The genome of a globally invasive passerine, the common myna, Acridotheres tristis.

In an era of global climate change, biodiversity conservation is receiving increased attention. Conservation efforts are greatly aided by genetic tools and approaches, which seek to understand patterns of genetic diversity and how they impact species health and their ability to persist under future climate regimes. Invasive species offer vital model systems in which to investigate questions regarding adaptive potential, with a particular focus on how changes in genetic diversity and effective population size interact with novel selection regimes. The common myna (Acridotheres tristis) is a globally invasive passerine and is an excellent model species for research both into the persistence of low-diversity populations and the mechanisms of biological invasion. To underpin research on the invasion genetics of this species, we present the genome assembly of the common myna. We describe the genomic landscape of this species, including genome wide allelic diversity, methylation, repeats, and recombination rate, as well as an examination of gene family evolution. Finally, we use demographic analysis to identify that some native regions underwent a dramatic population increase between the two most recent periods of glaciation, and reveal artefactual impacts of genetic bottlenecks on demographic analysis.

Genomic Tools in Biological Invasions: Current State and Future Frontiers.

Human activities are accelerating rates of biological invasions and climate-driven range expansions globally, yet we understand little of how genomic processes facilitate the invasion process. Although most of the literature has focused on underlying phenotypic correlates of invasiveness, advances in genomic technologies are showing a strong link between genomic variation and invasion success. Here, we consider the ability of genomic tools and technologies to (i) inform mechanistic understanding of biological invasions and (ii) solve real-world issues in predicting and managing biological invasions. For both, we examine the current state of the field and discuss how genomics can be leveraged in the future. In addition, we make recommendations pertinent to broader research issues, such as data sovereignty, metadata standards, collaboration, and science communication best practices that will require concerted efforts from the global invasion genomics community.

Species-wide genomics of kakapo provides tools to accelerate recovery.

The kakapo is a critically endangered, intensively managed, long-lived nocturnal parrot endemic to Aotearoa New Zealand. We generated and analysed whole-genome sequence data for nearly all individuals living in early 2018 (169 individuals) to generate a high-quality species-wide genetic variant callset. We leverage extensive long-term metadata to quantify genome-wide diversity of the species over time and present new approaches using probabilistic programming, combined with a phenotype dataset spanning five decades, to disentangle phenotypic variance into environmental and genetic effects while quantifying uncertainty in small populations. We find associations for growth, disease susceptibility, clutch size and egg fertility within genic regions previously shown to influence these traits in other species. Finally, we generate breeding values to predict phenotype and illustrate that active management over the past 45 years has maintained both genome-wide diversity and diversity in breeding values and, hence, evolutionary potential. We provide new pathways for informing future conservation management decisions for kakapo, including prioritizing individuals for translocation and monitoring individuals with poor growth or high disease risk. Overall, by explicitly addressing the challenge of the small sample size, we provide a template for the inclusion of genomic data that will be transformational for species recovery efforts around the globe.

Assembly of female and male hihi genomes (stitchbird; Notiomystis cincta) enables characterization of the W chromosome and resources for conservation genomics.

A high-quality reference genome can be a valuable resource for threatened species by providing a foundation to assess their evolutionary potential to adapt to future pressures such as environmental change. We assembled the genome of a female hihi (Notiomysits cincta), a threatened passerine bird endemic to Aotearoa New Zealand. The assembled genome is 1.06 Gb, and is of high quality and highly contiguous, with a contig N50 of 7.0 Mb, estimated QV of 44 and a BUSCO completeness of 96.8%. A male assembly of comparable quality was generated in parallel. A population linkage map was used to scaffold the autosomal contigs into chromosomes. Female and male sequence coverage and comparative genomics analyses were used to identify Z-, and W-linked contigs. In total, 94.6% of the assembly length was assigned to putative nuclear chromosome scaffolds. Native DNA methylation was highly correlated between sexes, with the W chromosome contigs more highly methylated than autosomal chromosomes and Z contigs. 43 differentially methylated regions were identified, and these may represent interesting candidates for the establishment or maintenance of sex differences. By generating a high-quality reference assembly of the heterogametic sex, we have created a resource that enables characterization of genome-wide diversity and facilitates the investigation of female-specific evolutionary processes. The reference genomes will form the basis for fine-scale assessment of the impacts of low genetic diversity and inbreeding on the adaptive potential of the species and will therefore enable tailored and informed conservation management of this threatened taonga (treasured) species.

