Haplotype networks
DnaSP was also used to prepare the sequence data for input into Network v5.0.0.0 in order to construct haplotype networks. The 16S data was exported in Rdf format with gaps/missing data not considered, whereas the coding regions (CO1 and EF-1ɣ) were exported with gaps considered. In order to accurately estimate the relationship between closely related Stasimopusindividuals of the same species (which have not yet undergone speciation), a haplotype network was constructed in Network. The Network software uses the method of Maximum Parsimony to select the shortest and least complex tree. The haplotype network was constructed using the median-joining (MJ) network algorithm. This allows for multistate data (A, T, G, C or N at any nucleotide position) . All settings for network calculation were left on default. The post-processing option of Maximum parsimony was used in order to remove all unnecessary median vectors.
Pie charts of haplotypes were plotted onto a map of the Karoo region using the coordinates of each locality. This allowed for the visualisation of the different haplotypes in geographic space.