Genetic and morphological analyses of historic and contemporary populations of western lowland gorilla: A multidisciplinary approach for the conservation of a critically endangered primate by Jaimie Morris Canterbury Christ Church University Thesis submitted for the Degree of Doctor of Philosophy of Biological Science 2020 Abstract This study investigates the morphology and genetic diversity of the critically endangered sub-species, the western lowland gorilla (Gorilla gorilla gorilla). Regional variation of a historic wild population was assessed morphologically and genetically, and genetic comparisons between this and a contemporary captive population were made to assess the genetic fitness of the contemporary population with the aim of assisting future conservation planning. Geometric morphometric analyses were applied to skulls and mandibles of both sexes in the historic population of gorillas to assess regional variation in relation to size and shape. No significant difference was found for regional size comparisons but shape variation between regions did find significant variation in skull morphology, particularly for males. MtDNA and nuclear markers were employed to detect regional differentiation in the historic population of gorillas, and to compare genetic diversity between historic and contemporary populations. The mtDNA results were hindered by nuclear insertions (numts) yet 30 sequences of the mitochondrial Control Region Hypervariable Region I (HVI) were obtained and haplogroups identified, which revealed potential differences in the historic distribution of haplogroups than current literature reports. Nuclear analysis based on microsatellites confirmed that all the gorillas used in this study were western lowland gorillas. Furthermore, the paternity of individuals in the contemporary population was confirmed. Comparisons between the historical population and the captive US population showed that genetic diversity of the contemporary population had been retained at similar levels to wild populations and the US captive population thus concluding that the contemporary population is genetically sustainable for the foreseeable future. ii Contents Abstract ii. Contents iii. List of tables vii. List of figures ix. List of appendices xvi. Dedication xvii. Acknowledgements xviii. Author declaration xx. Chapter 1 General Introduction 1 1.1 Structure and aims of thesis 1 1.2 An introduction to gorillas 2 1.2.1 Gorilla taxonomy 3 1.3 The western lowland gorilla 7 1.3.1 Gorilla distribution and population status 7 1.3.2 Physical traits and characteristics 9 1.3.3 Habitat and diet 9 1.3.4 Behaviour and social structure 10 1.3.5 Conservation status and population threats 10 1.4 The importance of natural history collections 11 1.5 The Powell-Cotton Museum natural history collection 12 1.6 Geographical Information Systems (GIS) and biodiversity mapping 16 1.7 Biodiversity mapping of the Powell-Cotton Museum data 19 1.7.1 Western lowland gorilla mapping 20 1.8 An introduction to geometric morphometrics 23 1.9 The role of wildlife parks/zoos in species conservation 27 1.10 The importance of genetic diversity 30 1.10.1 Mitochondrial DNA 31 1.10.2 Microsatellites 32 1.11 Regional variation of western lowland gorillas 33 1.12 The gorillas in this study 34 1.11 Summary 35 iii Chapter 2 Regional morphological variation of a historical population of western 36 lowland gorilla 2.1 Introduction 36 2.2 Aims 39 2.3 Hypotheses and predictions 39 2.4 Methods 40 2.4.1 Historical western lowland gorilla specimens 40 2.4.2 Geographical location of historical specimens 41 2.4.3 Digitisation of samples 43 2.4.4 Procrustes superimposition 45 2.4.5 Western lowland gorilla skull and mandible centroid size analyses 46 2.4.6 Western lowland gorilla skull and mandible shape analyses 46 2.5 Results 48 2.5.1 Tests for accuracy of landmark placement 48 2.5.2 Preliminary analyses of data 50 2.5.3 Regional variation in skull and mandible size of historical western lowland 53 gorillas 2.5.4 Regional variation in skull and mandible shape of historical western 55 lowland gorillas 2.6 Discussion 63 2.6.1 Skull and mandibular regional variation in western lowland gorillas 64 2.6.2 Further research 67 Chapter 3 Past and present populations of the western lowland gorilla: 69 Mitochondrial Hypervariable Region I 3.1 Introduction 69 3.2 Gorilla populations, past and present 70 3.3 Aims 72 3.4 Hypotheses and predictions 72 3.5 Methods 73 3.5.1 Sampling and DNA extraction 73 3.5.2 Mitochondrial DNA amplification and sequencing 77 3.5.3 Sequence alignment and genetic diversity analyses 79 3.5.4 Phylogenetic analyses 81 3.5.5 Detection and implications of numt sequences 83 3.6 Results 84 iv 3.6.1 Genetic diversity 84 3.6.2 Phylogenetic analyses 88 3.6.3 True mtDNA sequences 94 3.6.4 Genetic diversity of true mtDNA sequences 98 3.7 Discussion 104 3.7.1 Data analysis with numt sequences 105 3.7.2 True mtDNA data and phylogenetic relationships 106 3.7.3 