The saga of the American bison (Bison bison) is a well-known story of death, destruction, and greed circumvented by early conservationists. The foresight of 5 cattlemen and the Canadian and U.S. governments at the apex of the population bottleneck in the 1880s led to the eventual establishment of several federal bison populations, from which virtually all of the 300,000 extant bison are descended. A survey of 54 microsatellite loci spanning each autosomal and both sex chromosomes was used to compare levels of genetic variation among 10 of the 11 federal bison populations in the U.S. Although most populations contain moderate levels of genetic variation, the majority of genetic variation is contained within only 4 of the federal populations surveyed. The distribution and partitioning of genetic variation confirm historical records of founding lineages and transfers among populations. Previously published mitochondrial and nuclear markers were used to survey federal bison populations for evidence of domestic cattle introgression. While only 1 population was found to contain low levels of domestic cattle mitochondrial DNA, 7 of the 10 surveyed populations had detectable introgression of nuclear genes from domestic cattle. From this, 2 federal bison populations were identified that have both high levels of genetic variation and no evidence of introgression of domestic cattle genes. The data obtained from this study were used to examine consequences of past and present management practices in closed bison populations. In the case of the Texas State Bison Herd, observed chronic small population size, low levels of genetic variation, low natality rates, and high juvenile mortality rates combined with the results of population modeling indicate a high risk of extinction within the next 50 years unless new genetic variation is introduced into the herd. Alternatively, analysis of population substructure and nonrandom culling reveal the necessity for further investigation into the long-term effects of current management practices in the Yellowstone National Park bison population. This study illustrates that while bison may be considered a conservation success story, long-term survival of protected federal populations requires the development of effective genetic management strategies.

Population genetic methods can be employed to inform the conservation of a species in a number of ways. For instance, they can be used to determine if a species has gone through a genetic bottleneck (i.e. a drastic reduction in population size that results in reduced genetic variation), and also if a species exhibits local genetic structure, (i.e., whether there is population genetic structure among neighbouring populations of an otherwise widely distributed species). The objectives of this thesis were to investigate the population genetic structure and long-term effective population size of the recently rediscovered subspecies of the masked booby, the Tasman booby, Sula dactylatra tasmani, which unlike masked boobies ,which have a pantropical distribution and are widespread, are range restricted to three island groups in the North Tasman Sea. To achieve this, I apply population genetic methods to mitochondrial control region sequence data, and microsatellite genotype, along with morphometric data. I first examined the cross utility of 43 microsatellite loci developed for the blue-footed (S. nebouxii), red-footed (S. sula) and Peruvian (S. variegata) booby for a population genetic study in my focal subspecies, the Tasman booby. All of these loci amplified in the Tasman booby, and from these 13 independent polymorphic loci were found and used as nuclear data, along with mitochondrial sequence data, to estimate population genetic structure. I also used these two types of data to determine the effective population size of this subspecies, both recently and historically. I found strong population genetic structure from the mitochondrial sequence data, while the microsatellite genotype data revealed weak but significant population genetic structure. I suggest the differences in these two types of marker are most likely due to stochasticity in the mitochondrial genome and/or male-mediated gene flow. Combined, the mitochondrial and microsatellite data revealed the Tasman booby has existed at a relatively stable population size for the last 25,000 years, but estimates of the current effective population size of this subspecies were unreliable. From these combined data I recommend that the Tasman booby should be treated as a single management unit, and conservation efforts from Australia and New Zealand could benefit from communication regarding their management plans. Future work including both autosomal and sex-linked introns could help in resolving the presence or absence of male-mediated gene flow, and/or help estimate an accurate effective population size in this subspecies.

