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 Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries.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.

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.

Host phylogeny shapes viral transmission networks in an island ecosystem.

Virus transmission between host species underpins disease emergence. Both host phylogenetic relatedness and aspects of their ecology, such as species interactions and predator-prey relationships, may govern rates and patterns of cross-species virus transmission and hence zoonotic risk. To address the impact of host phylogeny and ecology on virus diversity and evolution, we characterized the virome structure of a relatively isolated island ecological community in Fiordland, New Zealand, that are linked through a food web. We show that phylogenetic barriers that inhibited cross-species virus transmission occurred at the level of host phyla (between the Chordata, Arthropoda and Streptophyta) as well as at lower taxonomic levels. By contrast, host ecology, manifest as predator-prey interactions and diet, had a smaller influence on virome composition, especially at higher taxonomic levels. The virus-host community comprised a 'small world' network, in which hosts with a high diversity of viruses were more likely to acquire new viruses, and generalist viruses that infect multiple hosts were more likely to infect additional species compared to host specialist viruses. Such a highly connected ecological community increases the likelihood of cross-species virus transmission, particularly among closely related species, and suggests that host generalist viruses present the greatest risk of disease emergence.

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.

Influence of management practice on the microbiota of a critically endangered species: a longitudinal study of kakapo chick faeces and associated nest litter.

BACKGROUND: The critically endangered kakapo is a flightless, nocturnal parrot endemic to Aotearoa New Zealand. Recent efforts to describe the gastrointestinal microbial community of this threatened herbivore revealed a low-diversity microbiota that is often dominated by Escherichia-Shigella bacteria. Given the importance of associated microbial communities to animal health, and increasing appreciation of their potential relevance to threatened species conservation, we sought to better understand the development of this unusual gut microbiota profile. To this end, we conducted a longitudinal analysis of faecal material collected from kakapo chicks during the 2019 breeding season, in addition to associated nest litter material. RESULTS: Using an experimental approach rarely seen in studies of threatened species microbiota, we evaluated the impact of a regular conservation practice on the developing kakapo microbiota, namely the removal of faecal material from nests. Artificially removing chick faeces from nests had negligible impact on bacterial community diversity for either chicks or nests (p > 0.05). However, the gut microbiota did change significantly over time as chick age increased (p < 0.01), with an increasing relative abundance of Escherichia-Shigella coli over the study period and similar observations for the associated nest litter microbiota (p < 0.01). Supplementary feeding substantially altered gut bacterial diversity of kakapo chicks (p < 0.01), characterised by a significant increase in Lactobacillus bacteria. CONCLUSIONS: Overall, chick age and hand rearing conditions had the most marked impact on faecal bacterial communities. Similarly, the surrounding nest litter microbiota changed significantly over time since a kakapo chick was first placed in the nest, though we found no evidence that removal of faecal material influenced the bacterial communities of either litter or faecal samples. Taken together, these observations will inform ongoing conservation and management of this most enigmatic of bird species.

ENVIRONMENTAL FACTOR INVESTIGATION OF EXUDATIVE CLOACITIS IN KAKAPO (STRIGOPS HABROPTILUS) ON WHENUA HOU (CODFISH ISLAND), NEW ZEALAND.

Kakapo (Strigops habroptilus) are critically endangered nocturnal parrots endemic to New Zealand. Exudative cloacitis is a disease only affecting the breeding population of Kakapo on Whenua Hou (Codfish Island), for which a consistent primary pathogenic organism involved has not been identified. This epidemiological study was conducted to identify the environmental factors contributing to the initiation of this disease in Kakapo by 1) producing and describing a case series; 2) mapping the geographic distribution of exudative cloacitis cases; 3) investigating the chemical characteristics of Kakapo roosting sites; and 4) assessing the effects of climatic factors on the incidence of exudative cloacitis each year. Soil samples from the Kakapo roost sites and other areas of the Whenua Hou were examined for pH, ammonium, and moisture contents. From 2002 to 2017, 22 sporadic cases of exudative cloacitis have been diagnosed and the disease distribution on Whenua Hou overlaps the Kakapo distribution. A mixed group of adults and juveniles was affected and there was no evidence of spatial or temporal clustering of the disease. Current findings on the chemical characteristics of Kakapo roosting sites do not show any evidence that these factors are involved in the initiation of the exudative cloacitis. Nevertheless, the results suggest that the ammonium and moisture levels of the roosts are worthy of more detailed study in future cases. We were not able to demonstrate any significant influence of broad measures of climate on the incidence of exudative cloacitis on Whenua Hou. Prospective data collection would help for a complete epidemiological investigation of this disease in future cases.

