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Item Population genetics and genomics of a marsupial species : analysis of native and invasive brushtail possum populations (Trichosurus vulpecula) : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Conservation Biology, Massey University, Manawatū Campus, New Zealand(Masey University, 2024-04-15) Pattabiraman, NimeshikaOne of the leading causes of global biodiversity decline is the introduction of invasive pest species that destroy native flora and compete with native fauna for food and other resources. Aotearoa New Zealand is one of the foremost countries in the world that has focussed on eradicating pest species and in particular exotic mammals from the archipelago, which lacks native, terrestrial mammals. The New Zealand Government recently set in train the ambitious task of removing all mustelids, rats, and possums from the terrestrial landscape by the year 2050. Brushtail possums (Trichosurus vulpecula) were introduced to Aotearoa New Zealand from Australia in the mid-1800s, after which they were translocated across the country and have become widespread, destroying indigenous habitat, eating native birds and invertebrates, and spreading bovine TB. Control efforts have seen possum numbers decline in the last two decades from close to 75 million in 2002 to 40 million in 2020. There is, however, a gap in the scientific understanding of possum populations with respect to their genetic composition and population structure across the country, and this knowledge could help us develop effective and dynamic management strategies to eradicate possums on a nationwide scale. In this thesis, I focus on three aspects of population structure and diversity of brushtail possums. First, I investigated a small geographical study area - The Kenepuru Peninsula - where I sought evidence of genetic correlations with geography, time and fur colour. I used two types of genetic markers that target the nuclear and mitochondrial regions of possum DNA with large population samples. In every case, it was determined that the possums comprised one freely interbreeding population at this scale. In particular I demonstrated that colour morphs associated with distinct subspecies in Australia, freely interbreed in New Zealand. I then increased the scale of sampling to include representation of populations across New Zealand and Australia, with the same genetic markers. This threw light on the heterogenous nature of possum diversity in New Zealand, and showed that even after ~110 generations, possums retained genetic separation among spatial groups. Additionally, the data showed evidence of multiple possum lineages across New Zealand that are derived from several Australian populations. High haplotype diversity in New Zealand suggests that the rapidly expanding population has retained novel haplotypes and the data thus far indicated a non-homogenous (metapopulation) distribution of possums without geographical concordance. As the project progressed, I was able to apply high-throughput genotyping-by-sequencing to generate a large genomic dataset. This dataset provided much more detail of the genotypic distribution of possums in Australia and among invasive metapopulations in New Zealand, as well as informing us of the relationship between them. This large, robust database of possum population structure and genetic diversity throughout Aotearoa New Zealand will support future studies in providing informed management decisions to eradicate brushtail possums.Item The molecular ecology of an understudied endemic marine isopod - Isocladus armatus : a thesis presented in partial fulfilment of the requirements for the degree of Master of Natural Science at Massey University, Albany, New Zealand(Massey University, 2019) Pearman, William SamuelThe study of populations and the adaptive significance of traits is a major theme in molecular ecology literature. In this thesis I present three lines of research that contribute to the understanding the molecular ecology of a species of New Zealand endemic marine isopod - Isocladus armatus (family: Sphaeromatidae). The goal of this thesis is to develop and utilize a framework to better understand the genomics of marine isopods from a range of genomic perspectives. The first primary chapter aims to assess two ways of enriching mitochondrial DNA from whole genome DNA, and to assemble this species mitochondrial genome. My research indicates that an atypical mitochondrial genome structure, widespread across Isopoda - but previously thought absent within Sphaeromatidae, is present within I. armatus suggesting that this trait has been maintained for an order of magnitude longer than previous estimates. The second primary chapter aims to describe and understand the genetic structure of populations for 8 locations around New Zealand, to understand connectivity and dispersal for I. armatus. Using a panel of 8,020 loci, I find high gene flow on a small spatial scale, while populations on a larger spatial scale exhibit a pattern of Isolation-By-Distance. Additionally, gene flow over one well known biogeographic barrier was much higher than between any other populations on a similar spatial scale, suggesting this barrier may not exhibit a strong effect on this species. Thus, my research indicates a need to revisit and study the way biogeographic barriers affect species with