Genetic diversity of populations of a Southern African millipede, Bicoxidens flavicollis (Diplopoda, Spirostreptida, Spirostreptidae)

Abstract

The African millipede genus Bicoxidens is endemic to Southern Africa, inhabiting a variety of regions ranging from woodlands to forests. Nine species are known within the genus but Bicoxidens flavicollis is the most dominant and wide spread species found across Zimbabwe. Bicoxidens flavicollis individuals have been found to express phenotypic variation in several morphological traits. The most commonly observed body colours are brown and black. In the Eastern Highlands of Zimbabwe body colour ranges from orange- yellow to black, individuals from North East of Harare have a green-black appearance and a range in size (75–110 mm). There is disparity in body size which has been noted with individuals ranging from medium to large and displaying variation in the number of body rings. Although much morphological variation has been observed within this species, characterization based on gonopod morphology alone cannot distinguish or define variation between phenotypically distinct individuals. Morphological classification has been found to be too inclusive and hiding significant genetic variation. Taxa must be re-assessed with the implementation of DNA molecular methods to identify the variation between individuals. This study aimed to detect genetic divergence of B. flavicollis due to isolation by distance of populations across Zimbabwe. The mitochondrial DNA 16S and 12S rRNA genes were used to detect levels of genetic variation as mitochondrial markers express high variability making them suitable for phylogenetic studies. Sequence analysis of the 16S rRNA gene resulted in the generation of 22 haplotypes, derived from 42 sequences with strong haplotype diversity (Hd > 0.9). Analysis of Molecular Variance (AMOVA) analysis determined that variation among the populations was significantly greater (> 80 %) than the variation occurring within populations (< 12 %). A high fixation index (FST = 0.88229) indicated a high level of population genetic differentiation. With analysis of the 16S rRNA gene, B. flavicollis individuals demonstrated both distinctive phylogeographic diversity and genetic similarity for specific regions within Zimbabwe. Phylogenetic analyses using the 12S rRNA gene provided evidence of a more distinct genetic structure between localities. Nineteen haplotypes were derived from 19 sequences, which indicated a genetically distinct population structure (Hd = 1.000). The AMOVA analysis demonstrated that variation among the populations was greater (> 60 %) than the variation occurring within populations (< 40 %), although both were quite high. A low fixation index (FST = 0.37466) suggests a predominantly homozygous population structure. Both genes indicated distinctly structured populations, whilst the 16S rRNA also suggested the existence of closely clustered populations based on PCoA analyses, which is further supported by the presence of admixed haplotypes. The results are significant for B. flavicollis as a genetically diverse species. The findings of this study can be considered for future comparative research within the genus Bicoxidens or against other geographically distant genera. Additional markers such as those of nuclear origin, can be used along with mitochondrial markers to investigate and identify more diplopods which exhibit this level of genetic divergence although belonging to a single species. Acquired knowledge and understanding from phylogeographic studies will provide researchers with greater taxonomic awareness.