Molecular Systematics and Conservation Genetics of Gliding Petaurids (Marsupialia: Petauridae) By Mansoureh Malekian (M.Sc.) A thesis submitted in the fulfilment of the degree of Doctor of Philosophy of Science (Ph.D.) in the Department of Ecology and Evolutionary Biology June 2007 Table of content Table of content II List of figures VII List of tables X Declaration XII Abstract XIII Acknowledgments XV 1 General introduction 1 1.1 Preamble 1 1.2 Introduction 1 1.3 Current taxonomy and distribution of gliding petaurids 2 1.4 Conservation status 3 1.5 Phylogeny and evolution of gliders 5 1.6 Phylogeography and population structure 6 1.7 Genetic variability and habitat fragmentation 7 1.8 Study aims 9 2 Molecular systematics of the genus Petaurus (Marsupialia: Petauridae) in Australia and New Guinea 11 2.1 Introduction 11 2.2 Material and methods 15 2.2.1 Taxa sampling 15 II 2.2.2 Genetic analyses 16 2.2.3 Choice of outgroups 16 2.2.4 DNA isolation, PCR- amplification and sequencing 17 2.2.5 Sequence analysis 18 2.2.6 Phylogenetic analyses 19 2.2.7 Molecular clock and divergence time estimates 20 2.3 Results 22 2.3.1 Sequence analyses 22 2.3.1.1 Mitochondrial regions 22 2.3.1.2 Nuclear marker 23 2.3.2 Phylogenetic analyses 24 2.3.2.1 Mitochondrial gene analyses 24 2.3.2.2 Nuclear gene analyses 25 2.3.3 Molecular clock and divergence time estimates 28 2.4 Discussion 31 2.4.1 Phylogenetic relationships 31 2.4.2 Divergence time and biogeography 35 3 Phylogeography of the sugar glider ( Petaurus breviceps ) in Australia 37 3.1 Introduction 37 3.2 Material and methods 39 3.2.1 Population sampling 39 3.2.2 PCR- amplification and sequence analyses 40 3.2.3 Phylogenetic analyses 40 3.2.4 Population structure 42 3.3 Results 42 3.3.1 Variation and distribution of haplotypes 42 3.3.2 Phylogenetic relationships 43 3.3.3 Population structure 44 3.3.3.1 Mitochondrial region 44 3.3.3.2 Omega-globin gene 49 III 3.4 Discussion 50 3.4.1 Phylogeography and genetic structure 50 3.4.2 Taxonomy 51 3.4.3 Implications for conservation 53 4 Nest box-use, social structure and mating system of P. breviceps 55 4.1 Introduction 55 4.2 Material and methods 57 4.2.1 Study area 57 4.2.2 Sampling methods 57 4.2.3 Comparison between small and large patches 60 4.2.4 DNA extraction and microsatellite analysis 60 4.2.5 Parentage analysis and mating system 61 4.2.5.1 Relatedness analysis 63 4.3 Results 64 4.3.1 Nest box occupancy 64 4.3.1.1 Species, Occupancy rate and pattern of use 64 4.3.1.2 Comparison between small and large patches 64 4.3.2 Parentage analyses 69 4.3.3 Mating system 74 4.3.4 Relatedness and kinship 75 4.4 Discussion 78 4.4.1 The effects of patch size on nest-box use, group size and structure 78 4.4.2 Mating system of P. breviceps 80 4.4.3 Relatedness and Kin structure of P. breviceps 81 4.4.4 Inbreeding avoidance 82 5 Genetic diversity and population structure of P. breviceps 84 5.1 Introduction 84 5.2 Materials and methods 86 IV 5.2.1 Study populations and molecular data 86 5.2.2 Genetic diversity 86 5.2.3 Population structure 87 5.3 Results 88 5.3.1 Genetic diversity 88 5.3.2 Population structure 90 5.4 Discussion 92 6 An extension to the known distribution of the squirrel glider ( Petaurus norfolcensis ) in Australia 95 6.1 Preamble 95 6.2 Introduction 95 6.3 Material and methods 97 6.3.1 Genetic investigation 97 6.3.2 Field investigations 98 6.3.3 Morphometric assessments 98 6.4 Results 100 6.4.1 Genetic investigation 100 6.4.2 Field investigation 101 6.4.3 Morphometric assessments 103 6.5 Discussion 107 7 Concluding discussion 109 7.1 Review of aims 109 7.2 Evolutionary relationships of Petaurus species 109 7.3 Phylogeography and population differentiation within P. breviceps in Australia. 110 V 7.4 Nest box-use social structure and mating system of P. breviceps in fragmented habitats 111 7.5 Genetic diversity and population structure of P. breviceps 112 7.6 Limitations of the study 113 7.7 Further research 113 References 116 Appendix 1 137 Appendix 2 139 Appendix 3 159 Appendix 4 168 Appendix 5 170 Appendix 6 188 Appendix 7 195 Appendix 8 208 VI List of figures Figure 2.1 Distribution map of the glider species in Australia and New Guinea. .......... 13 Figure 2.2 Current distribution of subspecies of P. breviceps in Australia and New Guinea............................................................................................................................. 15 Figure 2.3 Phylogenetic relationships of Petaurus species in comparison with the representatives from other genera of Petauridae, using Maximum Parsimony analyses of ND2 sequence data.......................................................................................................... 26 Figure 2.4 Phylogeny of the genus Petaurus based on combined mitochondrial ND2 and ND4 genes....................................................................................................................... 