Molecular analysis of the trophic interactions of British reptiles David Steven Brown September 2010 Thesis submitted for the degree of Doctor of Philosophy, Cardiff School of Biosciences, Cardiff University UMI Number: U517025 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U517025 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Declarations & Statements DECLARATION This work has not previously been accepted in substance for any degree and is not concurrently sjj&mitted in candidature for any degree. Signed ... .................................(candidate) Date: 30/09/2010 STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of ....fW ^ ....... (insert MCh, MD, MPhil, PhD etc, as appropriate) Signed .... (candidate) Date: 30/09/2010 STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources apaacknowledged by explicit references. Signed C4r\.r..*r..\r................................. (candidate) Date: 30/09/2010 STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed Crtrf.... jr.....W............................. (candidate) Date: 30/09/2010 STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access previously approved by the Graduate Development Committee. Signed (candidate) Date: 30/09/2010 Acknowledgements I would like to thank Bill Symondson for his supervision of my Ph.D. Thanks also go to the Herpetological Conservation Trust, the Countryside and Landscape Services of Caerphilly County Borough Council, the Flat Holm Project, Ecological Planning & Research Ltd, Cresswell Associates, Richard Griffiths and Brett Lewis for their assistance, advice and / or for providing access to land. Thanks to Simon Jarman for hosting my stay in Tasmania and for allowing me the opportunity to learn new techniques and enjoy some much-needed sunshine. Many thanks also to final year undergraduate project students Elizabeth Stewart and Katie Ebenezer for their useful contributions to my research. Special thanks go to fellow members of the “G10” lab and the BEPG whose advice, support, encouragement, guidance and, occasionally, consumables and reagents, made everything possible. In particular I would like to thank Catherine Eyre, Renata Medeiros, Daniel Read, Sigrid Mensch and Jo Lello. Finally I would like to thank my mum, dad and sister. Without their continual support and encouragement none of this would have been achievable. Thank you. This research was funded by the Natural Environment Research Council (NERC) with travel grants awarded from the Gillian Powell Memorial Travel Scholarship and the Society for Experimental Biology. Sequencing was performed by the NERC Biomolecular Analysis Facility (NBAF). Thesis Summary For this Thesis, molecular techniques were developed and applied in the analysis of the diet of British reptiles. Such approaches provide an opportunity to overcome the biases of conventional dietary analysis. Faeces were collected from smooth snakes ( Coronella austriaca), grass snakes (Natrix natrix) and slow worms ( Anguis fragilis) each month over two years. DNA was extracted and, through the use of PCR primers, cloning and pyrosequencing, seasonal and ontogenetic changes in their diet was investigated. The results establish, for the first time, that prey DNA is detectable in reptile faeces. Predation by slow worms on pulmonates and earthworms was much higher than previous studies have suggested, with seasonal patterns in predation but no ontogenetic ones. Predation on Arion slugs was higher in females in the spring and autumn than in males, suggesting preferential selection by females, possibly a result of differences in reproductive costs. Predation on earthworms was found to include deep-living species which only surface at night. This may have been a driving force in the evolution of nocturnal foraging behaviour in slow worms. The prevalence of a parasitic nematode of slow worms, Neoxysomatium brevicauditum, was also investigated using PCR primers. Prevalence was higher in males than females in April. While this might reflect different encounter rates between males and females, it may also be a function of testosterone-induced immunosuppression. Analysis of smooth snake diet confirmed that predation on reptiles was high but revealed increasing consumption of small mammals with age. Predation on reptiles by grass snakes was higher than expected and suggests competition may exist between smooth snakes and grass snakes at a juvenile stage. Overall, these results provide a clearer picture of the trophic networks some of Britain’s reptiles belong to, information pertinent to translocation and reintroduction programmes along with conservation management strategies. dedicated to my father Table of Contents Title Page i. Declarations ii. Acknowledgements iii. Thesis Summary iv. Tables of Contents vi-i. Chapter 1: Introduction...................................................................................... 