different life histories. The final primary chapter aims to understand the genetic basis for colour polymorphism in I. armatus, with the intention of understanding the adaptive significance and selective mechanism behind this trait. I use genome wide association approaches with a panel of 20,000 loci to answer these questions. I found that loci associated with Colour Polymorphism exhibited signatures of disruptive selection, contrary to initial hypothesis where I expected balancing selection to main colour polymorphism. I propose that substrate heterogeneity in Isocladus armatus’ habitat results in microhabitats, each of which imposes a selective pressure benefiting a specific morph type. The size of these microhabitats is so small that high levels of interbreeding between these microhabitats, and thus between morphs, results in the maintenance of polymorphism across the population.Item Landscape genetics for conservation management : brushtail possums (Trichosurus vulpecula) in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Conservation Biology at Massey University, New Zealand(Massey University, 2019) Pattabiraman, NimeshikaThe negative impact of brushtail possums (Trichosurus vulpecula) on New Zealand ecosystems became apparent soon after their introduction from Australia in 1858. Possums not only denude native vegetation but prey on native birds and invertebrates. They also carry bovine tuberculosis (TB) impacting the dairy industry and consequently the New Zealand economy. New Zealand possum populations have spread from several introduction sites and densities have increased. The resulting complex patterns of gene flow influences regional diversity, and potentially the effectiveness of control measures. Currently, ~100 million dollars are spent on 1080 management per year, mostly in response to Tb risk, but there is little information about the migration rates associated with resulting population density fluctuations. To determine whether the potential for intermixing between populations since their introductions could have caused a homogenizing effect on the genetic diversity across New Zealand, I began a detailed population genetic analysis by genotyping possums from 19 locations using nuclear microsatellites and mitochondrial DNA haplotyping from across the country to estimate population structure. Initial introductions of possums from multiple locations resulted in genetic and fur colour diversity but, in comparison to natural Australian populations, it appears that only a subset of genetic variants was brought to New Zealand from Australia. Mitochondrial sequence variation analyses showed overall high haplotype diversity with substantial differences among samples in haplotype frequencies, but with relatively low nucleotide diversity. Similarly, analysis of nuclear markers (microsatellite genotypes with Naïve Bayesian clustering) reveals that while there has been admixture between populations in various locations, indicated by shared genotypes, there are genetically distinct regional populations. Concordance of genetic and geographically distant sampling shows a well-developed population structure of possums across New Zealand. These results are also supported by pairwise Fst comparisons between all pairs of populations; although nearly all populations showed significant differences, there was no signature of isolation by distance as expected from their history of introductions. This study provides a foundation for further research into spatial structure of brushtail possums which will enable the effective targeting of management and is essential for modelling population recovery, disease spread, and potentially the emergence of toxin resistance. Predator-free 2050 is an ambitious objective considering current circumstances. In order to achieve its goals, even for the targeted species, we need to efficiently manage our resources and improve the accuracy of control measures to maintain long-term effects.Item Mitochondrial DNA diversity and variability in the Adélie penguin of Antarctica : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand(Massey University, 2003) Gibb, Gillian ClaireIn Antarctica, there are two distinct lineages of Adélie penguin (Pygoscelis adeliae) characterised by 8.3% divergence in mitochondrial DNA hypervariable region I (mt DNA HVR I). These two lineages are known as the Antarctic and Ross Sea lineages (A and RS respectively). This study aims to characterise aspects of mutation and variation as seen in HVR I of the Adélie penguin, by sequencing the DNA of individuals from different locations around Antarctica. The geographic distribution of the two lineages was examined in greater detail. A dramatic decrease in the RS lineage was discovered on the edge of the Ross Sea region of Antarctica. Because the two lineages have different geographic distributions, and are separated by 8.3% sequence divergence, this study also investigated the possibility that these two lineages were in fact cryptic species. Sequencing of mt DNA and microsatellite genotyping proved that individuals of the two lineages mate randomly and produce offspring. Recently, a rate of evolution based on serially preserved DNA from Adélie penguins was estimated at 0.96 substitutions/site/Million years. (0.53-1.43 s/s/Myr). This rate is four to seven times higher than previous avian control region evolution rates