The critically endangered orange fronted kākāriki!(Cyanoramphus malherbi)is an endemic parakeet restricted to three small breeding populations within the Hawdon, Hurunui and Poulter Valleys of North Canterbury, four translocated populations on offshore islands and a captive breeding facility in Christchurch. Seventeen polymorphic microsatellite loci were developed from next generation 454 sequencing of genomic DNA. At the commencement of this project, only birds sourced from the Hawdon and Hurunui populations had been used in the captive and translocated populations. To determine appropriate source populations for future translocation, this study used both nuclear and mitochondrial data to quantify the level of genetic diversity within, and the pattern of genetic differentiation among, the three remaining wild populations of C. malherbi. Six C. malherbi of known provenance from each of the three populations were genotyped at the 17 microsatellite loci and one microsatellite! loci previously developed for the closely related Forbe’s parakeet (C., forbesi), and sequenced at one mitochondrial (cytochrome b) and one nuclear exon (RAGT1). For each valley, the number of microsatellite alleles ranged from one to four per locus. Observed and expected heterozygosities ranged from 0.0 to 1.0 and from 0.17 to 0.74, respectively. The Poulter valley had the highest average allelic diversity (2.3) and the highest average observed and expected heterozygosities (0.40 & 0.38, respectively). Weak but significant population genetic structure was detected among valleys (FST= 0.06, F’ST=0.09,p=0.04). Pairwise FST estimates identified the Hurunui as being significantly different from both the Poulter and Hawdon Valleys (FST=0.11, p=0.01 and 0.061,p<0.01,respectively). In contrast, STRUCTURE analysis indicated that the three valleys comprise a single genetic cluster. Three cytochrome b haplotypes were identified, two of which were found in all three populations and one haplotype that was present in one Poulter valley individual only. Two RAG1 alleles were identified, both of which were shared by all three valleys. No significant population genetic structure was detected for either the cytochrome b or RAG 1 loci. These combined data suggest that the three North Canterbury valleys function as a metapopulation with some level of connectivity. The same nuclear and mitochondrial markers were used to determine the genetic distinctiveness of sympatric C. malherbi and C.auriceps North Canterbury populations and identify putative cryptic hybrids from the now extinct Hope Valley population. Based on both cytochrome b and microsatellite markers, C. malherbi and C.auriceps were found to be genetically distinct!(ϕST=p<0.01; FST=0.073,p< 0.01;K=2), and the two Hope Valley birds were confirmed to be hybrids. These findings lend support to the hypothesis that when one species is rare and the other abundant, limited hybridisation between sympatric populations of C.malherbi and C.auriceps is possible. Overall, this thesis has provided useful genetic tools and information to inform active conservation management of captive and translocated populations of C.malherbi. Based on my conclusions, I recommend the inclusion of individuals from each of the three source populations within the captive breeding and translocated populations to conserve current levels of genetic diversity. In addition, to ensure the genetic integrity of C.malherbi, I strongly recommend the use of these molecular methods to accurately identify all individuals prior to entering the captive breeding and translocation programmes. I also recommend the on going genetic monitoring and management of captive and translocated populations to guide future conservation management of this critically endangered kākāriki.

Freshwater mussel species in the genera Fusconaia and Pleurobema are particularly challenging to identify in the field. In this study, mussels from these genera were collected from the Green River, Kentucky for genetic and morphological analyses. I used molecular markers to detect any cryptic species within these genera and to test for genetic differentiation between two closely related nominal taxa P. rubrum and P. sintoxia using both mitochondrial (ND1, COI, 16S rRNA) and nuclear (ITS1) DNA sequences. After species identification, I used microsatellite DNA markers to estimate genetic diversity and effective population sizes (Ne) of species of Pleurobema. I used microsatellite primers that were developed for P. clava and P. pyriforme in previous studies, as well as microsatellites that I developed for P. plenum. Finally, I assessed morphological variation in my study species and developed dichotomous keys for the identification of both live mussels and shells. My results suggest that P. rubrum and P. sintoxia are the same species based on the mitochondrial DNA analyses, as there were few genetic differences between them. My results showed phylogenetically distinct lineages for F. flava, F. subrotunda, P. cordatum and P. plenum but no cryptic species were detected in the Green River. Current and contemporary Ne showed that these species have large population sizes that should allow for avoiding inbreeding and maintaining their evolutionary potential. Large genetic diversity as well as long-term effective population size could be the result of these species historically occurring as much larger assemblages that extended into the Ohio River and its numerous tributaries. The last objective was to assess morphometrical differences among these species. Using