An investigation of genetic heterogeneity in a biological sentinel species (Lumbricus rubellus) Robert Kevin Donnelly A submission presented in partial fulfilment of the requirements of the University of Glamorgan/Prifysgol Morgannwg for the degree of Doctor of Philosophy This research programme was carried out in collaboration with Cardiff University September 2011 Summary Studies have indicated the existence of several possible sources of genetic heterogeneity within the sentinel species Lumbricus rubellus that could compromise its suitability for ecotoxicological assessment. The species appears to consist of two genetically divergent cryptic lineages and has been demonstrated to display genetic adaptation towards contaminants within some populations. In order to investigate the cryptic lineages of L rubellus further a combined morphological and DNA barcoding approach was undertaken, with both the mitochondria! COI gene being sequenced and external morphological characters being assessed. Combined barcoding and morphological analysis confirmed the existence of the previously described genetic lineages of L. rubellus and highlighted a potential lineage-specific morphological trait. The effectiveness of this trait in field identification of the two lineages was tested in a blind trial. This indicated that the trait may be particularly effective in successfully identifying the individuals of one of these lineages. The two cryptic lineages were also analysed in a second study featuring cross-amplifying microsatellite loci. Both the sequencing and fragment analysis of these microsatellite loci strongly supported the existence of a high degree of reproductive isolation between the two lineages. Finally microsatellite markers were applied to test the hypothesis of genetic adaptation within L. rubellus populations located along an aerially-deposited nickel contamination gradient. No support was obtained for the existence of genetic adaptation within these populations. This could indicate that the most heavily contaminated sites represent demographic 'sinks' into which individuals immigrate from adjacent less-contaminated regions. The general implications of these studies are that the two lineages of L rubellus should be treated as separate species in future ecotoxicological trials given their high degree of genetic differentiation. An implication of the nickel study is that L. rubellus may display a greater capacity for tolerating toxic metals through phenotypic plasticity than previous studies have indicated. Acknowledgements I wish to thank the following people for their help over the course of my PhD. Firstly I would like to thank my supervisor Mike Bruford for both allowing me to use his laboratory and for his patience and unfaltering enthusiasm over the course of this study. This was much appreciated, particularly during those times when my own enthusiasm seemed to be fading. I must also thank John Morgan for teaching me a great deal about both earthworms and ecotoxicology and for collecting many hundreds of earthworms. Without John's sixth sense for finding worms this project would have been a lot more difficult. I must also thank George Harper for her supervision and help at the start of the PhD, and for developing some of the microsatellites that I used in the project. Finally I would like to thank Denis Murphy for his help as my second supervisor. Among the people at the University of Glamorgan who have helped me over the course of my PhD I would like to particularly thank Paul Hallas for being a great flat-mate, lab-mate and office- mate. I would also like to thank the following people at Glamorgan- Amie IMoone, Mark Partridge, Roy Wiles, Martin Powell, Sylvia Davies, Alvine Jones, Ed Morgan, Chris Lee, John Morton and anyone else I might have missed. Among the people at Cardiff University who have helped me over the course of my PhD I would like to particularly thank Gabi Juma who processed many of the earthworm samples analysed in the project, genotyped the worms in the nickel study to determine their lineage and determined the metal content of the soils. I am also grateful to Gabi for teaching me many molecular techniques at the start of the study. I must also thank Jane Andre and Andy King for their help and advice with some of the molecular work, Pete Kille for allowing me to use his laboratory and his advice on genetic barcoding and Pierfrancesco Sechi for genotyping the worms from Mid-Wales and England. I would also like to thank the following people at Cardiff- Adam, Alex, Asniza, Ben, Bill, Catherine, Coralie, Dave B, Dave S, Eddie, Ellie, Geoff, Isa-Rita, James P, James R, Jeff, Jo B, Jo C, Jo L, Jo S, Joan, Leila, Loys, Luca, Lucija, Maf B, Maf