Population Genetics of Common Carp (Cyprinus carpio L.) in the Murray-Darling Basin Gwilym David Haynes A thesis submitted to the Faculty of Veterinary Science, The University of Sydney, in fulfilment of the requirements for the Degree of Doctor of Philosophy March 2009 Declaration This thesis is submitted to the University of Sydney in fulfilment of the requirement for the Degree of Doctor of Philosophy The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. Apart from the assistance mentioned in the acknowledgements and the contribution of the research paper co-authors listed below, the work described in this thesis was executed by the author, who also had substantial input into planning of the projects. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution. Signature:……………………………………………. Date:…………………..... ii Contribution of Co-authors Chapter 3: Population genetics of a globally invasive species, common carp (Cyprinus carpio L.), in the Murray-Darling Basin, Australia: evidence for multiple introductions and genetic structure, with suggested management units Dr. D.M. Gilligan selected the sample sites, organised and coordinated the collection of fish samples from the Murray-Darling Basin and Prospect Reservoir and assisted in manuscript preparation. Dr. P. Grewe offered extensive technical advice, made genotyping equipment available and assisted with manuscript preparation. Prof. F. Nicholas supervised the project, contributed to extensive discussions concerning data analysis and interpretation, and performed extensive manuscript editing. Chapter 4: Invasive common carp (Cyprinus carpio L.) in Australia: origin of founding strains and population genetics of coastal waterways Dr. D.M. Gilligan selected the sample sites, organised and coordinated the collection of common carp samples from the Murray-Darling Basin, Prospect Reservoir, the Hunter and Hawkesbury-Nepean catchments and assisted in manuscript preparation. Dr. P. Grewe offered extensive technical advice, made genotyping equipment available and assisted with manuscript preparation. Prof. F. Nicholas supervised the project, contributed to extensive discussions concerning data analysis and interpretation, and performed extensive manuscript editing. Prof. C. Moran assisted with manuscript preparation and provided technical and analytical advice. Chapter 6: Rapid identification of maternal lineages in common carp (Cyprinus carpio L.) using real-time PCR and high resolution melt-curve analysis Dr. J. Gongora assisted with implementation of software analysis and manuscript preparation. iii Prof. F. Nicholas supervised the project, contributed to extensive discussions concerning data analysis and interpretation, and performed extensive manuscript editing. Dr. K.R. Zenger offered the original idea for the research paper and assisted with manuscript preparation. I certify that the above statement about my contribution to the research papers in this Ph.D. thesis is true and accurate, and give Gwilym Haynes full permission to submit these journal articles as part of his Ph.D. thesis. D.M. Gilligan Signature: Date:10 June 2008 P. Grewe Signature: Date: 30 May 2008. F. Nicholas Signature: Date:25 June 2008. C. Moran Signature:………………………… Date:…………………........ J. Gongora Signature:………………………… Date:…………………........ K.R. Zenger Signature: Date: 2 June 2008 iv Acknowledgements First and foremost, I would like to thank Prof. Frank Nicholas, who has been my primary supervisor throughout the duration of this project. His keen insight, objective reasoning and endless patience have helped me greatly in developing my research and critical thinking skills. I am also indebted to my associate supervisors Dr. Peter Grewe, Dr. Dean Gilligan and Prof. Chris Moran, without whom I would not have been able to complete this research. I would like to thank Dr. Klaus Kohlmann and Dr. Bernd Hänfling for supplying most of the “overseas” samples in my project; the Australian Koi Farm in Bringelly, NSW, for donating samples of Japanese koi carp; and Pets on Broadway in Camperdown, NSW, for donating samples of goldfish. I thank Leanne Faulks, Vanessa Carracher, Peter Boyd, Dean Hartwell, and Cameron McGregor from the NSW Department of Primary Industries, NSW; Ben Smith from the South Australian Research & Development Institute; Michael Hutchinson and Stephanie Backhouse from the Queensland Department of Primary Industries; Paul Brown from the Victorian Department of Primary Industries; and Dr. Jawahar Patil from CSIRO Marine Laboratories in Hobart for collecting samples from Australia. I am also very grateful to colleagues in the former Pest Animal Control CRC and its successor the Invasive Animals CRC, especially Tony