The genetic basis of the kakapo structural color polymorphism suggests balancing selection by an extinct apex predator.

The information contained in population genomic data can tell us much about the past ecology and evolution of species. We leveraged detailed phenotypic and genomic data of nearly all living kakapo to understand the evolution of its feather color polymorphism. The kakapo is an endangered and culturally significant parrot endemic to Aotearoa New Zealand, and the green and olive feather colorations are present at similar frequencies in the population. The presence of such a neatly balanced color polymorphism is remarkable because the entire population currently numbers less than 250 birds, which means it has been exposed to severe genetic drift. We dissected the color phenotype, demonstrating that the two colors differ in their light reflectance patterns due to differential feather structure. We used quantitative genomics methods to identify two genetic variants whose epistatic interaction can fully explain the species' color phenotype. Our genomic forward simulations show that balancing selection might have been pivotal to establish the polymorphism in the ancestrally large population, and to maintain it during population declines that involved a severe bottleneck. We hypothesize that an extinct apex predator was the likely agent of balancing selection, making the color polymorphism in the kakapo a "ghost of selection past."

Towards complete and error-free genome assemblies of all vertebrate species.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

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.

Influence of hand rearing and bird age on the fecal microbiota of the critically endangered kakapo.

The critically endangered New Zealand parrot, the kakapo, is subject to an intensive management regime aiming to maintain bird health and boost population size. Newly hatched kakapo chicks are subjected to human intervention and are frequently placed in captivity throughout their formative months. Hand rearing greatly reduces mortality among juveniles, but the potential long-term impact on the kakapo gut microbiota is uncertain. To track development of the kakapo gut microbiota, fecal samples from healthy, prefledged juvenile kakapos, as well as from unrelated adults, were analyzed by using 16S rRNA gene amplicon pyrosequencing. Following the original sampling, juvenile kakapos underwent a period of captivity, so further sampling during and after captivity aimed to elucidate the impact of captivity on the juvenile gut microbiota. Variation in the fecal microbiota over a year was also investigated, with resampling of the original juvenile population. Amplicon pyrosequencing revealed a juvenile fecal microbiota enriched with particular lactic acid bacteria compared to the microbiota of adults, although the overall community structure did not differ significantly among kakapos of different ages. The abundance of key operational taxonomic units (OTUs) was correlated with antibiotic treatment and captivity, although the importance of these factors could not be proven unequivocally within the bounds of this study. Finally, the microbial community structure of juvenile and adult kakapos changed over time, reinforcing the need for continual monitoring of the microbiota as part of regular health screening.

Prevalence of IgY antibodies against Erysipelothrix rhusiopathiae in a critically endangered parrot (kakapo, Strigops habroptilus) and associated responses to vaccination.

An enzyme-linked immunosorbent assay (ELISA) was developed to estimate levels of IgY antibody against the bacterium Erysipelothrix rhusiopathiae in serum samples collected from the critically endangered kakapo (Strigops habroptilus, Psittaciformes, Aves) before and after vaccination against this bacterium. Relative IgY antibody titres in pre-vaccination serum samples (n = 71 individual kakapo) were normally distributed with the exception of four outliers which displayed low IgY levels. Notably all four low IgY samples were collected from fledglings 3 - 6 months old. Pre-vaccination serum samples from nine nestlings <3 months old, seven of which were hatched in incubators and had no contact with either adult kakapo or their natural environment (e.g. soil), were found to have relatively high IgY levels, suggesting transfer of maternal IgY molecules to fledglings via the yolk. IgY levels in pre-vaccination serum samples from seven kakapo aged 25 - 30 months were also relatively high, suggesting that most kakapo naturally acquire anti- E.rhusiopathiae IgYs within their first 2 years. There was no evidence that vaccination increased the kakapo population's mean anti-E.rhusiopathiae IgY levels. However, there was a significant negative relationship between an individual bird's pre-vaccination IgY level and any subsequent increase following vaccination, suggesting that vaccination may only raise the IgY levels of birds with relatively low pre-vaccination IgY levels. A statistical model of the relationship between 'death from erysipelas' and sex, age and transfer from one to island sanctuary to another found that only transfer was significantly associated with death from erysipelas.

Non-invasive real-time genomic monitoring of the critically endangered kakapo.

We used non-invasive real-time genomic approaches to monitor one of the last surviving populations of the critically endangered kakapo (Strigops habroptilus). We first established an environmental DNA metabarcoding protocol to identify the distribution of kakapo and other vertebrate species in a highly localized manner using soil samples. Harnessing real-time nanopore sequencing and the high-quality kakapo reference genome, we then extracted species-specific DNA from soil. We combined long read-based haplotype phasing with known individual genomic variation in the kakapo population to identify the presence of individuals, and confirmed these genomically informed predictions through detailed metadata on kakapo distributions. This study shows that individual identification is feasible through nanopore sequencing of environmental DNA, with important implications for future efforts in the application of genomics to the conservation of rare species, potentially expanding the application of real-time environmental DNA research from monitoring species distribution to inferring fitness parameters such as genomic diversity and inbreeding.

Hidden impacts of conservation management on fertility of the critically endangered kakapo.