C, Milena, Mireille, Niall, Pablo, Pati, Renata, Rhys, Rui, Samrat, Sigrid, Steve, Tania, Tim, Vicky, Viola, Wendy, Xiang-Zhan and anyone else I might have missed. Finally I must thank my family and friends for their support and encouragement, particularly Mum, Dad, Pete and Tom. This thesis is dedicated to the memory of my Grandma, Peggy Grubb. "Electronic calculators can solve problems which the man who made them cannot solve; but no government subsidised commission of engineers and physicists could create a worm." Joseph Wood Krutch An investigation of genetic heterogeneity in a biological sentinel species (Lumbricus rubellus) Contents Chapter 1 General introduction 7 Lumbricus rubellus- general biology 8 Lumbricus rubellus as a sentinel species 9 The history of ecotoxicology 10 Sentinel species 11 Earthworms as sentinel species 13 Genetic homogeneity and sentinel species 15 Genetic adaptation 16 Cryptic variation 21 Molecular markers 22 Aims of the project 28 Chapter 2 Intraspecific variation in mtDNA and morphology within the genus Lumbricus 31 Introduction 31 Material and methods 34 Sample collection 34 DNA extraction, amplification and sequencing 34 Data analysis 35 Morphological analysis 36 Bind trial 36 Results 40 Molecular analysis 40 Morphological analysis 41 Blind trial 41 Discussion 49 Chapter 3 A comparison of microsatellite variation in cryptic lineages of Lumbricus rubellus 53 Introduction 53 Material and methods 57 Microsatellite sequencing 57 PCR amplification, purification and cloning 57 PCR amplification and sequencing of insert DNA 57 Microsatellite genotyping 59 Statistical analysis 61 Results 66 Fragment analysis 66 Linkage analysis and Hardy-Weinberg equilibrium 66 Population differentiation 66 Microsatellite flanking sequence variation 67 Microsatellite repeat region variation 68 Discussion 76 Chapter 4 Microsatellite analysis of Lumbricus rubellus populations along a gradient of nickel contamination 80 Introduction 80 Material and Methods 84 Sample collection 84 DNA extraction 84 Microsatellite genotyping 85 Statistical analysis 86 Results 95 Fragment analysis 95 Linkage analysis and Hardy-Weinberg equilibrium 96 Population differentiation 96 Population structure 98 Discussion 123 Chapter 5 General discussion 130 Background to study 130 Synthesis of the findings 131 Implications of the findings 135 Future directions 136 Bibliography 138 Appendix Appendix A- Site locations 166 Appendix B- Site information 167 Chapter 1. General introduction As an introduction to the thesis it is perhaps worth briefly describing how the focus of my studies changed over the course of this PhD. The initial focus of this PhD was to investigate the possible existence of genetic adaptation within populations of the earthworm species Lumbricus rubellus. This particular species is widely applied as a sentinel species to monitor the ecological effects of contaminants within studies of soil ecotoxicology. As a sentinel species it is important that the species displays genetic homogeneity between its populations (Hilty and Merenlander 2000). This means that experimental findings based upon one population of the species can be considered representative of all other populations. Concerns about the genetic adaptation of some L. rubellus populations to metal contamination have arisen following the demonstration of inherited resistance within populations from contaminated sites (Langdon et al. 1999, Langdon et al. 2001). The initial aim of this study was therefore to investigate the possible genetic adaptation of L. rubellus populations along a gradient of nickel contamination. This was attempted by conducting a population genetic analysis using microsatellite markers. Microsatellite analysis has indicated the possible existence of genetically adapted populations within studies of other species by revealing an increased degree of genetic differentiation between environmentally-stressed populations and unaffected populations (Plath et al. 2007, Tobler et al. 2008, Plath et al. 2010). However, shortly after the start of this study it was discovered that the 'species' of L. rubellus may actually comprise two distinct cryptic genetic lineages (King et al. 2008). The existence of such lineages could potentially complicate any studies of metal resistance given that comparative lineages in other annelid species have been found to differ markedly in their tolerance of contaminants (Sturmbauer et al. 1999, Linke-Gamenick et al. 2000). Differences in tolerance can also affect the distribution of such lineages across contaminated and non-contaminated sites (Sturmbauer etal. 1999). Following the discovery of these cryptic lineages within L. rubellus, the focus of the PhD was broadened into a wider consideration of genetic heterogeneity within the species that included studies of these