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.

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 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.

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.

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.

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.

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.

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.

Social and spatial effects on genetic variation between foraging flocks in a wild bird population.

Social interactions are rarely random. In some instances, animals exhibit homophily or heterophily, the tendency to interact with similar or dissimilar conspecifics, respectively. Genetic homophily and heterophily influence the evolutionary dynamics of populations, because they potentially affect sexual and social selection. Here, we investigate the link between social interactions and allele frequencies in foraging flocks of great tits (Parus major) over three consecutive years. We constructed co-occurrence networks which explicitly described the splitting and merging of 85,602 flocks through time (fission-fusion dynamics), at 60 feeding sites. Of the 1,711 birds in those flocks, we genotyped 962 individuals at 4,701 autosomal single nucleotide polymorphisms (SNPs). By combining genomewide genotyping with repeated field observations of the same individuals, we were able to investigate links between social structure and allele frequencies at a much finer scale than was previously possible. We explicitly accounted for potential spatial effects underlying genetic structure at the population level. We modelled social structure and spatial configuration of great tit fission-fusion dynamics with eigenvector maps. Variance partitioning revealed that allele frequencies were strongly affected by group fidelity (explaining 27%-45% of variance) as individuals tended to maintain associations with the same conspecifics. These conspecifics were genetically more dissimilar than expected, shown by genomewide heterophily for pure social (i.e., space-independent) grouping preferences. Genomewide homophily was linked to spatial configuration, indicating spatial segregation of genotypes. We did not find evidence for homophily or heterophily for putative socially relevant candidate genes or any other SNP markers. Together, these results demonstrate the importance of distinguishing social and spatial processes in determining population structure.

Genetic Kinship Analyses Reveal That Gray's Beaked Whales Strand in Unrelated Groups.

Some marine mammals are so rarely seen that their life history and social structure remain a mystery. Around New Zealand, Gray's beaked whales (Mesoplodon grayi) are almost never seen alive, yet they are a commonly stranded species. Gray's are unique among the beaked whales in that they frequently strand in groups, providing an opportunity to investigate their social organization. We examined group composition and genetic kinship in 113 Gray's beaked whales with samples collected over a 20-year period. Fifty-six individuals stranded in 19 groups (2 or more individuals), and 57 whales stranded individually. Mitochondrial control region haplotypes and microsatellite genotypes (16 loci) were obtained for 103 whales. We estimated pairwise relatedness between all pairs of individuals and average relatedness within, and between, groups. We identified 6 mother-calf pairs and 2 half-siblings, including 2 whales in different strandings 17 years and 1500 km apart. Surprisingly, none of the adults stranding together were related suggesting that groups are not formed through the retention of kin. These data suggest that both sexes may disperse from their mothers, and groups consisting of unrelated subadults are common. We also found no instances of paternity within the groups. Our results provide the first insights into dispersal, social organization, and the mating system in this rarely sighted species. Why whales strand is still unknown but, in Gray's beaked whales, the dead can tell us much about the living.

Building strong relationships between conservation genetics and primary industry leads to mutually beneficial genomic advances.

Several reviews in the past decade have heralded the benefits of embracing high-throughput sequencing technologies to inform conservation policy and the management of threatened species, but few have offered practical advice on how to expedite the transition from conservation genetics to conservation genomics. Here, we argue that an effective and efficient way to navigate this transition is to capitalize on emerging synergies between conservation genetics and primary industry (e.g., agriculture, fisheries, forestry and horticulture). Here, we demonstrate how building strong relationships between conservation geneticists and primary industry scientists is leading to mutually-beneficial outcomes for both disciplines. Based on our collective experience as collaborative New Zealand-based scientists, we also provide insight for forging these cross-sector relationships.

Transposable elements as agents of rapid adaptation may explain the genetic paradox of invasive species.

Rapid adaptation of invasive species to novel habitats has puzzled evolutionary biologists for decades, especially as this often occurs in the face of limited genetic variability. Although some ecological traits common to invasive species have been identified, little is known about the possible genomic/genetic mechanisms that may underlie their success. A common scenario in many introductions is that small founder population sizes will often lead to reduced genetic diversity, but that invading populations experience large environmental perturbations, such as changes in habitat and environmental stress. Although sudden and intense stress is usually considered in a negative context, these perturbations may actually facilitate rapid adaptation by affecting genome structure, organization and function via interactions with transposable elements (TEs), especially in populations with low genetic diversity. Stress-induced changes in TE activity can alter gene action and can promote structural variation that may facilitate the rapid adaptation observed in new environments. We focus here on the adaptive potential of TEs in relation to invasive species and highlight their role as powerful mutational forces that can rapidly create genetic diversity. We hypothesize that activity of transposable elements can explain rapid adaptation despite low genetic variation (the genetic paradox of invasive species), and provide a framework under which this hypothesis can be tested using recently developed and emerging genomic technologies.

