Conserving Amphibian Diversity: a Species Inventory and Gene Flow Studies in Fragmented Montane Forest, Mambilla Plateau, Nigeria
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Conserving amphibian diversity: a species inventory and gene flow studies in fragmented montane forest, Mambilla Plateau, Nigeria A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Ecology at the University of Canterbury by Denise Arroyo Lambaer University of Canterbury February 2015 Table of contents Acknowledgements . i Abstract . iii List of figures . vi List of tables . viii 1. General Introduction . 1 1.1 The problem . 1 1.2 Value of species inventories . 2 1.3 Population genetics . 4 1.4 Species of interest . 9 1.5 Criteria to select target species . 10 2. The amphibians and reptiles of Ngel Nyaki and Kurmin Danko Reserve, Mambilla Plateau, Nigeria . 12 Abstract . 12 2.1 Introduction . 13 2.2 Material and methods . 16 2.2.1 Study sites . 16 2.2.2 Sampling methods . 18 2.3 Results . 21 2.3.1 Species accounts . 21 2.3.1.1 Amphibia . 21 2.3.1.2 Reptilia . 33 2.3.2 Conservation status . 36 2.4 Discussion . 38 2.4.1 Conservation and management considerations . 39 3. An assessment and optimization of the Amplified Fragment Length Polymorphism (AFLP) method for the genetic characterization of four African anuran species . 42 Abstract . 42 3.1 Introduction . 43 3.1.1 Principle of the AFLP technique . 46 3.2 Material and methods . 50 3.2.1 Tissue samples . 50 3.2.2 Sample processing . 51 3.2.2.1 DNA extractions . 51 3.2.2.2 DNA preparation . 52 3.2.2.3 AFLP protocol . 53 3.2.3 Detection and scoring of AFLP markers . 56 3.2.4 Reproducibility and genotyping error . 58 3.3 Results . 59 3.3.1 DNA preparation and AFLP profiles . 60 3.3.2 Pre-selective and Selective PCR . 62 3.3.3 Reproducibility and genotyping error . 66 3.4 Discussion . 67 4. The genetic structure of four frog species of the Arthroleptidae from fragmented and continuous montane habitat in Ngel Nyaki and Kurmin Danko Forest Reserve . 72 Abstract . 72 4.1 Introduction . 73 4.1.1 Threats to the Nigerian montane forest . 73 4.1.2 Amphibians and habitat fragmentation . 74 4.1.3 Amphibian dispersal . 75 4.1.4 AFLP and genetic population structure . 77 4.1.5 Species of interest . 78 4.2 Hypothesis and predictions . 80 4.3 Material and methods . 81 4.3.1 Study area and field sampling . 81 4.3.2 DNA extraction and AFLP profiling . 83 4.4 Data analysis . 85 4.5 Results . 88 4.5.1 Cardioglossa schioetzi . 88 4.5.2 Leptodactylodon bicolor . 94 4.5.3 Astylosternus sp. 1 . 99 4.5.4 Astylosternus sp. 2 . 104 4.5.5 Comparing the genetic characterization of the four target species . 107 4.6 Discussion . 110 4.6.1 Genetic diversity . 111 4.6.2 Population genetic structure . 112 4.6.3 Genetic diversity and genetic population structure of polyploidy species . 118 4.6.4 Implications for conservation . 120 5. Discussion and future directions . 123 5.1 Base line data, the herpetofaunal inventory . 124 5.2 AFLP, molecular markers used for the population genetic study . 126 5.3 Advantages of AFLPs over other molecular markers for studies on genetic population structure . .. 128 5.4 Genetic population structure . 132 5.5 Importance of multispecies studies . 134 5.6 Conservation priorities and management plans . 135 5.7 Significance of the new findings . 138 References . 140 Appendix I. Modified CTAB method of DNA extraction (amphibian tissue) . 174 Appendix II. General frame for laboratory work . 176 Acknowledgements I am deeply grateful to my supervisors Professor Hazel Chapman, Dr. David Blackburn (California Academy of Sciences), especially to Dr. Marie Hale for her patience and infinite support. I would like to thank to each and all of the field assistants and staff from the Nigerian Montane Forest Project. I am extremely grateful to Thomas Patrick and Muhammed Adam (Hamadu) for their positive attitude even during the rainiest night in Kurmin Danko. Without them my field work would not have been as amusing and productive. During the lab work -likely the hardest but also the most rewarding part of this PhD- I had the unconditional assistance of Maggie Tisch and Craig Galilee to whom I am very grateful. Also, helping me in the lab were Thomas Evans and Claire Galilee. I would like to thank to Jenny Ladley for her support when preparing my field work, and Matt Walters for his amazing ideas when putting together posters and presentations related to my PhD project. Huge thanks to Matt Wallace for helping me in the first stage of my lab work, and to all members of the molecular lab group for their valuable feedback, especially to Tammy Steeves whom I admire due to her honesty. All my gratitude goes to my Mom and Dad who have given me their unconditional love and support ever since I decided to go to New Zealand to complete, so far, the greatest challenge of my life. Thank you to my brother for his helpful and equable advice, and special thanks to my sister who despite the distance was always with me giving me words of encouragement to keep going during the difficult stages of the development of this PhD. i I would like to thank my friends Lupe, Carol, Juliano, Nick, and Leighton for their friendship and support during a gloomy.