estimated by phylogenetic methods, and is more akin to rates of mutation determined by pedigree studies in other species such as humans. In the light of this higher direct estimate of the rate of evolution in Adélie penguins, this study also begins to determine a rate of mutation in Adélie penguins based on pedigree analysis. No new mutations were found, however three cases of inherited single point heteroplasmy were detected. The inclusion of heteroplasmy in mutation rate calculation is also addressed. One of the arguments as to why pedigree studies find a higher rate of mutation than phylogenetic studies is that pedigree studies preferentially find mutations at 'hot spots' in the DNA sequence. This study also seeks to characterise the distribution of variable sites in hypervariable region I in relation to the two mt DNA lineages, and also to geographic location. While the exact sites of variation differ between the two lineages, it was seen that the regions where variation was high or low is very similar in both lineages. This could be due to underlying physical constraints on DNA sequence variation. Looking towards future work in Adélie penguin mt DNA and an expansion of the studies undertaken here, the complete mitochondrial genome of the Adélie penguin was determined. This now provides the opportunity to estimate rates of change in the entire Adélie penguin mitochondrial genome, using ancient DNA from the extremely well preserved sub-fossil bones in Antarctica.Item Avian phylogeny and divergence times based on mitogenomic sequences : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand(Massey University, 2012) Slack, Kerryn ElizabethDespite decades of research using a variety of data sources (such as morphological, paleontological, immunological, DNA hybridization and short DNA sequences) both the relationships between modern bird orders and their times of origin remain uncertain. Complete mitochondrial (mt) genomes have been extensively used to study mammalian and fish evolution. However, at the very beginning of my study only the chicken mt sequence was available for birds, though seven more avian mt genomes were published soon after. In order to address these issues, I sequenced eight new bird mt genomes: four (penguin, albatross, petrel and loon) from previously unrepresented orders and four (goose, brush-turkey, gull and lyrebird) to provide improved taxon sampling. Adding these taxa to the avian mt genome dataset aids in resolving deep bird phylogeny and confirms the traditional placement of the root of the avian tree (between paleognaths and neognaths). In addition to the mt genomes, in a collaboration between paleontologists and molecular biologists, the oldest known penguin fossils (which date from 61- 62 million years ago) are described. These fossils are from the Waipara Greensand, North Canterbury, New Zealand, and establish an excellent calibration point for estimating avian divergence times. Bayesian analysis of the DNA sequence data, using the penguin calibration point plus two others, indicates a substantial radiation of modern bird orders in the Late Cretaceous (80 - 65 million years ago). Biotic interactions between modern birds and declining groups such as pterosaurs and early bird groups (e.g. Hesperornis and Ichthyornis) may thus have played an important role during this time.Item Nuclear and mitochondrial DNA evolution in Adélie penguins : studies of modern and ancient populations : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics, Allan Wilson Centre for Molecular Ecology and Evolution, Institute for Natural Sciences, Massey University, Auckland, New Zealand(Massey University, 2012) Beans Picón, Gabrielle AngelaThe Adélie penguin of Antarctica (Pygoscelis adeliae) breeds on the Antarctic continent and on offshore islands. Its evolutionary history has been, and its current biology remains, dependent on a range of climate variables. Over geological time, glacial warming and cooling periods have resulted in Adélie penguin populations decreasing and expanding. Therefore, understanding Adélie penguin population dynamics at a genetic level can provide insights into how the species responds to changing climates, one reason why Adélie penguins are an important natural model species. In addition, sub-fossil bone deposits of this species below modern and abandoned colonies provide an excellent source of ancient DNA that can bring a temporal dimension to population studies of the species. In combination, these attributes enable us to address some fundamental questions regarding evolutionary change. Making use of known mitochondrial DNA mutation rates and current population sizes, a positive and significant correlation between population size and modern mitochondrial control region diversity was detected. This finding supports the use of mitochondrial DNA for population inferences. Effective population sizes of breeding colonies are shown to have increased since the late Pleistocene. To extend current tools available for understanding Adélie penguins, six nuclear intron loci were recovered from a wide range of introns that can be applied to population genetics and phylogenetic studies of penguins. Five introns were used to investigate the persistence of the mitochondrial Antarctic (A) and Ross Sea (RS) lineages. No evidence for the existence of these lineages was found in the nuclear loci