2.4. Nuclear DNA sequencing and phylogenetic analysis

In addition to the previously published mtDNA data available for a subset of specimens selected to encompass all mtDNA lineages [19] plus possible hybrid individuals (determined with a combination of morphology and mtDNA), we sequenced a 1226 bp of recombination activating gene-1 (RAG1) exon in the N-terminal domain. Oligonucleotide primer pairs used in PCR amplification and sequencing are detailed in [35,36]. Sequence chromatograms were edited using Geneious v. 10.2.2 (Biomatters Ltd) to produce a single continuous sequence for each specimen.

RAG1 was found to follow the GTR + I + G model of substitution with no partitioning schemes using the corrected Akaike information criterion (AICc) on PartitionFinder2 on the CIPRES Science Gateway [24,25]. Bayesian analysis followed the same protocol as the previous section (see above), except using a GTR + I + G model with flat priors. In addition, a maximum-likelihood analysis was run using RAxML v. 8.2.11 [37] in Geneious v. 10.2.2. For that, a GTR + I + G model was used with 10 000 bootstrap replicates to determine branch support.