Tracing the introduction of the invasive common myna using population genomics.

The common myna (Acridotheres tristis) is one of the most invasive bird species in the world, yet its colonisation history is only partly understood. We identified the introduction history and population structure, and quantified the genetic diversity of myna populations from the native range in India and introduced populations in New Zealand, Australia, Fiji, Hawaii, and South Africa, based on thousands of single nucleotide polymorphism markers in 814 individuals. We were able to identify the source population of mynas in several invasive locations: mynas from Fiji and Melbourne, Australia, were likely founded by individuals from a subpopulation in Maharashtra, India, while mynas in Hawaii and South Africa were likely independently founded by individuals from other localities in India. Our findings suggest that New Zealand mynas were founded by individuals from Melbourne, which, in turn, were founded by individuals from Maharashtra. We identified two genetic clusters among New Zealand mynas, divided by New Zealand's North Island's axial mountain ranges, confirming previous observations that mountains and thick forests may form barriers to myna dispersal. Our study provides a foundation for other population and invasion genomic studies and provides useful information for the management of this invasive species.

The promise and challenges of characterizing genome-wide structural variants: A case study in a critically endangered parrot.

There is growing interest in the role of structural variants (SVs) as drivers of local adaptation and speciation. From a biodiversity genomics perspective, the characterization of genome-wide SVs provides an exciting opportunity to complement single nucleotide polymorphisms (SNPs). However, little is known about the impacts of SV discovery and genotyping strategies on the characterization of genome-wide SV diversity within and among populations. Here, we explore a near whole-species resequence data set, and long-read sequence data for a subset of highly represented individuals in the critically endangered kakapo (Strigops habroptilus). We demonstrate that even when using a highly contiguous reference genome, different discovery and genotyping strategies can significantly impact the type, size and location of SVs characterized genome-wide. Further, we found that the mean number of SVs in each of two kakapo lineages differed both within and across generations. These combined results suggest that genome-wide characterization of SVs remains challenging at the population-scale. We are optimistic that increased accessibility to long-read sequencing and advancements in bioinformatic approaches including multireference approaches like genome graphs will alleviate at least some of the challenges associated with resolving SV characteristics below the species level. In the meantime, we address caveats, highlight considerations, and provide recommendations for the characterization of genome-wide SVs in biodiversity genomic research.

Genomic signatures of inbreeding depression for a threatened Aotearoa New Zealand passerine.

For small and isolated populations, the increased chance of mating between related individuals can result in a substantial reduction in individual and population fitness. Despite the increasing availability of genomic data to measure inbreeding accurately across the genome, inbreeding depression studies for threatened species are still scarce due to the difficulty of measuring fitness in the wild. Here, we investigate inbreeding and inbreeding depression for the extensively monitored Tiritiri Matangi island population of a threatened Aotearoa New Zealand passerine, the hihi (Notiomystis cincta). First, using a custom 45 k single nucleotide polymorphism (SNP) array, we explore genomic inbreeding patterns by inferring homozygous segments across the genome. Although all individuals have similar levels of ancient inbreeding, highly inbred individuals are affected by recent inbreeding, which can probably be explained by bottleneck effects such as habitat loss after European arrival and their translocation to the island in the 1990s. Second, we investigate genomic inbreeding effects on fitness, measured as lifetime reproductive success, and its three components, juvenile survival, adult annual survival and annual reproductive success, in 363 hihi. We find that global inbreeding significantly affects juvenile survival but none of the remaining fitness traits. Finally, we employ a genome-wide association approach to test the locus-specific effects of inbreeding on fitness, and identify 13 SNPs significantly associated with lifetime reproductive success. Our findings suggest that inbreeding depression does impact hihi, but at different genomic scales for different traits, and that purging has therefore failed to remove all variants with deleterious effects from this population of conservation concern.

