Assessment of the impacts of anthropogenic hybridisation in a threatened non-model bird species through the development of genomic resources with implications for conservation

Interspecific hybridisation—the breeding between distinct species—can contribute to species extinction due to wasted reproductive potential, outbreeding depression, and introgression of genetic material mediated by backcrossing. Incomplete reproductive barriers can facilitate interspecific hybridisation as previously isolated species come into contact with one another. Interspecific hybridisation is relatively common among birds, but anthropogenic impacts that increase the incidence of such hybridisation between threatened native species and non-threatened species are of conservation concern due to the risks of genetic swamping, which at its most extreme may result in species extinction. While the impacts of interspecific hybridisation have previously been assessed using small numbers of genetic markers, new genomic sequencing developments now facilitate implementation of genome-wide reassessments providing greater resolution of analyses. The critically endangered kakī (black stilt; Himantopus novaezelandiae) is one such species that can benefit from these new genomic data. Anthropogenic habitat change and introduction of mammalian predators resulted in the decline of this Aotearoa New Zealand endemic wading bird during the 1900s. An intense population bottleneck resulting in an ephemeral sex-bias among the remaining kakī contributed to hybridisation with the self-introduced poaka (the Aotearoa New Zealand population of the Australian pied stilt; H. himantopus leucocephalus), a congeneric species previously thought to have diverged from a common ancestor with kakī one million years ago. Intensive conservation management including captive breeding for translocation and predator control has increased kakī numbers from ~23 adults in 1981 to approximately 169 wild adults in 2020. Previous genetic studies identified minimal evidence of introgression of poaka genetic material into kakī, and iv determined that moderate outbreeding depression in combination with stochastic processes likely limited introgression. These data informed the kakī captive breeding for translocation programme with the aim of maintaining genetic integrity. However, re-evaluation using genomic data was recommended for kakī. Using high-throughput sequencing techniques, I sequenced and assembled the first reference genomes for kakī and Australian pied stilts as tools for use in analyses of introgression. The kakī mitochondrial genome was also assembled to facilitate comparisons of contemporary and historic stilt diversity, showing that conservation management aimed at maximising genetic diversity has largely maintained mitochondrial diversity despite kakī decline, identifying three mitochondrial haplotypes present among contemporary kakī. Kakī and poaka are well-differentiated, and are estimated to have diverged from a common ancestor approximately 750,000 years ago based on Bayesian analysis of mitochondrial data. In addition, the analysis of high-resolution genomic markers generated from approximately 65% of contemporary wild kakī detected no introgression from poaka to kakī despite past hybridisation. These findings confirm the results of previous genetic analysis of introgression and the success of past conservation management. As kakī recovery continues, these combined findings will be used by the New Zealand Department of Conservation’s Kakī Recovery Programme to further maintain the genetic integrity of kakī. Overall, the genomic resources developed here have facilitated the transition from using genetic data to genomic data for kakī recovery, and contribute to our understanding of the impacts of anthropogenic hybridisation on a critically endangered taonga species

Reference Genomes from Distantly Related Species Can Be Used for Discovery of Single Nucleotide Polymorphisms to Inform Conservation Management

Threatened species recovery programmes benefit from incorporating genomic data into conservation management strategies to enhance species recovery. However, a lack of readily available genomic resources, including conspecific reference genomes, often limits the inclusion of genomic data. Here, we investigate the utility of closely related high-quality reference genomes for single nucleotide polymorphism (SNP) discovery using the critically endangered kakī/black stilt (Himantopus novaezelandiae) and four Charadriiform reference genomes as proof of concept. We compare diversity estimates (i.e., nucleotide diversity, individual heterozygosity, and relatedness) based on kakī SNPs discovered from genotyping-by-sequencing and whole genome resequencing reads mapped to conordinal (killdeer, Charadrius vociferus), confamilial (pied avocet, Recurvirostra avosetta), congeneric (pied stilt, Himantopus himantopus) and conspecific reference genomes. Results indicate that diversity estimates calculated from SNPs discovered using closely related reference genomes correlate significantly with estimates calculated from SNPs discovered using a conspecific genome. Congeneric and confamilial references provide higher correlations and more similar measures of nucleotide diversity, individual heterozygosity, and relatedness. While conspecific genomes may be necessary to address other questions in conservation, SNP discovery using high-quality reference genomes of closely related species is a cost-effective approach for estimating diversity measures in threatened species

