Demographic drivers of reproductive failure in a threatened bird: Insights from a decade of data.

Hatching failure affects up to 77% of eggs laid by threatened bird species, yet the true prevalence and drivers of egg fertilization failure versus embryo mortality as underlying mechanisms of hatching failure are unknown. Here, using ten years of data comprising 4,371 eggs laid by a population of a threatened bird, the hihi (Notiomystis cincta), we investigate the relative importance of infertility and embryo death as drivers of hatching failure and explore population-level factors associated with them. We show that of the 1,438 eggs that failed to hatch (33% of laid eggs) between 2010 and 2020, 83% failed due to embryo mortality, with the majority failing in the early stages of embryonic development. In the most comprehensive estimates of infertility rates in a wild bird population to date, we find that fertilization failure accounts for around 17% of hatching failure overall and is more prevalent in years where the population is smaller and more male biased. Male embryos are more likely to die during early development than females, but we find no overall effect of sex on the successful development of embryos. Offspring fathered by within-pair males have significantly higher inbreeding levels than extra-pair offspring; however, we find no effect of inbreeding nor extra-pair paternity on embryo mortality. Accurately distinguishing between infertility and embryo mortality in this study provides unique insight into the underlying causes of reproductive failure over a long-term scale and reveals the complex risks of small population sizes to the reproduction of threatened species.

A high-density linkage map reveals broad- and fine-scale sex differences in recombination in the hihi (stitchbird; Notiomystis cincta).

Recombination, the process of DNA exchange between homologous chromosomes during meiosis, plays a major role in genomic diversity and evolutionary change. Variation in recombination rate is widespread despite recombination often being essential for progression of meiosis. One such variation is heterochiasmy, where recombination rates differ between sexes. Heterochiasmy has been observed across broad taxonomic groups, yet it remains an evolutionary enigma. We used Lep-MAP3, a pedigree-based software that is efficient in handling large datasets, to generate linkage maps for the hihi or stitchbird (Notiomystis cincta), utilising information from >36 K SNPs and 36 families. We constructed 29 linkage maps, including for the previously unscaffolded Z chromosome. The hihi is an endangered passerine endemic to Aotearoa New Zealand that is sexually dimorphic and exhibits high levels of sexual conflict, including sperm competition. Patterns in recombination in the hihi are consistent with those in other birds, including higher recombination rates in micro-chromosomes. Heterochiasmy in the hihi is male-biased, in line with predictions of the Haldane-Huxley rule, with the male linkage map being 15% longer. Micro-chromosomes exhibit heterochiasmy to a greater extent, contrary to that reported in other birds. At the intra-chromosomal level, heterochiasmy is higher nearer to chromosome ends and in gene-rich regions. Regions of extreme heterochiasmy are enriched for genes implicated in cell structure. This study adds an important contribution in assessing evolutionary theories of heterochiasmy and provides a framework for future studies investigating fine-scale heterochiasmy.

Parental breeding decisions and genetic quality predict social structure of independent offspring.

Across the animal kingdom, newly independent juveniles form social associations that influence later fitness, mate choice and gene flow, but little is known about the ontogeny of social environments, particularly in wild populations. Here we test whether associations among young animals form randomly or are influenced by environmental or genetic conditions established by parents. Parents' decisions determine natal birth sites, which could affect who independent young initially encounter; secondly, mate choice determines genetic condition (e.g. inbreeding) of young and the parental care they receive, which can affect sociability. However, genetic and environmental factors are confounded unless related offspring experience different natal environments. Therefore, we used a long-term genetic pedigree, breeding records and social network data from three cohorts of a songbird with high extra-pair paternity (hihi, Notiomystis cincta) to disentangle (1) how nest location and relatedness contribute to association structure once juveniles disperse away from birth sites, and (2) if juvenile and/or parental inbreeding predicts individual sociability. We detected positive spatial autocorrelation: hihi that fledged closer by were more likely to associate even after dispersing, irrespective of genetic relatedness. Juvenile inbreeding did not predict sociability, but those raised by more inbred fathers formed more, stronger, associations, which did not depend on whether that male was the genetic parent or not. These results suggest that the natal environment created by parents, rather than focal genetic condition, establishes the foundation for social associations. Overall, we highlight how social inheritance may play an important role in population dynamics and evolutionary potential in wild animals.

Early-life telomere length predicts life-history strategy and reproductive senescence in a threatened wild songbird.

Telomeres are well known for their associations with lifespan and ageing across diverse taxa. Early-life telomere length can be influenced by developmental conditions and has been shown positively affect lifetime reproductive success in a limited number of studies. Whether these effects are caused by a change in lifespan, reproductive rate or perhaps most importantly reproductive senescence is unclear. Using long-term data on female breeding success from a threatened songbird (the hihi, Notiomystis cincta), we show that the early-life telomere length of individuals predicts the presence and rate of future senescence of key reproductive traits: clutch size and hatching success. In contrast, senescence of fledging success is not associated with early-life telomere length, which may be due to the added influence of biparental care at this stage. Early-life telomere length does not predict lifespan or lifetime reproductive success in this species. Females may therefore change their reproductive allocation strategy depending on their early developmental conditions, which we hypothesise are reflected in their early-life telomere length. Our results offer new insights on the role that telomeres play in reproductive senescence and individual fitness and suggest telomere length can be used as a predictor for future life history in threatened species.