Variation in shape and consistency of selection between populations of the threatened Hihi (Notiomystis cincta).

The shape and intensity of natural selection can vary between years, potentially resulting in a chronic reduction of fitness as individuals need to track a continually changing optimum of fitness (i.e., a "lag load"). In endangered species, often characterized by small population size, the lack of genetic diversity is expected to limit the response to this constant need to adjust to fluctuating selection, increasing the fitness burden and thus the risk of extinction. Here, we use long-term monitoring data to assess whether the type of selection for a key fitness trait (i.e., lay date) differs between two reintroduced populations of a threatened passerine bird, the hihi (Notiomystis cincta). We apply recent statistical developments to test for the presence or absence of fluctuation in selection in both the Tiritiri Matangi Island and the Karori sanctuary populations. Our results support the presence of stabilizing selection in Tiritiri Matangi with a potential moving optimum for lay date. In Karori our results favour a regime of directional selection. Although the shape of selection may differ, for both populations an earlier lay date generally increases fitness in both environments. Further, the moving optimum models of lay date on Tiritiri Matangi, suggesting that selection varies between years, imply a substantial lag load in addition to the fitness burden caused by the population laying too late. Our results highlight the importance of characterizing the form and temporal variation of selection for each population to predict the effects of environmental change and to inform management.

Genetic variance in fitness indicates rapid contemporary adaptive evolution in wild animals.

The rate of adaptive evolution, the contribution of selection to genetic changes that increase mean fitness, is determined by the additive genetic variance in individual relative fitness. To date, there are few robust estimates of this parameter for natural populations, and it is therefore unclear whether adaptive evolution can play a meaningful role in short-term population dynamics. We developed and applied quantitative genetic methods to long-term datasets from 19 wild bird and mammal populations and found that, while estimates vary between populations, additive genetic variance in relative fitness is often substantial and, on average, twice that of previous estimates. We show that these rates of contemporary adaptive evolution can affect population dynamics and hence that natural selection has the potential to partly mitigate effects of current environmental change.

Finding the adaptive needles in a population-structured haystack: A case study in a New Zealand mollusc.

Genetic adaptation to future environmental conditions is crucial to help species persist as the climate changes. Genome scans are powerful tools to understand adaptive landscapes, enabling us to correlate genetic diversity with environmental gradients while disentangling neutral from adaptive variation. However, low gene flow can lead to both local adaptation and highly structured populations, and is a major confounding factor for genome scans, resulting in an inflated number of candidate loci. Here, we compared candidate locus detection in a marine mollusc (Onithochiton neglectus), taking advantage of a natural geographical contrast in the levels of genetic structure between its populations. O. neglectus is endemic to New Zealand and distributed throughout an environmental gradient from the subtropical north to the subantarctic south. Due to a brooding developmental mode, populations tend to be locally isolated. However, adult hitchhiking on rafting kelp increases connectivity among southern populations. We applied two genome scans for outliers (Bayescan and PCAdapt) and two genotype-environment association (GEA) tests (BayeScEnv and RDA). To limit issues with false positives, we combined results using the geometric mean of q-values and performed association tests with random environmental variables. This novel approach is a compromise between stringent and relaxed approaches widely used before, and allowed us to classify candidate loci as low confidence or high confidence. Genome scans for outliers detected a large number of significant outliers in strong and moderately structured populations. No high-confidence GEA loci were detected in the context of strong population structure. However, 86 high-confidence loci were associated predominantly with latitudinally varying abiotic factors in the less structured southern populations. This suggests that the degree of connectivity driven by kelp rafting over the southern scale may be insufficient to counteract local adaptation in this species. Our study supports the expectation that genome scans may be prone to errors in highly structured populations. Nonetheless, it also empirically demonstrates that careful statistical controls enable the identification of candidate loci that invite more detailed investigations. Ultimately, genome scans are valuable tools to help guide further research aiming to determine the potential of non-model species to adapt to future environments.