Genetic Diversity Within and Among Populations of Black Robins on the Chatham Islands, New Zealand

Endemic island populations worldwide are at greater risk of extinction than similar mainland populations, in part due to the specific genetic threats faced by small populations, namely of loss of genetic diversity and inbreeding. Reduced genetic diversity limits the ability of populations to adapt to altered conditions, while unavoidable inbreeding reduces population fitness through the effects of inbreeding depression. Effective conservation management requires the understanding of these effects on populations of interest to adopt appropriate strategies to reduce such threats, and thereby ensure long-term population persistence. Through the use of next-generation sequencing, I isolated 11 polymorphic microsatellite loci to allow analysis of current levels of genetic diversity in the endangered Chatham Island black robin Petroica traversi. The black robin has a history of small population size, including a population bottleneck of a single breeding pair, prior to recovery of population size over the past 30 years. The species is currently limited to populations on two small islands, and likely has a high extinction risk due to unavoidable inbreeding in the recovering populations, and may have experienced loss of genetic diversity due to strong genetic drift within these small populations. I compared levels of genetic diversity in the black robin to that of its closest congener, the Chatham Island tomtit Petroica macrocephala chathamensis, to assess how the population history of the black robin has affected its genetic diversity. Additionally, I compared levels of diversity between island populations of each species, to determine whether the smaller populations experienced lower diversity and therefore greater extinction vulnerability. Genetic diversity was lower in the black robin than the tomtit, and lower in the smaller populations of both species. The detection of levels of genetic diversity in the tomtit similar to those of threatened species suggests population viability of this species of least concern may be lower than expected. The two island populations of black robin are thought to have been isolated from one another for 26 years, and so populations were genotyped to determine whether this isolation has resulted in population differentiation, despite the short period of isolation. The two populations show substantial genetic differentiation, indicating genetic drift has had strong independent effects on these isolated populations. Although the tomtit exists on three islands, there was no evidence of current dispersal between the two populations assessed, and there was a similar level of differentiation between these populations and the black robin populations. Over 30 years of observational data show the black robin to be socially monogamous, with no evidence of extra-pair breeding. However, assessment of the social pedigree using microsatellite genotyping found a conservative rate of extra-pair paternity of approximately 14%, and the existence of a low-level of intraspecific brood paternity could not be rejected. As yet, the reason for the evolution of a strategy of extra-pair paternity is unknown. From the results of this study, I recommend reciprocal translocations of black robins between island populations as a form of assisted gene flow to bolster genetic diversity of each population, and to reduce inbreeding in the smaller of the two populations. Furthermore, the establishment of a third population is recommended to minimise extinction vulnerability of this endangered species. As the black robin is not genetically monogamous, selection of individuals for translocation will require the use of molecular techniques to assess relatedness, rather than the social pedigree, to maximise success

Assessment of the impacts of anthropogenic hybridisation in a threatened non-model bird species through the development of genomic resources with implications for conservation