Gut microbiota of the threatened takahe: biogeographic patterns and conservation implications.

BACKGROUND: The Aotearoa New Zealand takahe (Porphyrio hochstetteri), once thought to be extinct, is a nationally threatened flightless rail under intensive conservation management. While there has been previous research into disease-related microbes in takahe, little is known about the microbes present in the gastrointestinal tract. Given the importance of gut-associated microbes to herbivore nutrition and immunity, knowledge of these communities is likely to be of considerable conservation value. Here we examined the gut microbiotas of 57 takahe at eight separate locations across Aotearoa New Zealand. RESULTS: Faecal samples, taken as a proxy for the hindgut bacterial community, were subjected to 16S rRNA gene amplicon sequencing using Illumina MiSeq. Phylogenetic analysis of > 2200 amplicon sequence variants (ASVs) revealed nine main bacterial phyla (Acidobacteriota, Actinobacteriota, Bacteroidota, Campilobacterota, Firmicutes, Fusobacteriota, Planctomycetota, Proteobacteria, and Verrucomicrobiota) that accounted for the majority of sequence reads. Location was a significant effect (p value < 0.001, 9999 permutations) that accounted for 32% of the observed microbiota variation. One ASV, classified as Lactobacillus aviarius, was present in all samples at an average relative abundance of 17% (SD = 23.20). There was strong evidence (p = 0.002) for a difference in the abundance of the genus Lactobacillus between locations. A common commensal bacterium previously described in takahe, Campylobacter spp., was also detected in most faecal samples. CONCLUSIONS: Location plays a pivotal role in the observed variation among takahe gut bacterial communities and is potentially due to factors such as supplemental feeding and medical treatment experienced by birds housed in captivity at one of the eight sampled sites. These data present a first glimpse of the previously unexplored takahe gut microbiota and provide a baseline for future microbiological studies and conservation efforts.

A single fungal strain was the unexpected cause of a mass aspergillosis outbreak in the world's largest and only flightless parrot.

Kakapo are a critically endangered species of parrots restricted to a few islands off the coast of New Zealand. Kakapo are very closely monitored, especially during nesting seasons. In 2019, during a highly successful nesting season, an outbreak of aspergillosis affected 21 individuals and led to the deaths of 9, leaving a population of only 211 kakapo. In monitoring this outbreak, cultures of aspergillus were grown, and genome sequenced. These sequences demonstrate that, very unusually for an aspergillus outbreak, a single strain of aspergillus caused the outbreak. This strain was found on two islands, but only one had an outbreak of aspergillosis; indicating that the strain was necessary, but not sufficient, to cause disease. Our analysis provides an understanding of the 2019 outbreak and provides potential ways to manage such events in the future.

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.

The Low-Diversity Fecal Microbiota of the Critically Endangered Kakapo Is Robust to Anthropogenic Dietary and Geographic Influences.

The critically endangered kakapo, an herbivorous parrot endemic to New Zealand, is subject to intensive management to increase its population size. Key aspects of the management program include supplementary feeding and translocation of kakapo between different predator-free islands to optimize the genetic composition of the breeding populations. While these practices have helped boost the kakapo population, their impact on the kakapo fecal microbiota is uncertain. Previous studies have found that the kakapo possesses a low-diversity fecal microbiota, typically dominated by Escherichia/Shigella spp. However, the question of whether the low diversity of the kakapo fecal microbiota is an inadvertent consequence of human interventions has yet to be investigated. To that end, we used high-throughput Illumina sequencing of 16S rRNA gene amplicons obtained from fecal material of 63 kakapo representing different diets, islands, and ages. Remarkably, neither supplementary feeding nor geographic location were associated with significant differences in the overall fecal microbial community structures of adult kakapo, suggesting that the kakapo's low-diversity fecal microbiota is both inherent to this species and robust to these external influences.