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Contact: [email protected] UNDERSTANDING THE SHINING RAMSHORN SNAIL, SEGMENTINA NITIDA: MORPHOLOGY, GENETICS AND BREEDING by Christopher Stephen Hobbs Canterbury Christ Church University Thesis submitted for the Degree of Doctor of Philosophy 2018 1 Abstract The Shining Ramshorn Snail, Segmentina nitida, is a rare freshwater snail found predominantly in drainage ditches along field margins and in marshland. It is experiencing marked declines in distribution in the United Kingdom (UK) and mainland Europe. The species was included in the IUCN Red Data Book for Invertebrates before a guideline change in 1994 and is included on the UK Biodiversity Action Plan (BAP) as a priority species for conservation. The BAP for S. nitida states that further research on the species required to inform reintroduction and translocation for its conservation. For this thesis, a modified sample evaluation method for Segmentina nitida was developed and evaluated. It increased sample assessment speed without significantly reducing accuracy in comparison to a traditional method of sample evaluation. Captive breeding of S. nitida was explored with the aim of developing simple breeding protocols that could provide stock for potential reintroduction of the species into historical locations. Breeding proved challenging due to fluctuations in water chemistry and subsequent high mortality rates. Geometric morphometric shape analyses were used to investigate variation in shell shape of the species across European populations from the UK, Germany, Poland and Sweden, and the Czech Republic. German and UK snails had similar shell morphologies, and Polish and Czech snails also clustered together morphologically, with the shape of Swedish snails being less distinct. Analysis of the population genetics of German, UK, Polish and Swedish populations using nuclear (ITS, microsatellites) and mitochondrial markers (COI) revealed two distinct lineages of S. nitida in Europe. One comprised of populations from Poland and Sweden (East), and one represented UK populations and a Swedish population (West) with the two lineages coexisting in Germany. These two lineages show no evidence of genetic admixture and can be delimited by both genetic markers and geometric morphometrics, indicating two evolutionarily distinct units, possibly equating to species. The genetic and shape differences between European populations has impacts the conservation of Segmentina nitida, especially in the UK, as previous descriptions of range may now be incorrect and the UK populations may be more significant globally than previously thought, if they are indeed S. nitida. Any future reintroduction plans in the UK and elsewhere would also need to take into account these genetic lineages, as they may result in the introduction of an invasive species or result in infertile offspring. 2 Acknowledgements This PhD project was made possible by the Section of Life Sciences at Canterbury Christ Church University, as part of my role as an academic instructor. The genetics work for this project was supported and made possible by the Natural Environment Research Council (NBAF1075). I’d like to thank all the staff & visitors in the NBAF laboratory at the University of Sheffield, especially Dr. Natalie dos Remedios, Dr. Gavin Horsburgh, and Dr. Deborah Dawson for the help and guidance throughout my stay there and their work on designing the microsatellite primers used in this study. My thanks to my supervisor Dr. Chris Harvey for his constant support, guidance, and comments throughout this project, and for the amazing amount of time spent reviewing and proof-reading my thesis. I would especially like to thank my partner Alice for her constant support, and putting up with the late nights, and long periods of time spent apart, I couldn’t have asked for more. Without her this thesis would never have been finished. I would like to acknowledge the assistance of Chloe Sadler of Kent Wildlife Trust for assistance with land access in the UK. Special thanks go to Dr. Rodrigo Vega for his help and support for both the geometric morphometrics and genetics analyses. I’d also like to thank the technician team at Canterbury Christ Church University for their help with the DNA extractions for the genetics. I would like to extend a special thank you to the chair of my supervisory committee, Prof. Georges Dussart for his constant support and discussions throughout the project. Several colleagues and volunteers participated in fieldwork