Interspecific hybridisation—the breeding between distinct species—can contribute to species extinction due to wasted reproductive potential, outbreeding depression, and introgression of genetic material mediated by backcrossing. Incomplete reproductive barriers can facilitate interspecific hybridisation as previously isolated species come into contact with one another. Interspecific hybridisation is relatively common among birds, but anthropogenic impacts that increase the incidence of such hybridisation between threatened native species and non-threatened species are of conservation concern due to the risks of genetic swamping, which at its most extreme may result in species extinction. While the impacts of interspecific hybridisation have previously been assessed using small numbers of genetic markers, new genomic sequencing developments now facilitate implementation of genome-wide reassessments providing greater resolution of analyses. The critically endangered kakī (black stilt; Himantopus novaezelandiae) is one such species that can benefit from these new genomic data. Anthropogenic habitat change and introduction of mammalian predators resulted in the decline of this Aotearoa New Zealand endemic wading bird during the 1900s. An intense population bottleneck resulting in an ephemeral sex-bias among the remaining kakī contributed to hybridisation with the self-introduced poaka (the Aotearoa New Zealand population of the Australian pied stilt; H. himantopus leucocephalus), a congeneric species previously thought to have diverged from a common ancestor with kakī one million years ago. Intensive conservation management including captive breeding for translocation and predator control has increased kakī numbers from ~23 adults in 1981 to approximately 169 wild adults in 2020. Previous genetic studies identified minimal evidence of introgression of poaka genetic material into kakī, and iv determined that moderate outbreeding depression in combination with stochastic processes likely limited introgression. These data informed the kakī captive breeding for translocation programme with the aim of maintaining genetic integrity. However, re-evaluation using genomic data was recommended for kakī. Using high-throughput sequencing techniques, I sequenced and assembled the first reference genomes for kakī and Australian pied stilts as tools for use in analyses of introgression. The kakī mitochondrial genome was also assembled to facilitate comparisons of contemporary and historic stilt diversity, showing that conservation management aimed at maximising genetic diversity has largely maintained mitochondrial diversity despite kakī decline, identifying three mitochondrial haplotypes present among contemporary kakī. Kakī and poaka are well-differentiated, and are estimated to have diverged from a common ancestor approximately 750,000 years ago based on Bayesian analysis of mitochondrial data. In addition, the analysis of high-resolution genomic markers generated from approximately 65% of contemporary wild kakī detected no introgression from poaka to kakī despite past hybridisation. These findings confirm the results of previous genetic analysis of introgression and the success of past conservation management. As kakī recovery continues, these combined findings will be used by the New Zealand Department of Conservation’s Kakī Recovery Programme to further maintain the genetic integrity of kakī. Overall, the genomic resources developed here have facilitated the transition from using genetic data to genomic data for kakī recovery, and contribute to our understanding of the impacts of anthropogenic hybridisation on a critically endangered taonga species

Genomic sequencing confirms absence of introgression despite past hybridisation between a critically endangered bird and its common congener

Genetic swamping resulting from interspecific hybridisation can increase extinction risk for threatened species. The development of high-throughput and reduced-representation genomic sequencing and analyses to generate large numbers of high resolution genomic markers has the potential to reveal introgression previously undetected using small numbers of genetic markers. However, few studies to date have implemented genomic tools to assess the extent of interspecific hybridisation in threatened species. Here we investigate the utility of genome-wide single nucleotide polymorphisms (SNPs) to detect introgression resulting from past interspecific hybridisation in one of the world’s rarest birds. Anthropogenic impacts have resulted in hybridisation and subsequent backcrossing of the critically endangered Aotearoa New Zealand endemic kakī (black stilts; Himantopus novaezelandiae) with the non-threatened self-introduced congeneric poaka (Aotearoa New Zealand population of pied stilts, Himantopus himantopus leucocephalus), yet genetic analyses with a limited set of microsatellite markers revealed no evidence of introgression of poaka genetic material in kakī, excluding one individual. We use genomic data for ~63% of the wild adult kakī population to reassess the extent of introgression resulting from hybridisation between kakī and poaka. Consistent with previous genetic analyses, we detected no introgression from poaka into kakī. These collective results indicate that, for kakī, existing microsatellite markers provide a robust, cost-effective approach to detect cryptic hybrids. Further, for well-differentiated species, the use of genomic markers may not be required to detect admixed individuals

Microsatellite records for volume 8, issue 2

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