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.

Fluctuating optimum and temporally variable selection on breeding date in birds and mammals.

Temporal variation in natural selection is predicted to strongly impact the evolution and demography of natural populations, with consequences for the rate of adaptation, evolution of plasticity, and extinction risk. Most of the theory underlying these predictions assumes a moving optimum phenotype, with predictions expressed in terms of the temporal variance and autocorrelation of this optimum. However, empirical studies seldom estimate patterns of fluctuations of an optimum phenotype, precluding further progress in connecting theory with observations. To bridge this gap, we assess the evidence for temporal variation in selection on breeding date by modeling a fitness function with a fluctuating optimum, across 39 populations of 21 wild animals, one of the largest compilations of long-term datasets with individual measurements of trait and fitness components. We find compelling evidence for fluctuations in the fitness function, causing temporal variation in the magnitude, but not the direction of selection. However, fluctuations of the optimum phenotype need not directly translate into variation in selection gradients, because their impact can be buffered by partial tracking of the optimum by the mean phenotype. Analyzing individuals that reproduce in consecutive years, we find that plastic changes track movements of the optimum phenotype across years, especially in bird species, reducing temporal variation in directional selection. This suggests that phenological plasticity has evolved to cope with fluctuations in the optimum, despite their currently modest contribution to variation in selection.

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.

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.

Systematic review of avian hatching failure and implications for conservation.

Avian hatching failure is a widespread phenomenon, affecting around 10% of all eggs that are laid and not lost to predation, damage, or desertion. Our understanding of hatching failure is limited in terms of both its underpinning mechanisms and its occurrence across different populations. It is widely acknowledged that rates of hatching failure are higher in threatened species and in populations maintained in captivity compared to wild, non-threatened species, but these differences have rarely been quantified and any broader patterns remain unexplored. To examine the associations between threat status, management interventions, and hatching failure across populations we conducted a phylogenetically controlled multilevel meta-analysis across 231 studies and 241 species of birds. Our data set included both threatened (Critically Endangered, Endangered, and Vulnerable) and non-threatened (Near Threatened and Least Concern) species across wild and captive populations, as well as 'wild managed' ('free-living') populations. We found the mean overall rate of hatching failure across all populations to be 16.79%, with the hatching failure rate of wild, non-threatened species being 12.40%. We found that populations of threatened species experienced significantly higher mean hatching failure than populations of non-threatened species. Different levels of management were also associated with different rates of hatching failure, with wild populations experiencing the lowest rate of hatching failure, followed by wild managed populations, and populations in captivity experiencing the highest rate. Similarly, populations that were subject to the specific management interventions of artificial incubation, supplementary feeding, and artificial nest provision displayed significantly higher rates of hatching failure than populations without these interventions. The driver of this correlation between hatching failure and management remains unclear, but could be an indirect result of threatened species being more likely to have lower hatching success and also being more likely to be subject to management, indicating that conservation efforts are fittingly being focused towards the species potentially most at risk from extinction. This is the most comprehensive comparative analysis of avian hatching failure that has been conducted to date, and the first to quantify explicitly how threat status and management are associated with the rate of hatching failure in a population. We discuss the implications of our results, focusing on their potential applications to conservation. Although we identified several factors clearly associated with variation in hatching failure, a significant amount of heterogeneity was not explained by our meta-analytical model, indicating that other factors influencing hatching failure were not included here. We discuss what these factors might be and suggest avenues for further research. Finally, we discuss the inconsistency in how hatching failure is defined and reported within the literature, and propose a standardised definition to be used in future studies which will enable better comparison across populations and ensure that the most accurate information is used to support management decisions.

Impact of exposure to urban air pollution on grey squirrel (Sciurus carolinensis) lung health.

The increased rate of global urbanisation has recently exacerbated the significant public health problem of traffic related air pollution. Despite the known significant impact on human health, little is known about the effects of air pollution on wildlife health. The lung is the primary target organ for the effects of exposure to air pollution, leading to lung inflammation, altering the lung epigenome, culminating in respiratory disease. In this study, we aimed to assess lung health and DNA methylation profiles in Eastern grey squirrel (Sciurus carolinensis) populations living across an urban-rural air pollution gradient. Squirrel lung health was assessed in four populations situated across the most polluted inner-city boroughs to the less polluted edges of Greater London. We also assessed lung DNA methylation across three London sites and a further two rural sites in Sussex and North Wales. Lung and tracheal diseases were present in 28% and 13% of the squirrels respectively. Specifically, focal inflammation (13%), focal macrophages with vacuolated cytoplasm (3%) and endogenous lipid pneumonia (3%). There was no significant difference in prevalence of lung, tracheal diseases, anthracosis (carbon presence) or lung DNA methylation levels between urban sites and urban and rural sites respectively or NO2 levels. BALT (Bronchus-Associated Lymphoid Tissue) was significantly smaller in the site with highest NO2 and contained the highest carbon loading compared to sites with lower NO2, however differences in carbon loading in between sites were not significant. High pollution site individuals also had significantly higher numbers of alveolar macrophages which suggests that grey squirrels are exposed to and respond to traffic-related air pollution and further research is needed to understand the impact of traffic-related air pollutants on wildlife health.