sequenced. A signature of historical population expansion, preceding the mitochondrial one, was detected. The utility of four introns in resolving penguin phylogenetic signals was also determined. Non-coding nuclear sequence of one intron were obtained from ancient sub-fossil remains of Adélie penguins using multiplex PCR enrichment, followed by second-generation sequencing of a barcoded library. A shift in haplotype frequencies was detected between ancient and modern intron sequences in Adélie penguins, despite a small sample size. In the future, advancing the current methodologies and extending sampling to additional introns as well as older samples, is likely to provide a new level of understanding of this remarkable species.Item DNA barcoding the birds of New Zealand : a thesis presented in fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular BioSciences at Massey University, Auckland, New Zealand(Massey University, 2011) Waugh, William John; Waugh, William JohnA comprehensive inventory of the life forms on earth is at the heart of any scientific study of evolution and biodiversity. The international "Barcode of Life" project is an attempt to identify the earth's biodiversity, at the species level, using short signature DNA sequences. The hypothesis underlying DNA barcoding is being comprehensively tested in different taxa. A database was constructed of DNA sequences from part of the mitochondrial gene cytochrome c oxidase subunit 1 for the avian fauna of New Zealand. To date, 833 sequences from 215 species have been added to this database, of which 628 sequences from 126 species are from native or endemic birds. This represents an average of 5 samples per species (minimum 1, maximum 18) for the latter group, which is the central focus of this thesis. Samples of species, from different geographical locations throughout New Zealand, have been collected to highlight any intraspecific nucleotide variation that may occur. Some samples analysed here were from historical specimens housed in museum collections and required specialised DNA extraction and amplification. These techniques were developed as part of the project and provide a means of collecting DNA barcodes where no modern material is available. In general, DNA barcoding proved effective at identifying avian species in New Zealand. However, some species were highlighted that contained distinct DNA barcode clusters, indicative of possible subspecies or cryptic species while in other cases two or more species that appear to be different share very similar DNA barcodes. Remains from aircraft birdstrikes were identified using this technique in order to inform wildlife management at airports around New Zealand. A review of and outlook for the uses of this technique are given.Item The mitochondrial genome of the little spotted kiwi : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biosciences, Massey University, Palmerston North, New Zealand(Massey University, 2010) Crimp, Elizabeth AnnThe complete mitochondrial genome of the little spotted kiwi, Apteryx owenii, has been sequenced and submitted to GenBank (acquisition number GU071052). A method was first developed to extract pure mitochondrial DNA from one millilitre of fresh blood; as birds have nucleated erythrocytes/red blood cells. The mitochondrial DNA was extracted from the isolated intact mitochondria and the genome was amplified by long-range PCR as 1- 4kb overlapping fragments. These fragments then became templates for the second, short-range, overlapping PCR amplifications and subsequent DNA sequencing. This procedure was first trialled using two millilitres of chicken blood before being successfully applied to the kiwi blood. The complete mitochondrial genome of the little spotted kiwi is ~ 17,020bp long. The gene order is the standard avian gene order first reported for chicken mitochondrial DNA. Phylogenetic relationships show the kiwi is part of the Australasian ratite group with the emu and cassowary. This mitochondrial sequence has been used as part of a larger study of the relationships of other ratite birds (such as moa, emu, cassowary, rhea and ostrich) and the weakly flying tinamou of South America. The implication of this analysis is that the ancestral paleognath was probably flying and that flight was lost multiple times during ratite evolution.Item Mammalian evolution and phylogeny from complete mitochondrial genomes : a thesis in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Molecular BioSciences at Massey University(Massey University, 2001) Lin, Yu-HsinThe evolutionary tree of mammals is being resolved quickly. Complete mitochondrial DNA sequences are valuable data for deep mammalian phylogenetic relationships. From this study, the use of long-range PCR followed by short-range PCR and sequencing was proven to be a successful strategy for sequencing complete mt-genomes. This method is more efficient and cheaper than current cloning approaches. This method is also able to avoid most of the nuclear mitochondrial copies. Long branch attraction is a problem confusing the deep mammalian phylogeny. By sequencing complete mt-genomes of key taxa (2 marsupials, 2 bats, a pika, a gymnure, a rodent and a shrew) to break up long branches, this study resolve some ambiguous relationships in mammalian phylogeny. The 8 mammalian mitochondrial DNA sequences from this study give additional support