Analysis of the Population Genetics and Polybrominated Diphenyl Ether (PBDE) Burdens of Otters in England and Wales: With Case Studies of Populations in South West England Submitted by Angela Pountney, to The University of Exeter as a thesis for the degree of Doctor of Philosophy in Biological Sciences, September 2008. This thesis is available for library use on the understanding that it is copyright material and that no quotation from the thesis may be submitted and approved without proper acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University. ……………………. 0 Abstract Otter populations declined drastically across many areas of England and Wales during the 1960s to 1980s. The main cause of this decline is thought to have been high concentrations of organic pollutants, in particular PCBs and dieldrin. Here we look at the health of the present day otter population, focussing on the numbers of otters, the genetic diversity of populations and investigating a possible new organic pollutant threat, polybrominated diphenyl ethers (PBDEs). A non-invasive spraint genotyping study of the otter population inhabiting the River Camel in Cornwall not only revealed that the river was capable of supporting a minimum number of 12 otters over a 9 month period, but gave insight into the ranges and genetic relationships of the individuals using the river system. A further population genetic study was carried out focussing on the River Itchen in Hampshire, a population which declined drastically to just a few isolated individuals before receiving otters through a captive breeding programme. Microsatellite genotyping of tissue samples showed the River Itchen population to be relatively diverse, indicating a successful population recovery, and haplotype analysis reveals that captive bred otters have successfully bred within the River Itchen population. However, haplotype analysis also indicates that the otters used to found the captive breeding programme were unlikely to have originated from a native British population. Concentrations of PBDEs in otters rival the high concentrations observed in many marine mammal species and are approaching the concentrations of PCBs and DDTs already observed in otters. The profile of the PBDE congeners found shows that lower congeners show relative concentrations similar to those observed in many other species of biota, with high BDE-47 dominating the profile and BDE-99 and -100 also found at significant concentrations. Otters also contain relatively high concentrations of the congeners BDE-153 and BDE-209, a trend generally typical of terrestrial top predators. In summary, the otter populations studied appear to be recovering well. However, increasing concentrations of PBDEs may cause problems for otter populations in the future. 1 Acknowledgments First of all I would like to thank my supervisors Jamie Stevens and Charles Tyler for their help and support throughout the project. In particular I would like to thank Charles for the faith he seems to have in me and I hope I haven’t let him down, and I would like to thank Jamie for always having his door open and for listening to all my worries along the way. I would also like to say a big thank you to Gareth Thomas for letting me work in his laboratory and for being very patient in teaching me pretty much everything I needed to know to carry out the PBDE analysis. I would like to say a big thank you to Tim Sykes, without whom the project would not have been possible. My heartfelt thanks go to Vic Simpson, who not only provided me with liver and kidney samples that he has painstakingly collected over the years, but offered me patience and advice, and along with his wife Jane, would always make me feel welcome when I visited. Paul Chanin, who seems to know everything and everyone there is to know in the world of mammals, and who’s help and advice was very much appreciated along the way. My thanks go to Liz Chadwick for allowing me to use the Cardiff sample collection and for putting her trust in me, and to Lyn Jenkins for the time she gave to me and the project. A BIG thank you to Graham Roberts, Kate Stokes and all of the collection volunteers who went out, sometimes at the crack of dawn, to go traipsing around on river banks collecting samples for me (and an apology to the Hampshire volunteers for the lack of results!). In particular I would like to thank Robert Hurrell, Tyson Jackson, Jon Evans and Chris Macham, all of whom I know put in particularly long hours on the riverbank. In addition I would like to thank Andrew Griffiths, Patrick Hamilton, Lucy Evans and Laura McDonagh for their help and advice with the molecular laboratory work. I would also like to thank Karen Coxon and Alison Hill for their help and advice during the first few stages of the project and Klaus-Peter Koepfli for help sequencing extra samples. This PhD was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), with Hampshire and Isle of White Wildlife Trust as a CASE partner. Extra funding was also provided by the UK Environment Agency. 2 Affiliated organisations and Universities include: UK Environment Agency (Tim Sykes, Lyn Jenkins) Department of Environmental Sciences, University of Lancaster (Gareth Thomas) Hampshire and Isle of Wight Wildlife Trust (Graham Roberts) Cornwall Wildlife Trust (Kate Stokes) Wildlife Veterinary Investigation Centre (Vic Simpson) Cardiff University Otter Project (Elizabeth Chadwick) 3 Table of Contents Abstract .............................................................................................................................1 Acknowledgments.............................................................................................................2 Table of Contents ..............................................................................................................4 List of Figures .................................................................................................................13 List of Tables...................................................................................................................19 1. Introduction.............................................................................................................23 1.1 Background .....................................................................................................23 1.1.1 The Eurasian Otter ( Lutra lutra ).............................................................23 1.1.1i) Natural History and Distribution ............................................................23 1.1.2 The Declines of the 1950s and 60s .........................................................27 1.1.2i) Otter Declines .........................................................................................27 1.1.2ii) Persecution.............................................................................................29 1.1.2iii) Habitat Destruction and Disturbance....................................................30 1.1.2iv) Road Deaths..........................................................................................32 1.1.2v) Otters and Mink .....................................................................................34 1.1.2vi) Nutrient Imbalances and Fish Stocks ...................................................35 1.1.2vii) Heavy Metals.......................................................................................37 1.1.3 Otter Declines and Chlorinated Hydrocarbons.......................................38 1.1.3i) DDTs ......................................................................................................38 1.1.3ii) Dieldrin..................................................................................................39 1.1.3iii) PCBs.....................................................................................................41 1.1.3iv) Conclusion............................................................................................46 1.1.4 Reintroductions.......................................................................................47 1.1.5 Monitoring Population Size ....................................................................49 1.1.5i) Why Monitor Otter Populations .............................................................49 1.1.5ii) Methods of Population Monitoring .......................................................49 1.1.6 Non-Invasive Genetic Census.................................................................51 1.1.6i) Non-Invasive Genetic Census Methodology..........................................51 1.1.6ii) Faecal Genotyping in Other Species .....................................................52 1.1.6iii) Sexing Faecal Samples.........................................................................53 1.1.7 Spraint Genotyping in Otters ..................................................................53 1.1.7i) Spraint Genotyping of Otters in South West England (Coxon et al. , 1999; Dallas et al. , 2003) ...................................................................................53 4 1.1.7ii) Comparisons with Other Census Methodologies (Arrendal et al. , 2007) .............................................................................................................................55 1.1.7iii) Inferring Levels of Genetic Relatedness (Hung et al. , 2004)...............55