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.

Maternally invested carotenoids compensate costly ectoparasitism in the hihi.

Dietary ingested carotenoid biomolecules have been linked to both improved health and immunity in nestling birds. Here, we test whether maternally invested egg carotenoids can offset the cost of parasitism in developing nestling hihi (Notiomystis cincta) from the bloodsucking mite (Ornithonyssus bursa). Our results reveal clear negative effects of parasitism on nestlings, and that maternally derived carotenoids compensate this cost, resulting in growth parameters and ultimate mass achieved being similar to nonparasitized young. Our results offer an unique example of a direct positive relationship between enhanced maternal investment of carotenoids and an ability to cope with a specific and costly parasite in young birds. As O. bursa infestations reduce population viability in hihi, our findings also highlight the importance of key nutritional resources for endangered bird populations to better cope with common parasite infestations.

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.

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.

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.

High genetic diversity in the remnant island population of hihi and the genetic consequences of re-introduction.

The maintenance of genetic diversity is thought to be fundamental for the conservation of threatened species. It is therefore important to understand how genetic diversity is affected by the re-introduction of threatened species. We use establishment history and genetic data from the remnant and re-introduced populations of a New Zealand endemic bird, the hihi Notiomystis cincta, to understand genetic diversity loss and quantify the genetic effects of re-introduction. Our data do not support any recent bottleneck events in the remnant population. Furthermore, all genetic diversity measures indicate the remnant hihi population has retained high levels of genetic diversity relative to other New Zealand avifauna with similar histories of decline. Genetic diversity (N(A) , alleles per locus, allelic richness, F(IS) and H(S) ) did not significantly decrease in new hihi populations founded through re-introduction when compared to their source populations, except in the Kapiti Island population (allelic richness and H(S) ) which had very slow post-re-introduction population growth. The N(e) /N(c) ratio in the remnant population was high, but decreased in first-level re-introductions, which together with significant genetic differentiation between populations (F(ST) & Fisher's exact tests) suggest that extant populations are diverging as a result of founder effects and drift. Importantly, simulations of future allele loss predict that the number of alleles lost will be higher in populations with a slow population growth, fewer founding individuals and with nonrandom mating. Interestingly, this species has very high levels of extra-pair paternity which may reduce reproductive variance by allowing social and floater males to reproduce a life history trait that together with a large remnant population size may help maintain higher levels of genetic diversity than expected.

Physiological factors influencing female fertility in birds.

Fertility is fundamental to reproductive success, but not all copulation attempts result in a fertilized embryo. Fertilization failure is especially costly for females, but we still lack a clear understanding of the causes of variation in female fertility across taxa. Birds make a useful model system for fertility research, partly because their large eggs are easily studied outside of the female's body, but also because of the wealth of data available on the reproductive productivity of commercial birds. Here, we review the factors contributing to female infertility in birds, providing evidence that female fertility traits are understudied relative to male fertility traits, and that avian fertility research has been dominated by studies focused on Galliformes and captive (relative to wild) populations. We then discuss the key stages of the female reproductive cycle where fertility may be compromised, and make recommendations for future research. We particularly emphasize that studies must differentiate between infertility and embryo mortality as causes of hatching failure, and that non-breeding individuals should be monitored more routinely where possible. This review lays the groundwork for developing a clearer understanding of the causes of female infertility, with important consequences for multiple fields including reproductive science, conservation and commercial breeding.

Sensitive males: inbreeding depression in an endangered bird.

Attempts to conserve threatened species by establishing new populations via reintroduction are controversial. Theory predicts that genetic bottlenecks result in increased mating between relatives and inbreeding depression. However, few studies of wild sourced reintroductions have carefully examined these genetic consequences. Our study assesses inbreeding and inbreeding depression in a free-living reintroduced population of an endangered New Zealand bird, the hihi (Notiomystis cincta). Using molecular sexing and marker-based inbreeding coefficients estimated from 19 autosomal microsatellite loci, we show that (i) inbreeding depresses offspring survival, (ii) male embryos are more inbred on average than female embryos, (iii) the effect of inbreeding depression is male-biased and (iv) this population has a substantial genetic load. Male susceptibility to inbreeding during embryo and nestling development may be due to size dimorphism, resulting in faster growth rates and more stressful development for male embryos and nestlings compared with females. This work highlights the effects of inbreeding at early life-history stages and the repercussions for the long-term population viability of threatened species.

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.