Canonical Variate Analysis, I found discernable morphological differences between the investigated species of Fusconaia and Pleurobema. The two Fusconaia species were morphologically different from the Pleurobema species. However, the Canonical Variate Analysis did not show differences among the Pleurobema species. I used decision tree analysis to develop a dichotomous tree, and random forest analysis was used to aid in the development of a dichotomous key by finding the most important diagnostic characters to distinguish these mussels. I then used the less subjective and easier to identify characters for the development of my dichotomous keys for live mussels and shells. However, both keys need to be tested in the field to determine their effectiveness. I could not separate P. rubrum and P. sintoxia mussels for morphometric analysis due to the lack of genetic differentiation and the inconsistent identification by the experts. However, I did describe a few individuals that look like P. rubrum and P. sintoxia to the eye of the experts. The description of these individuals matched previous descriptions of these mussels. Future studies need to assess taxonomic relationships among these species using genomics approaches, which might result in better node resolution. High genetic diversity and large effective population numbers for Pleurobema species suggest that these species' populations are genetically healthy. However, these results need to be interpreted carefully, and I therefore recommend additional studies to assess life history, habitat, host-fish availability, and current reproduction of these mussels in the Green River. / Doctor of Philosophy / Freshwater mussels offer important ecosystem services for humans to include water purification, nutrient storage and recycling, and mussels are part of the aquatic food web. In addition, freshwater mussels are indicators of ecosystem health. Because they rely on fish hosts to complete their complex life cycle, conservation of freshwater mussel species is particularly challenging. In this study, I focused my attention on freshwater mussel species commonly known as "pigtoes" which belong to the genera Fusconaia and Pleurobema. These species are difficult to distinguish morphologically even by experts. Hence, my study used molecular genetic markers to first identify these species. To assess the size and genetic health of these populations, I estimated genetic diversity and effective population number (Ne). Finally, I developed dichotomous keys to identify live mussel specimens and shells of molecularly identified mussels. My genetic results showed that there are five species of pigtoes in the Green River, which include Rough Pigtoe (Pleurobema plenum), Ohio Pigtoe (P. cordatum), Pink Pigtoe/Round Pigtoe (P. sintoxia/rubrum), Long-Solid (Fusconaia subrotunda), and Wabash Pigtoe (F. flava). A sixth pigtoe species, the endangered clubshell (Pleurobema clava), also is extant in the upper Green River but was not included in the analyses due to its rarity in the river. My results suggest that these mussel populations are large and healthy enough to survive and to adapt over time. The morphometric analysis using Canonical Variate Analysis (CVA) resulted in differentiation between F. flava and F. subrotunda. These two Fusconaia species also were morphologically different from the investigated species of Pleurobema. Results of this analysis suggest that species in the Genus Pleurobema remain difficult to differentiate from each other. The lack of genetic differentiation and the inconsistency in the experts' identification of P. rubrum and P. sintoxia, did not provide enough information to separate these two putative species for morphometric analysis. However, I was able to describe a few individuals that looked like typical examples of these two species to the eyes of the experts. Future taxonomic studies should use next-generation sequencing, which would likely result in a better resolution of evolutionary relationships. Large Ne values for each species suggest that these populations are genetically healthy. However, these results need to be considered together with results of future studies on life history, habitats, abundance, and reproduction of these species in the wild. Finally, the dichotomous mussel identification keys are intended to support more accurate identification of these species in the Green River, KY but need to be field-tested by mussel biologists

The use of genetic evidence to facilitate management outcomes for species of conservation or cultural concern can benefit from broadening the scope of inquiry. These efforts can include not only multiple geographic and genomic scales but also other academic disciplines and ways of knowing, which can identify unconventional drivers of genetic patterns. Genetic patterns revealed through such a broad approach can provide key information to managers regarding population differentiation, viability, isolation, and adaptive capacity, and can be incorporated into long-term precautionary management plans at local and regional scales. In this dissertation, I addressed several applied questions using multi-scale, interdisciplinary, and community-led approaches. The dramatic variation in habitat types and resource distribution in British Columbia, especially along the coastal to interior ecotone, allowed for investigation of potential genetic differentiation, landscape resistance, and local adaptation in two wide-ranging, omnivores. Grizzly bears (Ursus arctos) and black bears (Ursus americanus) in