in both the UK and Europe, and I would especially like to thank Dr. Phil Buckley and Dr. Chris Harvey for flying out to Germany and Sweden for fieldwork there. I would also like to thank Dr. Bartek Gołdyn and Jan Pröjts, Dr. Martin Haase for invaluable local knowledge and access to field sites in Poland, Sweden, and Germany, and Dr. Michal Horsák for providing samples from the Czech Republic. Thanks also go to Jon Ablett of NHM London, for access to the mollusc collection there. Finally, I’d like to thank my parents and my friends for their support, smiling and nodding politely whilst I ramble on about snail conservation, and providing me with more snail-themed paraphernalia than I’ll ever need. 3 Table of Contents ABSTRACT 2 ACKNOWLEDGEMENTS 3 TABLE OF CONTENTS 4 TABLE OF FIGURES 9 TABLE OF TABLES 13 CHAPTER 1- INTRODUCTION 17 1.1. Molluscan Classification 18 1.1.1 Phylum Mollusca 18 1.1.2 Class Gastropoda 18 1.1.3 Family Planorbidae 20 1.2. The Shining Ramshorn Snail, Segmentina nitida 24 1.2.1. Nomenclature 24 1.2.2. General Description of Segmentina nitida 25 1.2.3. Life Cycle of Segmentina nitida 28 1.2.4. Habitat of Segmentina nitida 29 1.2.5. Distribution and Decline of Segmentina nitida 33 1.2.6. Habitat Loss 36 1.2.7. Management of Drainage Ditches 37 1.2.8. Conservation of Segmentina nitida 38 1.3. Aims and Objectives of Study 40 CHAPTER 2- EVALUATING A QUICKER SAMPLING TECHNIQUE FOR SEGMENTINA NITIDA AND OTHER FRESHWATER GASTROPODS 41 2.1. Introduction 42 4 2.2. Materials and Methods 46 2.2.1. Field sites for the testing of quicker sample assessment method 46 2.2.2. Sampling procedure 49 2.2.3. Data Analysis and Statistics 53 2.3. Results 55 2.3.1. Comparison of quicker and traditional sample assessment methods for number of gastropod species and number of individuals 55 2.3.2. Comparison of ACFOR scores obtained using quicker and traditional methods 59 2.3.3. Comparison of species richness (Menhinick’s index) between quicker method and traditional method 59 2.3.4. Sampling time efficiency comparison of quicker and traditional sample assessment methods 61 2.4. Discussion 64 2.4.1. Detection of Segmentina nitida using quicker and traditional sampling methods 64 2.4.2. Comparison of ACFOR scores between quicker and traditional sample assessment methods 66 2.4.3. Sampling speed and detection of gastropod species 67 2.4.4. Relationship of Menhinick’s index between quicker and traditional sample assessment methods 69 2.4.5. Effectiveness of the quicker sample assessment method to detect gastropod populations across seasons 69 2.4.6. Limitations of the proposed quicker sample assessment method for freshwater gastropods 70 2.5. Conclusion 71 CHAPTER 3- EVALUATING LABORATORY BREEDING METHODS FOR SEGMENTINA NITIDA 73 3.1. Introduction 74 3.2. Materials and Methods 78 3.2.1. Breeding Segmentina nitida in Microcosms 78 3.2.2. Effect of Enteromorpha sp., natural sponge and paper as additional substrates on survival and fecundity of Segmentina nitida in microcosms 80 3.2.3. Breeding Segmentina nitida in mesocosms 83 3.2.4. Breeding Segmentina nitida in outdoor macrocosms 84 3.3. Results 87 3.3.1. Rearing of Segmentina nitida in microcosms. 87 3.3.2. Effect of substrate on survivability and fecundity of Segmentina nitida in microcosms 88 3.3.3. Rearing Segmentina nitida in mesocosms 91 3.3.4. Rearing Segmentina nitida in external macrocosms 92 5 3.4. Discussion 93 3.5. Conclusion 102 CHAPTER 4- GEOMETRIC MORPHOMETRIC ANALYSIS OF SEGMENTINA NITIDA SHELL MORPHOLOGY 103 4.1. Introduction 104 4.2. Materials and Methods 109 4.2.1. Acquisition of Segmentina nitida and Hippeutis complanatus shell samples for geometric morphometric analysis 109 4.2.2. Acquisition of Segmentina nitida samples for intraspecific geometric morphometric analysis 110 4.2.3. Photography of shells for two-dimensional geometric morphometric analysis 112 4.2.4. Digitisation and landmark placement for all geometric morphometric analysis 115 4.2.5. Data analysis and statistics comparing shell morphology of Segmentina nitida and Hippeutis complanatus 116 4.2.6. Data analysis and statistics comparing intraspecific shell morphology of European Segmentina nitida populations 118 4.3. Results 119 4.3.1. Geometric morphometric comparison of external shell shape of Segmentina nitida and Hippeutis complanatus 119 4.3.2. Geometric morphometrics analyses of shell shape variation in Segmentina nitida across Europe. 125 4.4. Discussion 134 4.4.1. Differences in shape between Hippeutis complanatus and Segmentina nitida 134 4.4.2.