The relevance of pedigrees in the conservation genomics era.

Over the past 50 years conservation genetics has developed a substantive toolbox to inform species management. One of the most long-standing tools available to manage genetics-the pedigree-has been widely used to characterize diversity and maximize evolutionary potential in threatened populations. Now, with the ability to use high throughput sequencing to estimate relatedness, inbreeding, and genome-wide functional diversity, some have asked whether it is warranted for conservation biologists to continue collecting and collating pedigrees for species management. In this perspective, we argue that pedigrees remain a relevant tool, and when combined with genomic data, create an invaluable resource for conservation genomic management. Genomic data can address pedigree pitfalls (e.g., founder relatedness, missing data, uncertainty), and in return robust pedigrees allow for more nuanced research design, including well-informed sampling strategies and quantitative analyses (e.g., heritability, linkage) to better inform genomic inquiry. We further contend that building and maintaining pedigrees provides an opportunity to strengthen trusted relationships among conservation researchers, practitioners, Indigenous Peoples, and Local Communities.

The design and application of a 50 K SNP chip for a threatened Aotearoa New Zealand passerine, the hihi.

Next-generation sequencing has transformed the fields of ecological and evolutionary genetics by allowing for cost-effective identification of genome-wide variation. Single nucleotide polymorphism (SNP) arrays, or "SNP chips", enable very large numbers of individuals to be consistently genotyped at a selected set of these identified markers, and also offer the advantage of being able to analyse samples of variable DNA quality. We used reduced representation restriction-aided digest sequencing (RAD-seq) of 31 birds of the threatened hihi (Notiomystis cincta; stitchbird) and low-coverage whole genome sequencing (WGS) of 10 of these birds to develop an Affymetrix 50 K SNP chip. We overcame the limitations of having no hihi reference genome and a low quantity of sequence data by separate and pooled de novo assembly of each of the 10 WGS birds. Reads from all individuals were mapped back to these de novo assemblies to identify SNPs. A subset of RAD-seq and WGS SNPs were selected for inclusion on the chip, prioritising SNPs with the highest quality scores whose flanking sequence uniquely aligned to the zebra finch (Taeniopygia guttata) genome. Of the 58,466 SNPs manufactured on the chip, 72% passed filtering metrics and were polymorphic. By genotyping 1,536 hihi on the array, we found that SNPs detected in multiple assemblies were more likely to successfully genotype, representing a cost-effective approach to identify SNPs for genotyping. Here, we demonstrate the utility of the SNP chip by describing the high rates of linkage disequilibrium in the hihi genome, reflecting the history of population bottlenecks in the species.

Who are you? A framework to identify and report genetic sample mix-ups.

Sample mix-ups occur when samples have accidentally been duplicated, mislabelled or swapped. When samples are subsequently genotyped or sequenced, this can lead to individual IDs being incorrectly linked to genetic data, resulting in incorrect or biased research results, or reduced power to detect true biological patterns. We surveyed the community and found that almost 80% of responding researchers have encountered sample mix-ups. However, many recent studies in the field of molecular ecology do not appear to systematically report individual assignment checks as part of their publications. Although checks may be done, lack of consistent reporting means that it is difficult to assess whether sample mix-ups have occurred or been detected. Here, we present an easy-to-follow sample verification framework that can utilise existing metadata, including species, population structure, sex and pedigree information. We demonstrate its application to a data set representing individuals of a threatened Aotearoa New Zealand bird species, the hihi, genotyped on a 50K SNP array. We detected numerous incorrect genotype-ID associations when comparing observed and genetic sex or comparing to relationships in a verified microsatellite pedigree. The framework proposed here helped to confirm 488 individuals (39%), correct another 20 bird-genotype links, and detect hundreds of incorrect sample IDs, emphasizing the value of routinely checking genetic and genomic data sets for their accuracy. We therefore promote the implementation and reporting of this simple yet effective sample verification framework as a standardized quality control step for studies in the field of molecular ecology.

