ResearchOnline@JCU This file is part of the following reference: Lind, Curtis E. (2009) Population genetics, phylogeography and the effects of aquaculture on genetic diversity of the silver-lipped pearl oyster, Pinctada maxima (Jameson) . PhD thesis, James Cook University. Access to this file is available from: http://eprints.jcu.edu.au/29608/ The author has certified to JCU that they have made a reasonable effort to gain permission and acknowledge the owner of any third party copyright material included in this document. If you believe that this is not the case, please contact [email protected] and quote http://eprints.jcu.edu.au/29608/ Population genetics, phylogeography and the effects of aquaculture on genetic diversity of the silver-lipped pearl oyster, Pinctada maxima (Jameson) Thesis submitted by Curtis E. Lind, BSc (Hons) May 2009 For the degree of Doctor of Philosophy, from the School of Marine and Tropical Biology, James Cook University, Townsville, Queensland, Australia Statement of Access I, Curtis E Lind, author of this work, understand that James Cook University will make this thesis available for use within the University Library and, via the Australian Digital Theses network, for use elsewhere. I understand that, as an unpublished work, a thesis has significant protection under the Copyright Act and; I do not wish to place any further restriction on access to this work. ____________________________________ ____________________ Signature Date 2 Statement of Sources Declaration I declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institution of tertiary education. Information derived from the published or unpublished work of others has been acknowledged in the text and a list of references is given. ____________________________________ ____________________ Signature Date 3 Statement on the Contribution of Others At the time of thesis submission, three manuscripts had been published in peer-reviewed academic journals. The contributions of co-authors to the respective manuscripts are outlined below: Manuscript from Chapter 2: Lind, C.E., Evans, B.S., Taylor, J.J.U., Jerry, D.R., 2007. Population genetics of a marine bivalve, Pinctada maxima, throughout the Indo-Australian Archipelago shows differentiation and decreased diversity at range limits. Molecular Ecology 16, 5193-5203. Contributions: CEL - Project design; sample collection; data generation & analysis; manuscript preparation BSE, JJUT, DRJ - Project design; sample collection; review of final manuscript Manuscript from Chapter 4: Lind, C.E., Evans, B.S., Knauer, J., Taylor, J.J.U., Jerry, D.R., 2009. Decreased genetic diversity and a reduced effective population size in cultured silver-lipped pearl oysters (Pinctada maxima). Aquaculture 286, 12-19. Contributions: CEL - Project design; sample collection; data generation & analysis; manuscript preparation BSE, JJUT - Project design; sample collection; review of final manuscript JK - Sample collection DRJ - Project design; review of final manuscript Manuscript from Chapter 5: Lind, C.E., Evans, B.S., Taylor, J.J.U., Jerry, D.R., The consequences of differential family survival rates and equalizing maternal contributions on the effective population size (Ne) of cultured silver-lipped pearl oysters, Pinctada maxima. Aquaculture Research. doi:10.1111/j.1365-2109.2009.0210.x Contributions: CEL - Project design; sample collection; data generation & analysis; manuscript preparation BSE, JJUT, DRJ - Project design; sample collection; review of final manuscript 4 Acknowledgements To have so many people generously give their time to assist, encourage and advise me over the last four years has left me with abundant gratitude. To this end, I am truly grateful to nobody more than my primary supervisor, Associate Professor Dean Jerry, for his patient guidance, support and genuine friendship - a considerable influence in my development. Wholehearted thanks are also extended to my other supervisors, Dr Brad Evans and Dr Joseph Taylor, for without their help this thesis would not have been achievable. I am indebted to Brad for his willingness to always listen to my many (often trivial) woes, despite us both regularly ending up scratching our heads in perplexity. I can also be certain that both the professional and social relationship I had, and continue to share, with Brad and Dean is universally envied by many a postgraduate student, something that I cherish and have never taken for granted. I also gratefully give thanks to Joseph, my supervisor from afar and an accomplished authority on pearl oysters, for his continual support and valuable advice throughout this project. Whoever has spent enough time in a molecular genetics lab will appreciate that a page full