for the 4 groupings (Xenarthrans, Afrotheria, Supraprimates and Laurasiatheria) of placental mammals from current molecular studies. Some of the ambiguous relationships of higher mammalian relationships also get improved resolution. Bats are a monophyletic group but megabats may be paraphyletic. Eulipotyphla is a monophyletic group and deepest in the Laurasiatheria. Rodents are monophyletic and apart from a problem with the tree shrew, are sister to lagomorphs (Glires). With the new gymnure complete mt-DNA available, the aberrant hedgehog mt-genome is returning to its traditional position in the placental tree and joins other Eulipotyphla (mole, shrew). This monophyletic Eulipotyphla is observed for the first time in the mammalian mitochondrial tree. The Erinaceidae (hedgehog and gymnure) and murid rodent seem to be under different processes of evolution and are attracted to the outgroups. By comparing trees without outgroups (unrooted trees) and with outgroups (rooted trees) and by constraining group(s) with unstable positions, the influence of marsupials/platypus outgroups on Erinaceidae and murid rodent can be investigated. The results from this study suggest that there is a long branch attraction problem between marsupials/platypus outgroups and murid rodent and Erinaceidae; the basal postions of Erinaceidae and murid rodent found in previous studies may be long branch attraction artifacts. The resolved mammalian tree will be the basis for further molecular studies for estimating the time of divergence of extant mammalian orders, for the prediction of protein secondary structure, for the processes of transition of nucleotides and amino acids sequences in the tree, etc. Having a resolved mammalian tree is not the end for this research, rather a pivotal step for understanding evolution in molecular level.Item Birds in a tree : a journey through avian phylogeny, with particular emphasis on the birds of New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics(Massey University, 2010) Gibb, Gillian ClaireTwo main themes to the avian research presented in this thesis are, 1. Deep resolution of birds generally, and 2. Investigation of specific aspects of the New Zealand avifauna. More specifically, this thesis covers phylogeny, and predictions about palaeognaths, pigeons, pelecaniforms and passerines. Significant progress is made in resolving the basal branches of Neoaves. This thesis examines whether the six-way basal Neoavian split of Cracraft (2001) is, in principle, resolvable. New mitochondrial genomes are added to improve taxon sampling, break up long branches, and allow testing of the prior assumptions of six Neoavian groups. This research shows the six-way split is resolvable, although more work is required for specific details. From a life-history perspective, it is interesting that the two bird-of-prey groups (falcons and buzzards) are very divergent, and may not be sister groups. Molecular dating supports major diversification of at least 12 Neoavian lineages in the Late Cretaceous. Additionally, novel avian mitochondrial gene orders are investigated and a hypothesis put forward suggesting gene conversion and stable intermediate forms allows an apparently rare event (gene rearrangement) to occur multiple times within Neoaves. One of Cracraft’s six groups, informally called the ‘Conglomerati’, is particularly difficult to resolve. The pigeons (Columbiformes) lie within the ‘Conglomerati’, and this chapter examines two aspects along the continuum of pigeon evolution. Firstly the large South Pacific fruit pigeon radiation is examined with mid-length mitochondrial sequences. This clade contains a third of all pigeon species, and has been very successful in island colonisation throughout South East Asia and the Pacific. Secondly, candidates for the closest relative of pigeons are tested using analysis of whole mitochondrial genomes. Highest support was found for the grouping of sandgrouse and pigeon, although they are clearly very divergent. Also within the ‘Conglomerati’ is the traditional order Pelecaniformes, and their close allies the Ciconiiformes. These orders (the P&C) are part of an adaptive radiation of seabird water-carnivores, including loons, penguins, petrels and albatrosses. This group is separate from the large shorebird water-carnivore group; although both appear to have begun radiating abut 70 million years ago. The tropicbird represents a separate, convergent life history and is not part of the Pelecaniformes, nor within the larger seabird water-carnivore group. Resolution of the basal phylogeny of oscine passerines is important for interpreting the radiation of this group out of the Australasian region. Many endemic New Zealand oscine passerines belong to ‘basal corvid’ lineages, but have not previously been investigated with mitochondrial DNA. This chapter shows that many ‘basal corvid’ lineages are actually ‘basal passerine’ lineages, and there is a discrepancy between nuclear Rag-1 phylogenies (the most commonly used gene in passerine phylogenetics) and other phylogenies, including mitochondrial, that requires further investigation. Taken as a whole, this thesis adds significantly to our understanding of the evolution of birds, and provides a foundation for future research, not only of phylogenetic relationships, but also of avian life history, long-term niche stability and macroevolution.