Genetic analyses of a Southern African millipede (Bicoxidens Attems 1928)

Abstract

Bicoxidens Attems, 1928 is an afrotropical millipede in the family Spirostreptidae which constitutes nine species. Based on the analysis of the male copulatory organs known as gonopods, Bicoxidens is monophyletic. However, gonopod morphology is central to millipede taxonomy, examination of gonopod morphology alone may not elucidate hidden genetic diversity thereby underestimating species richness in Bicoxidens. Analysis of genetic data as an additional source of taxonomic data has been noted to reveal genetic variation and flagged hidden species within millipedes. Hence there is a need to revise the morphological species definitions and phylogeny of Bicoxidens using both mitochondrial and nuclear DNA sequences. Furthermore, low vagility and habitat discontinuity may limit gene flow among Bicoxidens populations resulting differentiation and high genetic variation. In this regard, the sequences of the mitochondrial cytochrome oxidase 1 (COI) and 16S rRNA genes were used to delimit species, unravel hidden species and test the monophyly of Bicoxidens. In addition, the phylogeography and population genetic structure of B. flavicollis was investigated based on COI and 16S rRNA sequences. Genetic distances were computed using COI. Phylogenetic inferences were done based on Maximum likelihood and Bayesian inference analyses of the COI and 16S rRNA sequences individually and with the combined data set (COI+16S rRNA). The phylogeography and population genetic structure of B. flavicollis was inferred based on genetic diversity indices, population genetic differentiation estimate PhiPT, haplotype network analyses and maximum likelihood analyses of both COI and 16S rRNA. Bayesian population structure analyses was done using the COI data set only. Furthermore, Mantel’s isolation by distance among B. flavicollis specimens was tested. Interspecific genetic distances (> 0.075) based on the Juke-Cantor model supported the morphological species definitions in Bicoxidens. However, high intraspecific sequence differences in B. flavicollis (> 0.14), B. friendi (0.084) and B. brincki (> 0.188) suggest the presence of cryptic species. The possible presence of cryptic species was supported by the phylogenic analyses which recovered a paraphyletic Bicoxidens phylogram with B. flavicollis, B. brincki and B. friendi exhibiting divergent lineages. Bicoxidens flavicollis populations exhibited high genetic diversity and strong population differentiation which suggests restricted gene flow. Haplotype network and Maximum likelihood analyses revealed limited sharing of haplotypes and cryptic species in

populations at Mazowe and Nyanga, respectively. Bicoxidens flavicollis populations were grouped in two four genetic clusters. Isolation by distance was insignificant among B. flavicollis specimens. The results support the utility of both COI and 16S rRNA in species delimitation in Bicoxidens. There is a growing body of evidence which also suggests presence of hidden species in B. flavicollis, B brincki and B friendi. Furthermore effort to uncover more hidden species should be made to elucidate the species richness before conservation strategies can be implemented.