Four of the nine elements analyzed showed a significant difference in concentration (ug·g⁻¹) between the otolith core and edge for wild-caught fish from the Lachlan River (²⁴Mg, ⁸⁶Sr, ⁵⁵Mn, and ⁶⁶Zn; Student’s t-test p-values all < 0.01) (Figure 2, panel LL_edge vs. LL_core and UL_edge vs. UL_core).

The NMDS clearly separated the chemical signatures from the edges of otoliths from fish from the Lachlan River and those of the hatchery (YOY) samples (stress: 0.07) (Figure 3a). A PERMANOVA test with 999 bootstrapping using the Bray–Curtis dissimilarity index found a significant difference between the clusters (p-value = 0.001, F model = 76.35, ANOVA for homogeneity of multivariate dispersion p = 0.39), indicating a unique chemical signature for each locality. The analyses were then repeated on the lower Lachlan and upper Lachlan edge and core samples separately (Figure 3b,c). For the lower Lachlan, this revealed two distinct groups comprising core and edge samples (Figure 3b). The signature from the hatchery reference samples was more similar to the lower Lachlan core chemical signature than the edge signature of the lower Lachlan River individuals. In addition, a small group of individuals displayed similar core and edge signatures (Figure 3b), suggesting wild-born individuals from the lower Lachlan. Cluster analysis using elements significantly contributing to the core and edge variation (²⁴Mg, ⁸⁶Sr, ⁵⁵Mn, and ⁶⁶Zn) identified three primary groups (clusters) representing predominantly edge, core, and hatchery signatures. This analysis clearly identified six M. ambigua individuals for which core signatures were clustered with the edge signature indicating natural recruitment in the lower Lachlan catchment. Within the core and hatchery (YOY) groups, four individuals displayed core signatures that clustered with the hatchery reference group while the remaining individuals displayed core signatures that grouped separately (Figure 3d).

In the upper Lachlan River samples, the NMDS revealed three distinct groups comprising core, edge, and hatchery reference samples, but the cluster analysis using only those elements significantly contributing to the core and edge variation suggests more complex groupings within the data set (Figure 3e). The cluster analysis identified two primary clusters separating the core and edge measurements of the Lachlan River samples from the hatchery reference collections. Five tentative sub-clusters belonging to two major groups of Lachlan River samples and hatchery samples were evident; however, the groupings were less distinct within the clusters (Figure 3e). The results suggest none of the upper Lachlan samples were wild-born, while five individuals displayed a core signature that clustered with the hatchery reference group.

 

Our analysis revealed the presence of both hatchery-bred stocked and wild-born M. ambigua from the Lachlan River. Among the 44 fish collected from Lachlan River, a total of six M. ambigua were identified as wild-born fish (14%), while the rest of them were either hatchery-bred stocked fish or from unknown sources (Figure 4). The observation of annual rings in the otolith revealed that all six wild-born M. ambigua, sampled from the lower Lachlan, were spawned in 2002, 2007, 2009, 2014 (one fish each year), and 2010 (two fish). A comparison between the daily discharge data from the lower Lachlan (Figure 4) revealed no clear association between high flow events and the spawning year of the six wild-born fish.

The Diversity Arrays Technology (DArT) pipeline scored 5207 single nucleotide polymorphisms (SNPs) and 6956 SilicoDArT (presence-absence) markers after filtering based on polymorphic information content (>0.025). For the present study, we filtered out 2124 SNP markers based on the call ratio (0.90) and reproducibility average (0.90), resulting in a total of 3083 SNPs (null allele = 2.1%). Further filtering based on the Hardy–Weinberg equilibrium (HWE) was performed to remove markers under selection. Finally, 1481 SNPs were used for the present study. The same criteria (call ratio and polymorphic information content) were also used to filter out low-quality SilicoDArT markers. After filtering, 2902 (nNull allele = 5.7%) SilicoDArT markers were used for further analysis. Filtering for SilicoDArT loci under selection was not performed, as the allelic form of the loci is unknown (presence and absence of restriction fragment).

 

Despite a small cluster of hatchery reference individuals (hatchery YOY) (Figure 5a), the principal component analysis (PCA) suggests no specific clustering using SNP markers. High similarity among the Lachlan River samples and hatchery YOY and a lack of genetic structure means that the SNP markers could not conclusively distinguish the wild-born fish from the hatchery-bred stocked or unknown-source fish. We also performed a principal component analysis using only Lachlan River samples testing for differences between wild-born fish identified a priori and the rest from the otolith data (Figure 5b) and could find no genetic differences. The principal component analysis using SilicoDArT markers found a similar result, although it did indicate differences between the lower Lachlan fish (widely spread across PC1/Dim1 in the PCA plot) and upper Lachlan fish (a single cluster, which included the hatchery-bred YOY fish) (Figure 5c,d).