Supplementing the studbook. Using genetic analysis to complement a captive breeding programme of an endangered primate, Hylobates moloch. Katherine Stanbury A thesis submitted for the degree of Doctorate of Philosophy Writtle College University of Essex Date of submission January 2015 Abstract Genetic analysis of animals involved in captive breeding and reintroduction programmes can provide valuable information to aid in maintaining wild type genotypes and genetic variability. Hylobates moloch, also referred to as the silvery gibbon, is an Endangered primate species endemic to the Indonesian island of Java. As part of an overall conservation programme, a captive breeding and reintroduction programme is being organised. In order to aid both the management decisions within the breeding programme and success rates of re-introductions analyses at three genetic regions were carried out, with DNA extracted from non-invasively collated faecal samples. In order to assay if captively bred individuals were representative of their wild conspecifics, the population was split into two groups representative of wild born and captive born individuals. Genetic analyses at mitochondrial DNA hypervariable region-I (mtDNA HV-I), 15 microsatellite loci and the second exon of the major histocompatibility complex (MHC) DRB region, were carried out to ascertain genetic variability levels, levels of inbreeding, signs of selection and confirm the pedigree. Captive born individuals had markedly lower levels of variability at mtDNA HV-I, which was significant versus the wild group. The second neutral marker of microsatellites revealed no differentiation between wild and captive-born; moreover measures of standardised heterozygosity demonstrated a fairly high level of genomic variability overall. Pedigree analysis using the microsatellites produced information that differed from studbook entries. This was further supported by haplotypic data compiled from the MHC DRB exon 2 analysis. The MHC study revealed a total of 14 DRB alleles, 10 of which are from unknown lineages when compared to human and chimpanzees. As with microsatellites, no group differentiation between wild and captive has occurred but there are more rare i alleles present within wild individuals. In conclusion, whilst genetic variation is both high and shows no deviation from wild-born to captive-born at neutral microsatellite loci, care should be taken to maintain rare mtDNA haplotypes and MHC DRB alleles in future generations. ii Acknowledgements I would like to express very special thanks to Dr. Carlos de Luna, who introduced me to this project and served as my PhD supervisor. You recognised my passion for animal conservation whilst I was an animal science undergraduate and then encouraged me to pursue it further. In all the ups and downs on my PhD journey that then ensued you always believed in me. Thank you to Matthew Ford and Simon Jeffreys from Howletts and Port Lympne who collected the samples for this research. Thank you also to Dr. Joe Smith from Fort Wayne Childrens Zoo who also collected samples but for reasons beyond my control were not included within the study. I would also like to express special appreciation to Dr.Gaby Doxiadis from the Biomedical Primate Research Centre and her amazing team, Annemiek de Vos Rouweler and Nanine de Groot. Your help was invaluable for work carried out for the final experimental chapter. Thank you for allowing me to work in your laboratory and for sharing your expert knowledge in the subject. Finally, I have to thank my family and my friends. This project became my life and they stood by me and encouraged me every step of the way. To my parents, Val and Ken and my brother Stephen, your faith in me kept me motivated. To my husband, words can not express my thanks to you for all of your help, from making endless cups of coffee to proof reading. Your unwavering support was instrumental to my success. Lastly, to my son Noah. I dedicate this to you. iii Table of Contents Abstract i Acknowledgements iii List of Tables viii List of Figures x 1 Chapter one – Introduction ................................................................................... 1 1.1 The importance of conservation ................................................................ 1 1.2 Captive breeding and reintroduction programmes .................................. 3 1.2.1 The problems of inbreeding, outbreeding and adaptation ....................... 5 1.3 Hylobates moloch – the silvery gibbon ..................................................... 9 1.4 The aims of this study ............................................................................... 10 1.5 The objectives of this study ..................................................................... 11 2 Chapter two - Mitochondrial DNA analysis ......................................................... 14 2.1 Mitochondrial DNA – Form and Function ................................................ 14 2.1.1 Mitochondrial DNA as a molecular marker ............................................ 17 2.2 Materials and Methods .............................................................................. 20 2.2.1 Sample population ................................................................................. 20 2.2.2 DNA extraction ....................................................................................... 23 2.2.3 Hypervariable Region I (HV-I) PCR amplification .................................. 24 2.2.4 HV-I data analysis .................................................................................. 25 2.2.5 Statistical analysis ................................................................................. 26 2.3 Results ........................................................................................................ 31 2.3.1 Genetic diversity results ......................................................................... 32 2.3.2 The phylogeny of mtDNA HV-I within Captive and Wild haplotypes ...... 38 iv 2.4 Discussion .................................................................................................. 40 2.5 Conclusions ............................................................................................... 47 3 Chapter three - Analysis at the Nuclear DNA Level - Using Microsatellites to Estimate Genetic Diversity ....................................................................................... 49 3.1 The advantages and disadvantages of microsatellites in analysing genetic variation and ascertaining pedigree ................................................... 55 3.2 Materials and Methods .............................................................................. 61 3.2.1 Microsatellite analysis subjects .............................................................. 61 3.2.2 DNA extraction ....................................................................................... 62 3.2.3 Choice of microsatellite loci ................................................................... 62 3.2.4 Microsatellite PCR optimisation ............................................................. 66 3.2.4.1 Final PCR conditions ........................................................................ 67 3.2.5 Statistical analysis of microsatellites ...................................................... 69 3.2.5.1 Viewing allele sizes – electropherogram analysis ............................ 69 3.2.5.2 Identification of possible genotyping errors ...................................... 70 3.2.5.3 Measures of genetic diversity ........................................................... 70 3.2.5.4 Measures of genetic variation between the two groups ................... 74 3.2.5.5 The pedigree of Hylobates moloch ................................................... 75 3.3 Results of Microsatellite genetic diversity and pedigree analysis ....... 76 3.3.1 Genetic diversity measures at the group and locus level ....................... 77 3.3.2 Pedigree analysis .................................................................................. 84 3.4 Discussion – Does genomic DNA diversity follow the same patterns observed in mitochondrial DNA within the Hylobates moloch groups? ....... 86 3.4.1 Genomic diversity of Hylobates moloch ................................................. 91 v 2, 3.4.2 Values of SCALED Mean d , Standardized Heterozygosity (SH) and Internal Relatedness (IR), to analyse inbreeding levels .................................... 94 3.5 Conclusions ............................................................................................... 98 4 Chapter four - Analysis of an adaptive marker Class II of the Major Histocompatibility Complex .................................................................................... 100 4.1.1 Choice of region for analysis – The MHC class II DRB Exon 2 ........... 101 4.1.2 Hypotheses of driving forces behind MHC variation ............................ 102 4.1.3 The value of analyses of MHC class II genes for captive breeding and reintroduction programmes ............................................................................. 106 4.1.4 Choice of MHC DRB typing method .................................................... 110 4.2 Materials and Methods ............................................................................ 113 4.2.1 Sample population ............................................................................... 113 4.2.2 DNA extraction ....................................................................................