The global freshwater fish fauna is severely affected by numerous anthropogenic stressors such as habitat modification, fragmentation, invasion, overfishing, pollution, and climate change. Stock enhancement of wild fisheries using hatchery-bred fish is a widespread practice in freshwater fisheries, mostly for recreational fishing as well as conservation action. Although stock enhancement has been shown to have positive impacts on fish abundance, a number of risk factors have also been identified. These include increased competition within the population, disease, displacement of wild stock, alterations in sex ratios, and genetic consequences such as reduced heterozygosity, altered genetic structure, and reduced effective population size. Frequent genetic and ecological assessment is key to managing the adverse effects of stocking and designing proper management practices. In this thesis, I addressed several gaps in our understanding of the effect of long-term stocking of two Australian native freshwater fishes. The Lachlan River in the Murray-Darling Basin (MDB), Australia, is used as a case study to address the gaps. Here, I addressed the effect of stocking on two aspects of population structure: (i) genetics and breeding and (ii) sex ratios of Australian endemic golden perch (Macquaria ambigua) (Richardson 1845) and Murray cod (Maccullochella peelii) (Mitchell 1838). In chapters 2 and 3, I presented evidence of a deeper understanding of the potential effect of stocking on genetic diversity and population genetic structure, along with the natural recruitment status of the existing golden perch population in the Lachlan River. Chapter 4 of my thesis emphasises on developing tools for comparative genomic analysis using genome-wide sequence data from golden perch. Finally, in chapter 5, I reported the karyotype and mode of sex determination in golden perch and Murray cod to enhance our understanding of sex ratios in these species. I investigated the genetic diversity and population genetic structure of golden perch and Murray cod using Single Nucleotide Polymorphism (SNP) markers, with a focus on detecting any subpopulation level genetic structuring that will provide insight to understand the effect of stock enhancement. The results show that two distinct genetic clusters for both golden perch and Murray cod in the Lachlan River. Analyses revealed the probable cause of such genetic differentiation is more likely to be family relationships driven by stock enhancement rather than geographic barriers. Combining the outcomes of the genetic analysis and current knowledge gaps, I further investigated the natural recruitment status of golden perch in the Lachlan River using otolith microchemistry analysis (Chapter 3). The otolith microchemistry data provided evidence of a limited natural recruitment in the Lachlan River golden perch population and the co-existence of wild-born and hatchery-bred stocked fish. In addition, I investigated the effectiveness of genetic markers to infer the natal origin of golden perch. The two types of genetic markers investigated, co-dominant SNPs and dominant SilicoDArT markers, could not reliably distinguish natal origin. In addition to population genetic analyses, I developed a computational pipeline to utilise Diversity Arrays Technology (DArTseq) derived SNP data for comparative genome analysis and presented it in Chapter 4. Usually, only one nucleotide of a 69 bp (base pairs) sequence acts as the data point in DArTseq derived SNPs for population genetic analysis. This leaves 68 bp sequences unused and I asked the question whether these unused sequences can be utilized for comparative genome analysis. Using these unused sequences of 6,776 SNPs from 90 golden perch, I generated a hypothetical golden perch genome. This hypothetical genome represented 0.1-0.3% of the actual genome and used this hypothetical genome to identify sequence homologies against 12 model fish genomes available in the Ensembl genome browser. I identified sequence homologies for 17 evolutionarily conserved genes along with an ancestral evolutionarily conserved syntenic block (euteleostomi Block_210). The proportion (approximately 12%) and types (DNA transposon, LTR and non-LTR retrotransposon) of repetitive sequences and the hierarchical pattern of the number of sequence homologies with phylogenetically close species indicate the validity and reproducibility of the approach. Maintaining a balanced sex ratio is a critical yet often overlooked aspect of stock enhancement. A skewed sex ratio in a natural population can have an adverse effect on both genetics and breeding, such as sex-specific selection, limited natural recruitment due to inadequate mate choice, low effective population size and reduced heterozygosity. However, sex ratio analysis is challenging in the early stages of the life cycle, particularly for those fishes, such as golden perch that do not have sexual dimorphism. Hence, genetic sexing is a potential tool for such species. In chapter 5 of this thesis, I investigated the karyotype to elucidate modes of sex determination of golden perch and Murray cod using molecular cytogenetic tools such as fluorescent staining, c-banding, comparative genome hybridisation (CGH), fluorescence in situ hybridisation (FISH) of repetitive DNAs and methylation landscapes of chromosomes. Results from this study suggest both golden perch and Murray cod have a conserved karyotype 2n=48 and have a male heterogametic (XX/XY) sex chromosome system. Comparative karyotype analysis revealed probable sex chromosome turnover in the study species. These results will assist future studies focusing on the genetic sexing of golden perch and Murray cod, such as identification of sex-linked markers, as well as understanding sex chromosome evolution within Australian freshwater fishes. Stock enhancement is an effective conservation approach. However, there are significant knowledge gaps concerning genetic impacts. This thesis addresses a number of these knowledge gaps, including the genetic effect of stocking on populations and natural recruitment, and provides new tools to address key issues, including natal origin and modes of sex determination. Although this thesis is focused on two Australian freshwater fishes, golden perch and Murray cod, the research presented here has broader implications for fish conservation globally. These issues are summarised in Chapter 6.
|Date of Award||2021|
|Supervisor||MD Tariq Ezaz (Supervisor) & Fiona Dyer (Supervisor)|