatus), consistent with their recent divergence and/or possible hybridization with each other.ConclusionsMolecular phylogenetic analyses suggest that Pleistocene-era climatic oscillations have played an important role in the speciation of this largely forest-dwelling group. Our results also reveal the most well supported species phylogeny for the subfamily to date, but also highlight several areas of inconsistency between our current understanding of duiker taxonomy and the evolutionary relationships depicted here. These findings may therefore prove particularly relevant to future conservation efforts, given that many species are presently regulated under the Convention for Trade in Endangered Species.

African duikers in the subfamily Cephalophinae (genera Cephalophus, Philantomba and Sylvicapra) constitute an important target for DNA barcoding efforts because of their importance to the bushmeat trade and protection under the Convention for International Trade in Endangered Species (CITES). Duikers also make a challenging test case of barcoding methods due to their recent diversification, substantial intra-specific genetic variation and high species richness. However, no study to date has evaluated how well DNA barcoding methods can be used to delineate all of the taxa within this group. To address this question, cytochrome c oxidase subunit 1 (COX1) sequences from all eighteen species within this subfamily and an outgroup taxon (genus Tragelaphus) were used to build a neighbor-joining tree, identify species-specific diagnostic synapomorphies, and determine whether species exceed a given pair-wise genetic distance threshold commonly employed in DNA barcoding studies. Tree-based analyses of the data indicate that several species within two clusters of closely related taxa consistently failed to form reciprocally monophyletic clades and similarly lack species-specific synapomorphies. Furthermore, one additional taxon failed to constitute a diagnosable clade and another occupied an unresolved position in the tree. Of the two genetic distance criteria evaluated, the 3% threshold was far more effective in delimiting species than a threshold level based on the ratio of inter-to intra-specific distances. However, neither approach could effectively delineate all sister species. While the taxonomy of this group might be open to question, the fact that barcodes consistently failed to differentiate several currently recognized sister taxa challenges the routine application of this approach in forensic studies of duiker species. Future barcoding work of this group should always include a complete taxonomic sampling and strive to include a broader geographic sampling of sequence diversity than has been carried out to date. Lastly, this work highlights the need to re-examine