the area now known as British Columbia (BC) also hold high cultural value. Additionally, bears and people have been cohabitating and sharing resources on this landscape for millennia, prompting investigations of how this relationship and shared landscape might have shaped both. This relationship is reflected in Indigenous-led long term bear monitoring on the central coast of BC by the Nuxalk, Haíɫzaqv, Kitasoo/Xai’xais, Gitga’at, and Wuikinuxv First Nations. In the bear system of the central coast and larger BC there are management opportunities for the integration of local and regional monitoring, intergovernmental collaboration, and using genetic data to re-assert Indigenous-led management goals. Finally, given that black bear populations of the area contain a single genetic variant responsible for creating white phase or Spirit bear individuals, relevant genetic evidence that can be considered in the management of bears in BC ranges from a single genetic variant to genome-wide investigations of local adaptation across the coastal to interior ecotone. In my first data-driven chapter (Chapter 2), I used microsatellite markers to examine the pattern of genetic structure and its potential drivers among grizzly bears on the central coast of BC. We incorporated potential landscape resistance factors informed by relevant literature. Also recognizing the dramatic changes in Indigenous settlement density following European colonization and the potential for genetic markers to reflect historical patterns, we estimated resistance surfaces reflective of both pre and post colonization periods. Although no resistance surface explained a consequential proportion of genetic differentiation, we found a significant spatial overlap between Indigenous language families and the three bear genetic groups. We suspect that this pattern reflects a similar response of bears and people to unknown resource and geographic discontinuities across the landscape. This work contributes to the emerging intersection of landscape genetic and biocultural scholarship that includes non-traditional landscape factors at multiple temporal scales and considers parallel responses of wildlife and people to the landscapes they share. Additionally, given that current Provincially-designated management unit boundaries misalign to spatial patterns shown by genetic groups this research contributes detailed and actionable management implications. In Chapter 3, I used similar genetic methods to identify patterns of genetic differentiation for black bears of the central coast. In contrast with the three genetic groups of grizzly bears, we found eight genetic groups of black bears at a similar scale. This pattern likely reflects the differences in home range sizes and foraging ecology between these species. We also identified groups with low genetic diversity, with two of these groups containing high frequencies of the Spirit bear allele. We additionally found that wide waterways corresponded to genetic differentiation between groups and areas of lower than average estimated migration. We provide management recommendations based on these results that focus on balancing sufficient gene flow to ensure long-term viability of isolated and genetically depauperate genetic groups with ensuring that the rare Spirit bear variant is not swamped by an influx of genetic material. With this work, we show that linear landscape features other than roads (i.e. waterways) can provide resistance to even highly mobile species and that more gene flow is not always optimal for all scales of genetic conservation. In my third data chapter (4), I broadened the investigation of grizzly bear genetic differentiation from the scale of the central coast used in Chapter 2 to the ecotone spanning coastal and interior BC. Using whole genome resequencing, we additionally expanded the scale of genetic data to identify potential signatures of local adaptation. We found two broad-scale genetic groups corresponding to coastal and interior populations admixed along valleys that bisect the North to South Coast Mountain Range. We additionally identified potential signatures of local adaptation in genes associated with growth, development of muscle and bone, and immunity in the coastal genetic group, as well as those related to DNA repair and growth inhibition in the interior group. The functions of these candidate genes broadly align with morphological differences observed between larger coastal bears with consistent access to salmon and smaller interior bears with intermittent access to protein resources and exposure to more extreme environmental conditions. In a management context, this work highlights vulnerabilities to rapid environmental or resource changes in potentially locally adapted populations, and supports management efforts to protect connectivity via valleys that bisect the Coast Mountain Range. Finally, I summarize and discuss the conceptual and management contributions of this work and opportunities for future research (Chapter 5; dissertation conclusion). I highlight the contribution of Indigenous stewardship partners and their traditional and local ecological knowledge in defining, shaping, and expanding the scope of this dissertation, as well as applying management- relevant results. Our research methods and findings support the inclusivity of broad scientific and non-scientific communities and knowledge in genetic research and the application of genetic research to local management. / Graduate / 2022-09-14