Replicated analysis of the genetic architecture of quantitative traits in two wild great tit populations.

Currently, there is much debate on the genetic architecture of quantitative traits in wild populations. Is trait variation influenced by many genes of small effect or by a few genes of major effect? Where is additive genetic variation located in the genome? Do the same loci cause similar phenotypic variation in different populations? Great tits (Parus major) have been studied extensively in long-term studies across Europe and consequently are considered an ecological 'model organism'. Recently, genomic resources have been developed for the great tit, including a custom SNP chip and genetic linkage map. In this study, we used a suite of approaches to investigate the genetic architecture of eight quantitative traits in two long-term study populations of great tits--one in the Netherlands and the other in the United Kingdom. Overall, we found little evidence for the presence of genes of large effects in either population. Instead, traits appeared to be influenced by many genes of small effect, with conservative estimates of the number of contributing loci ranging from 31 to 310. Despite concordance between population-specific heritabilities, we found no evidence for the presence of loci having similar effects in both populations. While population-specific genetic architectures are possible, an undetected shared architecture cannot be rejected because of limited power to map loci of small and moderate effects. This study is one of few examples of genetic architecture analysis in replicated wild populations and highlights some of the challenges and limitations researchers will face when attempting similar molecular quantitative genetic studies in free-living populations.

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.

Genome mapping in intensively studied wild vertebrate populations.

Over the past decade, long-term studies of vertebrate populations have been the focus of many quantitative genetic studies. As a result, we have a clearer understanding of why some fitness-related traits are heritable and under selection, but are apparently not evolving. An exciting extension of this work is to identify the genes underlying phenotypic variation in natural populations. The advent of next-generation sequencing and high-throughput single nucleotide polymorphism (SNP) genotyping platforms means that mapping studies are set to become widespread in those wild populations for whom appropriate phenotypic data and DNA samples are available. Here, we highlight the progress made in this area and define evolutionary genetic questions that have become tractable with the arrival of these new genomics technologies.

Partitioning of genetic variation across the genome using multimarker methods in a wild bird population.

The underlying basis of genetic variation in quantitative traits, in terms of the number of causal variants and the size of their effects, is largely unknown in natural populations. The expectation is that complex quantitative trait variation is attributable to many, possibly interacting, causal variants, whose effects may depend upon the sex, age and the environment in which they are expressed. A recently developed methodology in animal breeding derives a value of relatedness among individuals from high-density genomic marker data, to estimate additive genetic variance within livestock populations. Here, we adapt and test the effectiveness of these methods to partition genetic variation for complex traits across genomic regions within ecological study populations where individuals have varying degrees of relatedness. We then apply this approach for the first time to a natural population and demonstrate that genetic variation in wing length in the great tit (Parus major) reflects contributions from multiple genomic regions. We show that a polygenic additive mode of gene action best describes the patterns observed, and we find no evidence of dosage compensation for the sex chromosome. Our results suggest that most of the genomic regions that influence wing length have the same effects in both sexes. We found a limited amount of genetic variance in males that is attributed to regions that have no effects in females, which could facilitate the sexual dimorphism observed for this trait. Although this exploratory work focuses on one complex trait, the methodology is generally applicable to any trait for any laboratory or wild population, paving the way for investigating sex-, age- and environment-specific genetic effects and thus the underlying genetic architecture of phenotype in biological study systems.

Genomic dissection of variation in clutch size and egg mass in a wild great tit (Parus major) population.

Clutch size and egg mass are life history traits that have been extensively studied in wild bird populations, as life history theory predicts a negative trade-off between them, either at the phenotypic or at the genetic level. Here, we analyse the genomic architecture of these heritable traits in a wild great tit (Parus major) population, using three marker-based approaches - chromosome partitioning, quantitative trait locus (QTL) mapping and a genome-wide association study (GWAS). The variance explained by each great tit chromosome scales with predicted chromosome size, no location in the genome contains genome-wide significant QTL, and no individual SNPs are associated with a large proportion of phenotypic variation, all of which may suggest that variation in both traits is due to many loci of small effect, located across the genome. There is no evidence that any regions of the genome contribute significantly to both traits, which combined with a small, nonsignificant negative genetic covariance between the traits, suggests the absence of genetic constraints on the independent evolution of these traits. Our findings support the hypothesis that variation in life history traits in natural populations is likely to be determined by many loci of small effect spread throughout the genome, which are subject to continued input of variation by mutation and migration, although we cannot exclude the possibility of an additional input of major effect genes influencing either trait.

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.

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.