MtDNA analyses
The whole and half cytb datasets consisted of 1141 and 601 base pairs for 267 and 293 individuals respectively. Maximum Likelihood (ML) was run on the two cytb datasets with RAxML producing trees with likelihood scores of -3145.296216 and -1595.307571, and the rapid bootstrap search for both analyses finishing at 650 and 500 replicates respectively (simplified tree for whole cytb in Fig. 6; detailed trees for both whole and half cytb in Figs. S3 and S4 respectively).
For the full cytb dataset, the deeper phylogenetic relationships were moderately well-supported and largely similar to those found for SNPs. The KN clade was sister to the KE clade and both were sister to a composite KS/KW clade, comprising three distinctive sub-lineages referable to taxon KS plus well-supported, sister sub-lineages for KW and KWm (Figs. 6a, S3). Consistent with their hybrid status, KSxKE individuals displayed either KS-derived or KE-derived haplotypes (ratio 14:15, Fig. S4). For the non-hybrid taxa, an individual’s mtDNA clade membership was concordant with their SNP/allozyme primary lineage identification in all instances apart from six KS fish, all from three northern MDB rivers (sites 65, 66, and 68; Gwydir, Namoi, and Macquarie Rivers).
Mapping the distribution of the major cytb lineages (Fig. 6) clearly demonstrates that KE-derived haplotypes have intruded into the KSxKE hybrid zones as identified using our nuclear datasets (Fig. 1 and shaded in Fig. 6) plus are present in the pure KS populations from some upper MDB rivers. It also reveals that haplotypes from two rare KS-lineages, otherwise characteristic of the Border Rivers in the upper MDB (sites 59, 62, and 63) have also spread across the drainage divide into the upper Clarence River (site 41) and far downstream into the KSxKE hybrid zone in the lower Murray.