Systematics and Biogeography of Three Widespread Australian Freshwater Fish Species. by Bernadette Mary Bostock B.Sc. (Hons) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Deakin University February 2014 i ABSTRACT The variation within populations of three widespread and little studied Australian freshwater fish species was investigated using molecular genetic techniques. The three species that form the focus of this study are Leiopotherapon unicolor, Nematalosa erebi and Neosilurus hyrtlii, commonly recognised as the three most widespread Australian freshwater fish species, all are found in most of the major Australian drainage basins with habitats ranging from clear running water to near stagnant pools. This combination of a wide distribution and tolerance of a wide range of ecological conditions means that these species are ideally suited for use in investigating phylogenetic structure within and amongst Australian drainage basins. Furthermore, the combination of increasing aridity of the Australian continent and its diverse freshwater habitats is likely to promote population differentiation within freshwater species through the restriction of dispersal opportunities and localised adaptation. A combination of allozyme and mtDNA sequence data were employed to test the null hypothesis that Leiopotherapon unicolor represents a single widespread species. Conventional approaches to the delineation and identification of species and populations using allozyme data and a lineage-based approach using mitochondrial 16S rRNA sequences were employed. Apart from addressing the specific question of cryptic speciation versus high colonisation potential in widespread inland fishes, the unique status of L. unicolor as both Australia’s most widespread inland fish and most common desert fish also makes this a useful species to test the generality of current biogeographic hypotheses relating to the regionalisation of the Australian freshwater fish fauna. The results of this study based upon comprehensive and congruent allozyme and 16s mtDNA data sets, failed to identify any cryptic diversity of ii taxonomic significance within the species Leiopotherapon unicolor. Thus, this species retains its position as Australia’s most widespread freshwater fish species. An unexpected outcome of this study, especially given the number of populations of the target species sampled, was finding evidence suggesting the existence of two sympatric cryptic species among the five L. aheneus individuals sampled from the Fortescue River in Western Australia. This highlights the value of genetic data for identifying cryptic species. The second research chapter (Chapter 3) uses similar techniques, with the addition of a second mtDNA gene region, cytochrome b, to test the null hypothesis that Nematalosa erebi represents a single widespread species. This study also aimed to examine the phylogeographic structure of N. erebi within and among major Australian drainage basins; with a view to investigating the historical connections between these basins and to examine previously established hypotheses of biogeographical associations among them. Based on the results of this study, there is strong support to reject the null hypothesis that N. erebi is a single widespread species. The results of this study support the recognition of a divergent N. erebi lineage in three rivers of the Western Australian Pilbara region. In addition to this, the data revealed a degree of river basin phylogeographic structuring between N. erebi populations in Australia, suggesting that the two major inland drainages, the Murray-Darling Basin and the Lake Eyre Basin, are not biogeographically closely associated to each other. Rather, the Murray-Darling Basin is more closely allied with the eastern coastal drainages across the Great Dividing Range. The results also indicate that the Finke River may have been isolated from the remainder of the Lake Eyre Basin catchment for a significant period of time. iii The third and final research chapter (Chapter 4) aimed to confirm the taxonomic position of Neosilurus hyrtlii found in Australian freshwater drainages to determine whether or not this widespread species is actually a single species or, in fact a complex of species. The same molecular techniques employed in Chapter 3 were also utilised in this study. The results indicate that whilst most populations of N. hyrtlii are composed of a single widespread species; there is strong evidence to support the existence of at least two sibling species in Western Australia. Relationships among catchments inferred from the phylogenetic analyses are largely in agreement with the findings of Chapter 3, the main exception being that of the Finke River population which, rather than showing evidence of isolation, shows a close affinity to Lake Eyre Basin drainages. The findings of this research provide a strong foundation to support further investigation of the proposed cryptic speciation and significant divergence within the Pilbara Region of Western Australia. iv CONTENTS ABSTRACT ............................................................................................................I CONTENTS ......................................................................................................... IV LIST OF FIGURES ............................................................................................. VI LIST OF TABLES ............................................................................................VIII ACKNOWLEDGEMENTS ................................................................................. X CHAPTER 1. .......................................................................................................... 1 1.1 AUSTRALIAN FRESHWATER FISHES ................................................ 1 1.2 AUSTRALIAN FRESHWATER SYSTEMS ........................................... 5 1.3 MOLECULAR GENETICS .................................................................... 11 1.3.1 Mitochondrial DNA .......................................................................... 11 1.3.2 Allozyme Electrophoresis ................................................................. 12 1.4 BIOGEOGRAPHY .................................................................................. 15 1.5 PHYLOGENETICS ................................................................................. 19 1.5.1 Application of phylogenetics to Australian Fish taxonomy ............. 21 1.6 RESEARCH OBJECTIVES AND THESIS FORMAT .......................... 24 CHAPTER 2. ........................................................................................................ 26 2.1 INTRODUCTION ................................................................................... 26 2.2 MATERIALS AND METHODS ............................................................. 30 2.2.1 Sample Collection ............................................................................. 30 2.2.2 Allozyme Electrophoresis ................................................................. 33 2.2.3 DNA Extraction and Amplification .................................................. 36 2.3 RESULTS ................................................................................................ 38 2.3.1 Allozyme Data .................................................................................. 38 2.3.2 Mitochondrial Data ........................................................................... 47 2.4 DISCUSSION .......................................................................................... 50 CHAPTER 3. ........................................................................................................ 56 3.1 INTRODUCTION ................................................................................... 56 3.2 MATERIALS AND METHODS ............................................................. 60 3.2.1 Sample Collection ............................................................................. 60 3.2.2 Allozyme Electrophoresis ................................................................. 63 3.2.3 DNA Extraction and Amplification .................................................. 66 3.3 RESULTS ................................................................................................ 69 3.3.1 Allozyme Data .................................................................................. 69 3.3.2 Mitochondrial Data ........................................................................... 78 3.4 DISCUSSION .......................................................................................... 81 CHAPTER 4. ........................................................................................................ 86 4.1 INTRODUCTION ................................................................................... 86 4.2 MATERIALS AND METHODS ............................................................. 89 4.2.1 Sample Collection ............................................................................. 89 v 4.2.2 Allozyme Electrophoresis ................................................................. 93 4.2.3 DNA Extraction and Amplification .................................................. 96 4.3 RESULTS .............................................................................................. 100 4.3.1 Allozyme Data ................................................................................ 100 4.3.2 Mitochondrial Data ........................................................................