MOLECULAR CHARACTERIZATION AND SOME ENVIRONMENTAL FACTORS INFLUENCING DISTRIBUTION OF THE ENDANGERED AND ENDEMIC Gulella taitensis IN TAITA HILLS, KENYA MWAURA NJERI ANN, (BSc) Reg. No. I56/10628/2006 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE (GENETICS) IN THE SCHOOL OF PURE AND APPLIED SCIENCES OF KENYATTA UNIVERSITY APRIL 2010 ii DECLARATION This thesis is my original work and has not been presented for a degree in any other university or any other award. Mwaura Njeri Ann, (BSc) Reg. No. I56/10628/2006 Signature………………………… Date………………. We confirm that the work reported in this thesis was carried out by the candidate under our supervision. Prof. Douglas Ndiritu Department of Plant and Microbial Science Kenyatta University P.O Box 43844- 00100, Nairobi Signature…………………………. Date………………. Dr. Charles N. Lange Department of Invertebrate Zoology, National Museums of Kenya P. O. Box 40658-00100, Nairobi Signature………………………….. Date………………………. Dr. Kem Githui Department of Molecular Genetics, National Museums of Kenya P. O. Box 40658-00100, Nairobi Signature…………………………. Date………………. iii DEDICATION I dedicate this work to my daughter Hope Gathoni, nephews Nduba, Berac, Neem and, my Niece Wairimu, my husband Francis and all my other family members. iv ACKNOWLEDGEMENT I acknowledge the grace of God which has enabled me to come this far, His grace has been sufficient for me. I wish to acknowledge Critical Ecosystem Partnership Fund (CEPF) who sponsored this research, The Nature Kenya office which facilitated the logistics, National Museums of Kenya and especially Molecular Genetics and Entomology Sections where lab work was carried out, Kenyatta University, KEFRI, Forest office and Mr Muigai District Forest Officer, Wundanyi. I am indebted to my supervisors Prof. Nderitu, Dr.Lange and Dr. Githui, Mr.Reuben and driver who assisted me during field work. My mother Mary, sisters Hellen, Grace, Rosemary and Njeri and Dr. Kamiri Nduba, Harun and Francis my husband, who always encouraged and gave me moral support during my low moments, I will always be grateful. To all my friends who gave me moral support and made this work possible I will always be grateful. v TABLE OF CONTENTS DECLARATION ........................................................................................................... ii DEDICATION ............................................................................................................. iii ACKNOWLEDGEMENT ............................................................................................ iv TABLE OF CONTENTS ............................................................................................... v LIST OF TABLES ....................................................................................................... vii LIST OF FIGURES ................................................................................................... viii ACRONYMS AND ABBREVIATIONS ..................................................................... ix ABSTRACT ................................................................................................................... x CHAPTER ONE ............................................................................................................ 1 1.0 INTRODUCTION ................................................................................................... 1 1.1 Background .............................................................................................................. 1 1.2 Problem statement and justification ......................................................................... 3 1.3 Research questions ................................................................................................... 3 1.4 Hypotheses ............................................................................................................... 4 1.5 Objectives ................................................................................................................ 4 1.5.1 General objective .................................................................................................. 4 1.5.2 Specific objectives ................................................................................................ 4 CHAPTER TWO ........................................................................................................... 5 2.0 LITERATURE REVIEW ........................................................................................ 5 2.1 Land snails in East Africa ........................................................................................ 5 2.2 Taita hills forest ....................................................................................................... 6 2.3 Land snail and forest calcium .................................................................................. 7 2.4 Land snail diet and behavior .................................................................................... 9 2.5 Economic importance of land snails ...................................................................... 10 2.6 Molecular systematics ............................................................................................ 12 2.6.1 Amplification of microsatellites ......................................................................... 13 CHAPTER THREE ..................................................................................................... 17 3.0 MATERIALS AND METHODS ........................................................................... 17 3.1 The study area ........................................................................................................ 17 3.2 Methods.................................................................................................................. 19 3.2 1 Site selection ....................................................................................................... 19 3.2.2 Sampling Gulella taitensis .................................................................................. 19 3.2.3 Preservation of snail for DNA extraction ........................................................... 20 3.2. 4 DNA extraction .................................................................................................. 20 3.2.5 Gel electrophoresis.............................................................................................. 21 3.2.5.1 Agarose gel electrophoreses ............................................................................ 21 3.2.5.2 Acrylamide gel electrophoresis ........................................................................ 22 3.2.6 PCR ..................................................................................................................... 22 3.2.7 Determination of environmental factors ............................................................. 23 3.2.8 Litter depth and percentage cover ....................................................................... 23 3.2.9 Percentage canopy cover ..................................................................................... 24 3.2.10 Trees density, dead fallen wood density ........................................................... 24 3.3 Data analysis .......................................................................................................... 24 CHAPTER FOUR ........................................................................................................ 25 4.0 RESULTS .............................................................................................................. 25 4.1 Genetic diversity of G. taitensis............................................................................. 25 4.2 Distribution of G. taitensis ..................................................................................... 29 4.3 Correlation analysis of environmental variables with snail ................................... 33 vi 4.4 Regression analysis ................................................................................................ 36 CHAPTER FIVE ......................................................................................................... 38 5.0 DISCUSSION ........................................................................................................ 38 5.1 Molecular characteristics of G. taitensis ................................................................ 38 5.2 Distribution of G. taitensis in Taita hills ............................................................... 38 5.3 Influence of environmental factors on distribution of G. taitensis ........................ 40 CHAPTER SIX ............................................................................................................ 44 6.0 CONCLUSION AND RECOMMENDATIONS .................................................. 44 6.1 Conclusion ............................................................................................................. 44 6.2 Recommendations .................................................................................................. 45 CHAPTER SEVEN ..................................................................................................... 47 7.0 REFERENCE ......................................................................................................... 47 CHAPTER EIGHT ...................................................................................................... 54 8.0 Appendices ............................................................................................................