Sample collection
All sampling was approved by the University of Canberra Committee for Ethics in Animal Experimentation (approval codes CEAE 13-06, 15-06 and 20180442) and undertaken under the following state wildlife collecting licences: New South Wales P07/0007-5.0 and P18/0027-1.1; Queensland 168221, 191126 and 212524; South Australia S115 and ME9902959; Victoria RP1146 and RP1344. Fish were ethically euthanized using either AQUI-STM or dilute clove oil, snap frozen in liquid nitrogen, and deposited in the SA Museum’s Australian Biological Tissues Collection.
We sampled throughout range of H. klunzingeri for a total of 120 sites (Fig. 1, Table 1). All river names used herein are cross-referenced to site numbers in Table 1. Each molecular dataset has a different number of populations and individuals but overlap between marker types was high. The SNP dataset consisted of 106 sites and 204 individuals, allozymes had 80 sites and 233 individuals and cytbincluded 101 sites and 293 individuals (Table 1). The cytbdataset includes all H. klunzingeri sequences from Thacker et al. (2007). Two versions of the cytb dataset were constructed, one for the whole gene with 267 individuals, and a second one for the first 601 base pairs to accommodate an additional 26 individuals from four locations (marked in Table 1 with an asterisk) that provided additional phylogeographic insights within the MDB using these larger sample sizes. For ease of presentation, individuals are identified throughout by their final taxon assignment, namely pure taxa KN, KE, KS, and KW (reflecting their various compass orientations), plus assorted hybrid/admixed combinations thereof (KEm = KE from the MDB, KWm = KW from the MDB, and KSxKE = hybrids between KS and KE).