The Bechstein’s bat, Myotis bechsteinii, is known as one of Britain’s most elusive mammals. Critical information on the species is lacking, hindering evidence-based conservation and management in a human-dominated landscape. In this thesis, I used a combination of molecular and landscape approaches to assess the genetic health and population genetic structure of M. bechsteinii and understand how the British landscape affects the species habitat and its connectivity. I also aimed to develop new molecular tools, such as non-invasive genetic sampling and molecular ageing, which could then be used to better monitor the species. Data from nuclear markers (microsatellites) showed high levels of genetic diversity and little inbreeding across the species range, though genetic diversity was slightly lower in Britain than in mainland Europe. Bayesian and spatial Principal Components (sPCA) analysis showed a clear separation between British and European populations. This analysis also revealed that in Europe the Italian population south of the Alps was found to constitute a different group from other sites. In Britain, there was genetic structuring between the northern and southern part of the species range. Despite there being little genetic divergence in mitochondrial DNA (mtDNA) sequences throughout most of Europe, the mtDNA patterns in Britain confirmed this separation of northern and southern populations. Such genetic structuring within Britain — in the absence of any obvious physical barriers — suggested that other features such as landuse may limit gene-flow. To better understand how the species interacts with 4 the British landscape, I used a landscape genetic approach, habitat suitability modelling using presence-only data and a landscape connectivity analysis. The negative association of M. bechsteinii presence with distance from woodland was identified as the main variable determining habitat suitability, while the landscape genetics results highlighted the importance of woodlands for gene flow. M. bechsteinii habitat was highly fragmented and only showed good connectivity if the species was able to disperse over 5,000 m. These results subsequently highlight the importance of woodlands not only for providing suitable habitat, but also in maintaining genetic connectivity between populations. Then, I investigated the use of non-invasive capture-mark-recapture (CMR) and demographic history models to estimate the population size and changes of M. bechsteinii. Bat droppings were collected below roosting sites of a single colony. After species identification, the 123 droppings belonging to M. bechsteinii were genotyped at nine DNA microsatellite loci in order to differentiate all individuals. All microsatellites showed very low amplification rates indicating low quality samples. However, at a larger scale, the use of population demographic models to assess effective population size variation using a dataset of 260 bats of the British population gave an estimate of the effective population size of 6,569 (CI: 5,307-8,006) and suggested that the British population of Myotis bechsteinii is stable and possibly expanding. Finally, I developed an epigenetic assay to estimate the age of individual bats. For this, I measured DNA methylation on bats of known age at seven CpG sites from three genes. All CpG sites from the tested genes showed a significant relationship between DNA methylation and age and provided reliable age estimates. 5 The findings presented in this thesis show that despite exhibiting high levels of genetic diversity throughout its range, the genetic structure, habitat and connectivity of M. bechsteinii populations is highly influenced by woodlands. It also offers a novel method to monitor the species by developing an assay which can provide information on the age structure of an entire colony from a single sampling session. Such approaches are much needed in the field of conservation and could in the future help preserve a wider range of species.

Minnows of the genus Dionda (Cyprinidae, Teleostei) inhabit spring-fed streams in the southwestern United States and Mexico. Five nominal species of Dionda (D. argentosa, D. diaboli, D. episcopa, D. nigrotaeniata and D. serena) are found in streams and rivers in central and west Texas. Because Dionda require clean, flowing water, they serve as aquatic indicator species of biological impacts of drought and human water use. Consequently, the ecological and conservation status of species of Dionda are important relative to monitoring habitat deterioration. This study used genetic data from geographic samples of the five nominal species of Dionda in Texas waters to document the conservation-genetics status of populations in each species. Fish were collected in cooperation with the Texas Parks and Wildlife Department and the U.S. Fish and Wildlife Service. Data from 585 base pairs of the mitochondrially encoded, protein coding ND-5 gene and from 21 to 33 nuclear-encoded microsatellites were used to assess genetic variation, population structure, historical demography, and genetic effective size of samples of each of the five species. The sample from Independence Creek, initially assumed to be D. episcopa because of its location, was found to be D. argentosa. Results of genetic assays indicate that each geographic sample in each species should be treated as a separate population and managed in a way that preserves the natural diversity found within each species. Genetic data revealed that all of the populations evaluated may be compromised genetically and should be monitored further.