Genetic diversity and structure of true data 107 3.7.4 Captive and historic populations 109 Chapter 4 Microsatellite genotyping of historic and contemporary populations of 113 western lowland gorillas 4.1 Introduction 113 4.2 Aims 114 4.3 Hypotheses and predictions 115 4.4 Methods 115 4.4.1 DNA extraction, amplification and genotyping 115 4.4.2 Assessing genotyping errors 121 4.4.3 Linkage disequilibrium and the Hardy-Weinberg equilibrium 122 4.4.4 Genetic diversity analysis 123 4.4.5 Genetic structure of populations 126 4.4.6 Genetic bottlenecks, effective population size, migration and mutation 129 4.5 Results 130 4.5.1 Geographic regions and sampling 130 4.5.2 Genotyping errors, data quality checks and paternity testing 131 4.5.3 Hardy-Weinberg equilibrium and linkage disequilibrium 133 4.5.4 Genetic diversity 133 4.5.5 Genetic structure 142 4.5.6 Genetic bottlenecks, effective population size and migration 154 4.6 Discussion 156 4.6.1 Reliability of the data and pre-analysis observations 156 4.6.2 Species and subspecies determination and population structure 158 4.6.3 Regional genetic diversity of the historic western lowland gorillas 164 4.6.4 Genetic diversity and demographic comparisons of the contemporary and 165 historic western lowland gorillas 4.6.5 Conservation implications 168 v Chapter 5 General Discussion 172 5.1 Main findings and conservation implications 172 5.2 Complementary or contradictory? A summarised synthesis of findings 180 5.3 Future work and impact case studies 183 5.4 General conclusions 185 References 186 Appendix 1 219 Appendix 2 221 Appendix 3 222 Appendix 4 226 Appendix 5 228 vi List of tables Table 1.1. Summarised hypotheses of gorilla taxonomy based on morphological research. Table 1.2. Currently accepted gorilla taxonomy. Table 1.3. Summary of gorilla subspecies diets. Table 1.4. Examples from the gorilla field notes recorded by Fred Merfield from the Powell-Cotton Museum collection. Table 2.1. Analysis of variance (ANOVA) results for landmark placement using centroid size (transformed with natural logarithm) of western lowland gorillas. Table 2.2. Summary of results for sampling adequacy and tests of sphericity for four datasets of western lowland gorillas. Table 2.3. Total variance of principal components based on eigenvalues for all four datasets of western lowland gorillas. Table 2.4. Analysis of variance (ANOVA) results for centroid size (transformed by natural logarithm) of western lowland gorillas. Table 2.5. Multivariate analysis of variance (MANOVA) and pairwise tests for regional variation in shape for western lowland gorillas. Table 3.1. Summary of DNA polymorphism and genetic diversity of the 11 groups of gorillas. Table 3.2. Summary of DNA polymorphism and genetic diversity of captive and wild populations of gorillas. Table 3.3. Summary of R2 and P-values for the 11 groups of gorillas. Table 3.4. Pairwise FST differences using the distance method for the 11 groups of gorillas. Table 3.5. Genetic distance matrix for the 11 groups of gorillas based on mitochondrial Hypervariable Region I (HVI) sequences. Table 3.6. Pairwise differences among gorilla groups based on mitochondrial Hypervariable Region I (HVI) DNA sequences. Table 3.7. R2 statistics for gorilla groups based on mitochondrial Hypervariable Region I (HVI) DNA sequences. vii Table 3.8. Genetic diversity of haplogroups and haplo-subgroups of gorillas based on mitochondrial Hypervariable Region I (HVI) DNA sequences. Table 3.9. FST pairwise differences of haplogroups and haplo-subgroups of gorillas based on mitochondrial Hypervariable Region I (HVI) DNA sequences. Table 3.10. R2 values of haplogroups and subgroups of gorillas based on mitochondrial Hypervariable Region I (HVI) DNA sequences. Table 4.1. Panel of 10 microsatellite loci used for this study (based on Bradley et al. 2000). Table 4.2. Thermocycler protocols tested for the amplification of 10 microsatellite loci. Table 4.3. Genotype scoring data for Louna for two methods of DNA extraction (tissue and FTA card). Table 4.4. Data quality checks for known family groups. Male (sire) alleles are indicated in blue and female (dam) in pink. Table 4.5. Paternity testing of Kwimba’s infants between two sires (Djanghou and Kisane). Green alleles indicate a mismatch in paternity. Table 4.6. Number of alleles (NA), allelic richness (AR), effective number of alleles (Ae), observed heterozygosity (HO), individual heterozygosity (HI) and expected heterozygosity (HE) by loci for the contemporary and historic populations and subgroups of gorillas. Table 4.7. Mean results of genetic diversity for populations of gorillas for number of alleles (Na), allelic richness (AR), effective number of alleles (Ae), observed heterozygosity (HO), expected heterozygosity (HE) and individual heterozygosity (HI). Results for the effective population size (Ne), number of migrants (Nm), θ (under the step-wise mutation model) and the inbreeding coefficient (FIS) are also included.