of adjectives is only the beginning, and that sometimes the smallest word of advice can be the difference between a fantastic weekend and no weekend at all. I therefore give many thanks to Dr Carolyn Smith-Keune, for all her time, help and knowledge, particularly in the early years, as I‟m sure I would be still sitting there bewildered without it. I must also thank my fellow after- hours specialist, Adrian McMahon, for his help (and counsel!) over the years and for sharing the bedlam of „high-throughput‟ genotyping with me. I would also like to acknowledge all the assistance in the lab given by Dr Martin Elphinstone, Dr Brad Evans, Christian de Santis and Renate Kvingedal - thanks a lot. I am hugely appreciative of the all the effort from Dr Joseph Taylor, Dr Jens Knauer and the Penyabangan hatchery staff at Atlas South Sea Pearl Ltd. / P.T. Cendana Indopearls for the facilitation and care-taking of my experimental oysters in Bali. It goes without saying that this research simply could not have proceeded without their help and co-operation. For assistance 5 arranging or collecting samples, I also thank the following people: Geoff McGlauchlin (Arrow Pearls), Tam Nguyen, Paul Southgate, K. Takami (Kazu Pearls), Aiman Wang and Dave Williams (Coral Sea Pearls). Finally, I must thank my family, close friends and dear partner, Anna, who have all supported and encouraged me from the very beginning until the end of this project. It is oftentimes the unspoken support that is given when no one really wants to hear your plight which makes the world of difference, and in the end, has got me over the line. I am graciously thankful for this. 6 Abstract The silver/gold-lipped pearl oyster, Pinctada maxima (Jameson 1901), is one of the most important pearl producing species throughout Southeast Asia and northern Australia. The commercial production of high value “South Sea” pearls is based on the culture of P. maxima, and stands to benefit substantially from the implementation of long-term selective breeding programs. Industry-wide interest towards genetic improvement of P. maxima is rapidly growing, however, several fundamental issues must be addressed before selection commences. To achieve sustained response to selective breeding, it is essential that adequate genetic diversity is present within the population; yet currently there is little known about how the culture process affects diversity in P. maxima. This thesis addresses key issues concerning the capture and maintenance of genetic diversity in cultured P. maxima populations, as well as identifying and understanding patterns of genetic structure and diversity distribution throughout its natural range. As the source of broodstock to create base populations for selective breeding, it is important to understand the genetic properties of wild P. maxima populations. Analysis of microsatellite and mitochondrial DNA (mtDNA) variation in wild P. maxima populations throughout its natural distribution revealed a gradient in genetic diversity across its range, with decreasing levels of variation seen in peripheral populations when compared to those situated more centrally (i.e. central Indonesia). Significant genetic structuring and differentiation was also observed amongst populations, and is attributed to historic and contemporary biogeographic influences. Comparisons between wild and cultured P. maxima populations indicate a high level of genetic erosion has occurred in hatchery-propagated populations, with effective populations sizes (Ne) as low as 3.5 and reductions in microsatellite variation as high as 44% occurring as early as two generations beyond wild progenitors. The practice of mass spawning was identified as a major factor in the reduction of diversity, although diversity was not necessarily maintained when a more controlled spawning approach was utilised. Subsequent investigation using DNA parentage analyses revealed highly variable broodstock contributions have played a significant 7 role towards an increase in genetic relatedness and low Ne in cultured P. maxima and is likely to be exaggerated by variable survival rates amongst different pearl oyster families. Upon further investigation and experimentation, it was determined that highly variable family survival will affect Ne in communally reared P. maxima and the practice of equalising family sizes in order to reduce family size variance (and maximise Ne) may only become consistently beneficial once further progress is made towards understanding and then reducing variation in family survival rates. Culture practices related to variation in growth, such as size grading, culling and mass- selection were assessed for the ability to contribute to diversity losses. It was