Peacock, Brad Tucker, Wayne Fulton Kylie Hall and Diane Holloway, for providing my scholarship, for facilitating my research, and for encouraging my participation in CRC activities. I thank Lee Miles and Dr. Jaime Gongora for assistance with calling genotypes; Lee Ann Rollins, Dr. Jaime Gongora and Dr. Kyall Zenger for assistance with manuscript preparation; and Zung Doan for technical support. Finally, I would like to thank my friends and family in my hometown of Adelaide, in Sydney, in New Zealand and around the world. Their kind support has been invaluable. v Funding support was provided by the Fisheries R&D Corporation, the Murray-Darling Basin Commission, the Invasive Animals Cooperative Research Centre (formerly the Pest Animal Control CRC), the NSW Department of Primary Industries and the University of Sydney. vi Abstract Common carp (Cyprinus carpio L.) are a highly invasive species of freshwater fish in Australia. Native to Eurasia, they can be separated into 3-4 different subspecies and innumerable aquaculture and ornamental strains. They have been introduced into Australia on a number of occasions and were established in the Murray-Darling Basin (MDB), Australia’s largest and most important river system, by the 1920s. The release of a new aquaculture strain in the late 1960s, followed by extensive flooding in the mid 1970s, resulted in an explosion of common carp numbers. They are now the dominant species in this river system, and cause extensive ecological damage by competing with native freshwater species and by their feeding mode, in which they suck up mud, filter it through their gill rakers and expel water and fine particles through their gill opening. This feeding mode has been linked to increases in water turbidity, algal blooms, damage to river banks, loss of aquatic vegetation, alterations to the trophic cascade of ecosystems and declines in native fish. However, the effects of carp are difficult to discern from other factors degrading waterways and affecting native fish, such as flow regulation, irrigation and land clearing. There is substantial public interest in the control of common carp. Australians find them unpalatable, considering them too bony and their flesh poor in taste. Subsequently, they are undesirable for recreational fishing and few commercial markets exist in Australia. In addition, as mentioned above, they are suspected of exerting a detrimental effect on the aquatic environment. In fact, carp are currently considered by fisheries biologists as the worst freshwater pest fish in many of the countries where they have been introduced. The cost of management in Australia has been estimated at a total of $15.8 million annually, with $2 million spent on research, $2 million on management, and $11.8 million on remediation of environmental impacts. Previous population genetic studies on carp in Australia identified four strains: Prospect, Boolara, Yanco and Japanese koi. Interbreeding has been recorded between the Yanco and Boolara strain, and there is no reason to believe that it cannot occur between the other carp strains also. Hybridisation between carp and goldfish (Carassius auratus) has also been detected in the MDB, but the level of introgression between the two species has not been quantified. Some genetic structuring of carp within the MDB has been identified previously, although there was little clear pattern to this structuring. vii The main aims of this Ph.D. study were: 1. to characterise the population genetic structure and level of genetic diversity of carp in the MDB; 2. to discern the history of introduction and spread of carp in the MDB; 3. to identify barriers to gene flow in the MDB, and from this data propose management units for control programs. In addition, a number of side projects were also initiated with the following aims: 4. to discern the origin of the different strains of common carp that have been introduced into Australia; 5. to investigate the population genetics of three carp populations in separate waterways on the east coast of Australia; 6. to optimise PCR of microsatellite loci in both carp and goldfish; 7. to characterise the level of introgression between feral carp and goldfish in the MDB; and 8. to develop a protocol for the screening of sequence variants in the mitochondrial control region using real-time PCR and high-resolution melt-curve analysis technology. Common carp were collected from every major river in the MDB. In rivers with major dams, carp were collected from both above and below these impoundments. Additionally, feral carp were collected from Prospect Reservoir (source of the Prospect strain) in the Sydney Basin; Japanese koi carp and domestic mirror-scaled carp