Understanding the dynamics of functional genetic variability in small populations can have important implications in their conservation. I screened the variation of MHC II gene in Galapagos mockingbirds to evaluate the evolutionary forces that shaped the genetic variation. I found out that genetic drift affected the MHC variation together with a specific form of natural selection. Although the MHC is supposed to be under a pathogen-mediated selection I found no evidence for this theory in the mockingbird study system.

<p> Climate change is ostensibly one of the greatest modern selective pressures, and species with sensitive life histories or physiologies must adapt, migrate, or buffer its effects to persist. Some 15&ndash;37% of species are expected to be endangered or extinct by 2050. The most vulnerable include habitat specialists, local endemics, and species with low intrinsic growth rates. Yosemite toads (<i>Anaxyrus canorus</i>) are one such alpine endemic, having been extirpated from up to 69% of their historical range. Several features of their natural history make them vulnerable: small population sizes, high larval mortality, infrequent breeding, and specialized, patch-limited habitat prone to premature desiccation. In addition to their role as ecosystem flagships, Yosemite toads provide a model system for the many other specialists with similar life histories that are challenged by environmental change. The goal of this dissertation is to understand how historical evolutionary processes such as lineage divergence and secondary admixture, along with current levels of genetic connectivity, are expected to shape the future of Yosemite toad persistence in the face of climate change. The first chapter reconstructs phylogeographic patterns of lineage formation and fusion during repeated bouts of Pleistocene glaciation, and showcases a role for refugia in ecological divergence. The second chapter examines three contact zones as replicate tests of the hypothesis that loci associated with incipient speciation are distinct from those that readily cross ancient lineage boundaries. The third chapter models modern genetic connectivity as a network of environmental and climatic interactions, using a novel approach that incorporates phylogeographic structure. The fourth chapter forecasts the future selective pressure of climate change, and predicts where connectivity may be a mitigating force to restore genetic diversity. My dissertation provides an example of how conservation strategies can incorporate the many temporal processes (ancient, recent, and current) that have shaped current genetic diversity patterns, and use a &ldquo;total evidence&rdquo; approach to predict future adaptive potential.</p><p>

Abstract The effects of habitat loss and fragmentation on the genetic structure and vulnerability of populations strongly depend on the behaviour of a particular species. In this thesis, I examined the effects of forest fragmentation on genetic population structure with the aim of identifying and evaluating the different genetic and behavioural factors important for species conservation and management on different geographical scales. The species studied were the mound building red wood ants Formica lugubris and F. aquilonia, and a lekking bird, the capercaillie, Tetrao urogallus. Habitat loss and fragmentation affected the genetic structure in both wood ants and capercaillie. In general, the effects were related to the time since fragmentation and to the level of habitat loss and isolation from the other existing populations. The loss of genetic diversity due to population fragmentation was less observable than the differences in population structure. The response to habitat fragmentation was further dependent on species characteristics such as dispersal and mating behaviour. Sociality affected the genetic vulnerability of wood ant populations by decreasing gene diversity, increasing inbreeding depression and restricting gene flow between subpopulations. The results on the capercaillie in turn suggested that lekking behaviour restricts dispersal of both sexes, thus elevating the occurrence of inbreeding between individuals. The present study provided important information on species conservation and management in terms of better understanding species' biology and behaviour, as well as increased knowledge concerning the genetic issues that should be taken into account when planning conservation actions. By examining the genetic structure of the species it was possible to clarify the conservation status including the effective population size, the question of origin, and the genetic vulnerability (genetic diversity, inbreeding and inbreeding depression) of the populations and/or species. Overall, the results emphasised the importance of preserving the effective population size and the connectivity of habitat patches when planning species specific management strategies. There were great differences in conservation needs among the species, which should be taken into account especially in local management actions.