Mitochondrial Sequencing and haplotype network
Mitochondrial DNA haplotypes were obtained from a subset of 67
individuals from the five genetic clusters. PCR reactions were performed
using a Taq-Pro COMPLETE kit (Denville Scientific, CB4065-4) targeting a
partial region of the COI gene with the Lep50 primer set from Folmer et
al. (1994) and the thermocycler profile from Herbert et al. (2003). PCR
products were cleaned using an EXOSAP-IT kit (ThermoFisher, 78201.1.ML)
and prepared for sequencing using a BigDye Terminator v.3.1 Cycle
Sequencer Kit (Applied Biosystems, 4337454). Sanger sequencing was done
using an Applied Biosystems 3500 Genetic Analyzer. Chromatograms were
cleaned and aligned using the program Geneious v.9.1 (Kearse et al.,
2012).
A haplotype network analysis was conducted using the 67 COI sequences
obtained in this study combined with 218 C. variipennis complex
sequences previously collected (M. Hopken unpublished data). Sequences
were aligned in MEGA v.10.1.8 (Kumar, Stecher, Li, Knyaz, & Tamura,
2018) and trimmed to 546 bp to ensure all sequences contained identical
lengths. A median-joining analysis was performed using NETWORK v.5.0.1.0
(Bandelt, Forster, & Rohl, 1999). Specimens collected in this study
were assigned a color based on the results from the SNP clustering
analyses while the remaining samples were left unassigned. All
individuals were used to calculate the mean uncorrectedp- divergence between and within the different groupings inferred
from the haplotype network using MEGA.