Expanding the conservation genomics toolbox: Incorporating structural variants to enhance genomic studies for species of conservation concern.

Structural variants (SVs) are large rearrangements (>50 bp) within the genome that impact gene function and the content and structure of chromosomes. As a result, SVs are a significant source of functional genomic variation, that is, variation at genomic regions underpinning phenotype differences, that can have large effects on individual and population fitness. While there are increasing opportunities to investigate functional genomic variation in threatened species via single nucleotide polymorphism (SNP) data sets, SVs remain understudied despite their potential influence on fitness traits of conservation interest. In this future-focused Opinion, we contend that characterizing SVs offers the conservation genomics community an exciting opportunity to complement SNP-based approaches to enhance species recovery. We also leverage the existing literature-predominantly in human health, agriculture and ecoevolutionary biology-to identify approaches for readily characterizing SVs and consider how integrating these into the conservation genomics toolbox may transform the way we manage some of the world's most threatened species.

Genomic data of different resolutions reveal consistent inbreeding estimates but contrasting homozygosity landscapes for the threatened Aotearoa New Zealand hihi.

Inbreeding can lead to a loss of heterozygosity in a population and when combined with genetic drift may reduce the adaptive potential of a species. However, there is uncertainty about whether resequencing data can provide accurate and consistent inbreeding estimates. Here, we performed an in-depth inbreeding analysis for hihi (Notiomystis cincta), an endemic and nationally vulnerable passerine bird of Aotearoa New Zealand. We first focused on subsampling variants from a reference genome male, and found that low-density data sets tend to miss runs of homozygosity (ROH) in some places and overestimate ROH length in others, resulting in contrasting homozygosity landscapes. Low-coverage resequencing and 50 K SNP array densities can yield comparable inbreeding results to high-coverage resequencing approaches, but the results for all data sets are highly dependent on the software settings employed. Second, we extended our analysis to 10 hihi where low-coverage whole genome resequencing, RAD-seq and SNP array genotypes are available. We inferred ROH and individual inbreeding to evaluate the relative effects of sequencing depth versus SNP density on estimating inbreeding coefficients and found that high rates of missingness downwardly bias both the number and length of ROH. In summary, when using genomic data to evaluate inbreeding, studies must consider that ROH estimates are heavily dependent on analysis parameters, data set density and individual sequencing depth.

Polygenic basis for adaptive morphological variation in a threatened Aotearoa | New Zealand bird, the hihi (Notiomystis cincta).

To predict if a threatened species can adapt to changing selective pressures, it is crucial to understand the genetic basis of adaptive traits, especially in species historically affected by severe bottlenecks. We estimated the heritability of three hihi (Notiomystis cincta) morphological traits known to be under selection (nestling tarsus length, body mass and head-bill length) using 523 individuals and 39 699 single nucleotide polymorphisms (SNPs) from a 50 K Affymetrix SNP chip. We then examined the genetic architecture of the traits via chromosome partitioning analyses and genome-wide association scans (GWAS). Heritabilities estimated using pedigree relatedness or genomic relatedness were low. For tarsus length, the proportion of genetic variance explained by each chromosome was positively correlated with its size, and more than one chromosome explained significant variation for body mass and head-bill length. Finally, GWAS analyses suggested many loci of small effect contributing to trait variation for all three traits, although one locus (an SNP within an intron of the transcription factor HEY2) was tentatively associated with tarsus length. Our findings suggest a polygenic nature for the morphological traits, with many small effect size loci contributing to the majority of the variation, similar to results from many other wild populations. However, the small effective population size, polygenic architecture and already low heritabilities suggest that both the total response and rate of response to selection are likely to be limited in hihi.

A comparison of pedigree, genetic and genomic estimates of relatedness for informing pairing decisions in two critically endangered birds: Implications for conservation breeding programmes worldwide.