1 1.1 Overview 2 1.2 Background - the status of, and threats to, British reptiles 2 1.3 Distribution of British reptiles 6 1.4 Diet of British reptiles 10 1.4.1 Grass snake (Natrix natrix) diet 11 1.4.2 Smooth snake ( Coronella austriaca) diet 13 1.4.3 Adder {Vipera berus) diet 16 1.4.4 Diet overlap amongst snakes 18 1.4.5 Slow worm (Anguis fragilis) diet 19 1.5 Understanding and measuring trophic interactions 20 1.6 Molecular techniques for the detection of prey 25 1.7 Aims 28 1.8 Thesis layout 29 1.9 References 31 Chapter 2 : Method development and preliminary analysis......................... 43 2.1 Introduction 44 2.2 Study animals and husbandry 44 2.3 Faecal extraction kits 45 2.4 Primer development 47 vi 2.4.1 Primer design 47 2.4.2 Mouse- and chicken-specific primers 47 2.4.3 Universal primers 51 2.4.3.1 18S primers 51 2.4.3.2 COI primers 54 2.5 Detection of prey DNA in faeces 54 2.6 Detection of a pulse of prey in faeces 57 2.7 Preliminary cloning of slow worm prey 59 2.8 Fieldwork 62 2.8.1 Study sites 62 2.8.2 Sampling regime 63 2.8.3 Descriptive statistics of smooth snakes and slow worms66 2.9 References 68 tapter 3 ; Methods o f preventing predator bias in P C R.................................... 71 3.1 Abstract 72 3.1 Introduction 73 3.1.1 Taxa-specific primers 73 3.1.2 Universal primers 78 3.1.2.1 Removal of predator DNA 81 3.2 Method development and results 89 3.2.1 Extraction and sequencing of slow worm DNA 89 3.2.2 Restriction enzymes 89 3.2.2.1 Post-PCR restriction enzyme digest 90 3.2.2.2 Secondary PCR following enzyme digestion 93 3.2.2.3 Pre-PCR enzyme digestion 93 3.2.3 PCR inhibition 95 3.2.3.1 Slow worm primers 95 3.2.3.2 Testing specificity of slow worm primers 96 3.2.3.3 Blocking primers 96 3.2.3.4 Non-target testing of blocking primers 97 3.2.3.5 Cloning of artificial mixtures of slow worm non-target “prey” DNA 99 3.3 Discussion 100 3.4 References 102 Chapter 4 : Pyrosequencing ofprey DNA in reptile faeces reveals benefit of nocturnal hunting.............................................................................................................................. 112 4.1 Abstract 114 4.2 Introduction 116 4.3 Methods 121 4.3.1 Field study 122 4.3.2 Earthworm group-specific primers 124 4.3.3 Data analysis 125 4.4 Results 126 4.5 Discussion 128 4.6 References 133 Chapter 5 : Seasonal, ontogenetic and sex-related differences in the diet of slow worms .............................................................................................................................................. 143 5.1 Abstract 144 5.2 Introduction 146 5.3 Methods 149 5.3.1 Field sites and faecal DNA extraction 149 5.3.2 Multiplex optimization 150 5.3.3 Primer screening 152 5.3.4 Statistics 152 5.4 Results 152 5.4.1 Predation on earthworms 153 5.4.2 Predation on pulmonates 154 5.5 Discussion 162 5.5.1 Earthworms 163 5.5.2 Pulmonates 164 5.6 Conclusions 166 5.7 References 167 Chapter 6 : First record o/Neoxysomatium brevicaudatumin the UK through the non- invasive sampling of Anguis fragilis: complementary morphological and molecular detection.......................................................................................................................... 174 6.1 Abstract 176 6.2 Introduction 176 6.3 Methods 177 6.3.1 Morphological identification 177 6.3.2 Molecular identification 177 6.3.3 Molecular screening 179 6.4 Results 179 6.5 Discussion 181 6.6 References 182 Chapter 7: Molecular detection of sex and age-biased nematode prevalence in slow worms (Anguis fragilis)............................................................................................... 184 7.1 Abstract 186 7.2 Introduction 187 7.3 Methods 191 7.3.1 Study sites and faecal collection