27 Figure 2.5 Distribution map of the mitochondrial lineages within P. breviceps in Australia and New Guinea.............................................................................................. 29 Figure 2.6 ω-globin gene tree inferred using a partitioned mixed-model in MrBayes... 30 Figure 2.7 50% posterior probability Bayesian consensus tree using all three genes (ND2 , ND4 and ω-globin gene) with model partitioning, implemented in MRBAYES.31 Figure 2.8 The distribution of morphological subspecies and evolutionary lineages from genetic data obtained in the present study. ............................................................ 35 Figure 3.1 Omega-globin gene tree of P. breviceps inferred using a partitioned mixed- model in MrBayes........................................................................................................... 46 Figure 3.2 Maximum Parsimony tree of combined mtDNA ( ND2 and ND4 ) from P. breviceps in Australia ..................................................................................................... 47 Figure 3.3 Unrooted network of mtDNA haplotypes from P. breviceps haplotypes inferred using statistical parsimony and associated nested clade design........................ 48 Figure 3.4 Haplotype network of ω-globin gene generated under 95% statistical limit of parsimony........................................................................................................................ 49 Figure 3.5 Distribution of the current subspecies of P. breviceps in Australia and the two mtDNA clades found in the current study. .............................................................. 53 VII Figure 4.1 Location of 23 patches surveyed in this study. ............................................. 59 Figure 4.2 Seasonal use of nest boxes by P. breviceps ................................................... 65 Figure 4.3 The percentage of the total of nest box checks where a nest box was occupied by at least one P. breviceps individual in five small and five large patches. ................. 66 Figure 4.4 The mean number of individual P. breviceps per nest box in small and large patches. ........................................................................................................................... 66 Figure 4.5 Frequency histogram of the number of P. breviceps inhabiting nest boxes at one time in small and large patches. ............................................................................... 67 Figure 4.6 The mean number of reproductively active adult male and female P. breviceps per nesting group in small and large patches.................................................. 68 Figure 4.7 The mean number of juvenile and sub-adult male and female P. breviceps per nesting group in small and large patches.................................................................. 68 Figure 4.8 Average coefficient of relatedness of nesting adult individuals in nest groups ........................................................................................................................................ 76 Figure 4.9 Average coefficient of relatedness for nesting individuals nesting together. 77 Figure 5.1 Log likelihood probability of data (Ln P(X/K) as a function of K for P. breviceps samples from 16 populations.......................................................................... 92 Figure 6.1 Distribution map of P. norfolcensis in Australia prior to this study. ............ 95 Figure 6.2 Sketch map of upper south-east of South Australia. ..................................... 96 Figure 6.3 Parameters used in morphometric analysis ................................................... 99 Figure 6.4 Neighbour-Joining phylogram from 700 base pairs of ND2 sequenced from four Australian gliding petaurid species and suspected P. norfolcensis from Bordertown and Western Flat.. ......................................................................................................... 101 Figure 6.5 The first live suspected squirrel glider in South Australia. ......................... 102 Figure 6.6 Roadside habitat in Western Flat................................................................ 103 VIII Figure 6.7 Bivariate plot of Condylobasal length (CBL) over maximum zygomatic breadth (MZB) for P. breviceps and P. norfolcensis .................................................... 105 Figure 6.8 Relationship between the first (PCA1) and second (PCA2) components of the Principle Component Analysis for P. breviceps and P. norfolcensis. .......................... 105 Figure 6.9 Relationship between the first (PCA1) and second (PCA2) components