Conservation management strategies for many highly threatened species include conservation breeding to prevent extinction and enhance recovery. Pairing decisions for these conservation breeding programmes can be informed by pedigree data to minimize relatedness between individuals in an effort to avoid inbreeding, maximize diversity and maintain evolutionary potential. However, conservation breeding programmes struggle to use this approach when pedigrees are shallow or incomplete. While genetic data (i.e., microsatellites) can be used to estimate relatedness to inform pairing decisions, emerging evidence indicates this approach may lack precision in genetically depauperate species, and more effective estimates will likely be obtained from genomic data (i.e., thousands of genome-wide single nucleotide polymorphisms, or SNPs). Here, we compare relatedness estimates and subsequent pairing decisions using pedigrees, microsatellites and SNPs from whole-genome resequencing approaches in two critically endangered birds endemic to New Zealand: kaki/black stilt (Himantopus novaezelandiae) and kakariki karaka/orange-fronted parakeet (Cyanoramphus malherbi). Our findings indicate that SNPs provide more precise estimates of relatedness than microsatellites when assessing empirical parent-offspring and full sibling relationships. Further, our results show that relatedness estimates and subsequent pairing recommendations using PMx are most similar between pedigree- and SNP-based approaches. These combined results indicate that in lieu of robust pedigrees, SNPs are an effective tool for informing pairing decisions, which has important implications for many poorly pedigreed conservation breeding programmes worldwide.

Can threatened species adapt in a restored habitat? No expected evolutionary response in lay date for the New Zealand hihi.

Many bird species have been observed shifting their laying date to earlier in the year in response to climate change. However, the vast majority of these studies were performed on non-threatened species, less impacted by reduced genetic diversity (which is expected to limit evolutionary response) as a consequence of genetic bottlenecks, drift and population isolation. Here, we study the relationship between lay date and fitness, as well as its genetic basis, to understand the evolutionary constraints on phenology faced by threatened species using a recently reintroduced population of the endangered New Zealand passerine, the hihi (Notiomystis cincta). A large discrepancy between the optimal laying date and the mode of laying date creates a strong selection differential of -11.24. The impact of this discrepancy on fitness is principally mediated through survival of offspring from hatchling to fledgling. This discrepancy does not seem to arise from a difference in female quality or a trade-off with lifetime breeding success. We find that start of breeding season depends on female age and average temperature prior to the breeding season. Laying date is not found to be significantly heritable. Overall, our research suggests that this discrepancy is a burden on hihi fitness, which will not be resolved through evolution or phenotypic plasticity. More generally, these results show that threatened species introduced to restored habitats might lack adaptive potential and plasticity to adjust their phenology to their new environment. This constraint is also likely to limit their ability to face future challenges, including climate change.

Little Adaptive Potential in a Threatened Passerine Bird.

Threatened species face numerous threats, including future challenges triggered by global change. A possible way to cope with these challenges is through adaptive evolution, which requires adaptive potential. Adaptive potential is defined as the genetic variance needed to respond to selection and can be assessed either on adaptive traits or fitness [1]. However, a lack of high-quality data has made it difficult to rigorously test adaptive potential in threatened species, leading to controversy over its magnitude [1-3]. Here we assess the adaptive potential of a threatened New Zealand passerine (the hihi, Notiomystis cincta) based on two populations: (1) the sole remaining natural population, on the island of Te Hauturu-o-Toi, and (2) a reintroduced population with a long-term dataset (intensively monitored for 20 years) based on the island of Tiritiri Matangi. We use molecular information (reduced representation genome sequencing, on both populations), as well as long-term phenotypic and fitness data from the Tiritiri Matangi population, to find (1) a lack of molecular genetic diversity at a genome-wide level in both populations, (2) low heritability of traits under selection and (3) negligible additive genetic variance of fitness in the Tiritiri Matangi population. In combination, these results support a lack of adaptive potential in this threatened species. We discuss our findings within the context of other passerines and methods for assessing adaptive potential, as well as the impact of these results on conservation practice, for the hihi and species of conservation concern in general.

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 kaki/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 kaki 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.