Background: Animal conservation often requires intensive management actions to improve reproductive output, yet any adverse effects of these may not be immediately apparent, particularly in threatened species with small populations and long lifespans. Hand-rearing is an example of a conservation management strategy which, while boosting populations, can cause long-term demographic and behavioural problems. It is used in the recovery of the critically endangered kakapo (Strigops habroptilus), a flightless parrot endemic to New Zealand, to improve the slow population growth that is due to infrequent breeding, low fertility and low hatching success. Methods: We applied Bayesian mixed models to examine whether hand-rearing and other factors were associated with clutch fertility in kakapo. We used projection predictive variable selection to compare the relative contributions to fertility from the parents' rearing environment, their age and previous copulation experience, the parental kinship, and the number of mates and copulations for each clutch. We also explored how the incidence of repeated copulations and multiple mates varied with kakapo density. Results: The rearing status of the clutch father and the number of mates and copulations of the clutch mother were the dominant factors in predicting fertility. Clutches were less likely to be fertile if the father was hand-reared compared to wild-reared, but there was no similar effect for mothers. Clutches produced by females copulating with different males were more likely to be fertile than those from repeated copulations with one male, which in turn had a higher probability of fertility than those from a single copulation. The likelihood of multiple copulations and mates increased with female:male adult sex ratio, perhaps as a result of mate guarding by females. Parental kinship, copulation experience and age all had negligible associations with clutch fertility. Conclusions: These results provide a rare assessment of factors affecting fertility in a wild threatened bird species, with implications for conservation management. The increased fertility due to multiple mates and copulations, combined with the evidence for mate guarding and previous results of kakapo sperm morphology, suggests that an evolutionary mechanism exists to optimise fertility through sperm competition in kakapo. The high frequency of clutches produced from single copulations in the contemporary population may therefore represent an unnatural state, perhaps due to too few females. This suggests that opportunity for sperm competition should be maximised by increasing population densities, optimising sex ratios, and using artificial insemination. The lower fertility of hand-reared males may result from behavioural defects due to lack of exposure to conspecifics at critical development stages, as seen in other taxa. This potential negative impact of hand-rearing must be balanced against the short-term benefits it provides.

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.

Population genomics of the critically endangered kakapo.

The kakapo is a flightless parrot endemic to New Zealand. Once common in the archipelago, only 201 individuals remain today, most of them descending from an isolated island population. We report the first genome-wide analyses of the species, including a high-quality genome assembly for kakapo, one of the first chromosome-level reference genomes sequenced by the Vertebrate Genomes Project (VGP). We also sequenced and analyzed 35 modern genomes from the sole surviving island population and 14 genomes from the extinct mainland population. While theory suggests that such a small population is likely to have accumulated deleterious mutations through genetic drift, our analyses on the impact of the long-term small population size in kakapo indicate that present-day island kakapo have a reduced number of harmful mutations compared to mainland individuals. We hypothesize that this reduced mutational load is due to the island population having been subjected to a combination of genetic drift and purging of deleterious mutations, through increased inbreeding and purifying selection, since its isolation from the mainland ∼10,000 years ago. Our results provide evidence that small populations can survive even when isolated for hundreds of generations. This work provides key insights into kakapo breeding and recovery and more generally into the application of genetic tools in conservation efforts for endangered species.

Inheritance of telomere length in a bird.

Telomere dynamics are intensively studied in human ageing research and epidemiology, with many correlations reported between telomere length and age-related diseases, cancer and death. While telomere length is influenced by environmental factors there is also good evidence for a strong heritable component. In human, the mode of telomere length inheritance appears to be paternal and telomere length differs between sexes, with females having longer telomeres than males. Genetic factors, e.g. sex chromosomal inactivation, and non-genetic factors, e.g. antioxidant properties of oestrogen, have been suggested as possible explanations for these sex-specific telomere inheritance and telomere length differences. To test the influence of sex chromosomes on telomere length, we investigated inheritance and sex-specificity of telomere length in a bird species, the kakapo (Strigops habroptilus), in which females are the heterogametic sex (ZW) and males are the homogametic (ZZ) sex. We found that, contrary to findings in humans, telomere length was maternally inherited and also longer in males. These results argue against an effect of sex hormones on telomere length and suggest that factors associated with heterogamy may play a role in telomere inheritance and sex-specific differences in telomere length.

Thirty polymorphic microsatellite loci from the critically endangered kakapo (Strigops habroptilus).

Thirty polymorphic microsatellite loci were developed from the critically endangered kakapo (Strigops habroptilus), using an enriched genomic library. Characterization of loci using 90 kakapo revealed an average of 3.3 alleles per locus (range: 2-5) and an average expected heterozygosity of 0.47 (range: 0.17-0.70). The probability of identity (7.2 × 10(-15) ) and probability of exclusion (0.999999) demonstrate that these loci are a highly informative marker set that can aid the genetic management of the kakapo.

Sex allocation theory aids species conservation.

Supplementary feeding is often a key tool in the intensive management of captive and threatened species. Although it can increase such parameters as breeding frequency and individual survival, supplementary feeding may produce undesirable side effects that increase overall extinction risk. Recent attempts to increase breeding frequency and success in the kakapo Strigops habroptilus using supplementary feeding inadvertently resulted in highly male-biased chick sex ratios. Here, we describe how the inclusion of sex allocation theory has remedied this conservation dilemma. Our study is the first to manipulate chick sex ratios in an endangered species by altering maternal condition and highlights the importance of incorporating evolutionary theory into modern conservation practice.