Evaluating DNA barcoding criteria using African duiker antelope (Cephalophinae) as a test case

Abstract

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

A multi-locus species phylogeny of African forest duikers in the subfamily Cephalophinae: evidence for a recent radiation in the Pleistocene

“…Interestingly, Bayesian analysis of the nuclear data provides support for a sister relationship between C. nigrifrons and the C. natalensis/C. harveyi clade, indicating that C. nigrifrons and C. rufilatus may not be sister taxa, as previously mitochondrial analyses suggest [22-24]. …”

“…Past attempts to reconstruct the evolutionary history of the Cephalophinae have met with considerable challenge [22-24], likely owing to the recent and rapid radiation of this group [21,22]. Using a combination of mitochondrial and nuclear markers, the present study provides the most well supported phylogeny to date.…”

“…Inclusion of nuclear data further substantiates the lack of any clear genetic distinction between these two taxa and is consistent with a history of either recent or on-going hybridization and/or incomplete lineage sorting. Given the results of the present study, it seems unlikely that any mitochondrial or nuclear marker will be able to differentiate these two taxa, posing a challenge to the regulation of the bushmeat trade [24,47] or wildlife monitoring studies of field collected feces [23]. Further work should explore patterns of range-wide population genetic variation between these two taxa in order to better understand their species status and potential for hybridization.…”

“…The cyt b gene fragment from C. jentinki was amplified according to published primers and protocols [23]. Similarly, most (n = 31) of the COX1 sequences were previously published [24,52] and an additional five samples were amplified according to published protocols [24]. To test for the potential presence of non-functional nuclear translocated copies of mitochondrial DNA (Numts), each of the mitochondrial gene sequences were translated to amino acids in the program MEGA v3.…”

“…To date, support for these lineages has been based solely on single mitochondrial gene genealogies that may not accurately reflect the evolutionary history of this group. Furthermore, the position of the two remaining taxa, C. adersi and C. zebra , remains unresolved and appears to be highly labile between studies [22-24]. …”