Transitions to captivity often produce population bottlenecks. On the one hand, bottlenecks increase inbreeding and decrease effective population size, thus increasing extinction risk. On the other hand, elevated homozygosity associated with inbreeding may purge deleterious mutations. Previous empirical studies of purging in captive breeding programs have focused on phenotypic measurements. We test natural selection's ability to purge deleterious mutations following an extreme population bottleneck by analyzing patterns of genetic diversity in wild and captive-bred individuals of the Lord Howe Island stick insect, Dryococelus australis. Dryococelus australis has been bred in captivity for two decades, having passed through an extreme bottleneck-only two mating pairs with few new additions since then. The magnitude of the bottleneck together with high female fecundity but low offspring recruitment set up nearly ideal conditions for the purging of deleterious mutations. As expected, captive-bred individuals had a greater number of long runs of homozygosity compared with wild individuals, implying strong inbreeding in captivity which would facilitate purging in homozygous regions. Stop-codon mutations were preferentially depleted in captivity compared with other mutations in coding and noncoding regions. The more deleterious a mutation was predicted to be, the more likely it was found outside of runs of homozygosity, implying that inbreeding facilitates the expression and thus removal of deleterious mutations, even after such an extreme bottleneck and under the benign conditions of captivity. These data implicate inbreeding and recessive deleterious mutation load in fitness variation among captive and wild D. australis.

Abstract Livestock guardian dogs (LGDs) are increasingly used to protect livestock from predators, but their effects on the distribution and behaviour of wild predators are mostly unknown. A key question is whether LGDs exclude predators from grazing land, or if predators continue to use areas with LGDs but modify their behaviour in ways that reduce impacts on livestock. We studied effects of LGDs (Maremma sheepdogs) on distribution and behaviour of red foxes Vulpes vulpes in north‐eastern Victoria, Australia. We mapped the activity of LGDs across the study areas using GPS tracking and measured fox activity using remote cameras. We also measured risk‐sensitive foraging in foxes to test if they reduced feeding time at sites regularly used by LGDs. Foxes occurred throughout areas occupied by LGDs, but their probability of detection was negatively related to the probability of LGD presence. Foxes extracted fewer food items from experimental food stations in proportion to the intensity of local activity of LGDs. This indicates that, though foxes overlapped with LGDs, they responded to risk of encountering LGDs by allocating less time to foraging. While LGDs do not necessarily exclude wild predators from areas used for livestock production, they can have strong effects on predator behaviour. Reduction in time allocated to foraging in areas regularly used by LGDs could lead to suppression of hunting behaviour and therefore a reduction in attacks on livestock. The flexible response of predators to LGDs should facilitate coexistence of wild predators with livestock farming, by allowing predators to continue to use areas occupied by livestock while still preventing attacks on those livestock. Our results therefore strengthen the case for use of LGDs in the conservation of predators threatened by conflict with farming. Suppression of hunting behaviour should also mean that prey species experience reduced rates of predation on farmland with LGDs. This effect could be valuable for conservation of threatened species of prey.

Captive breeding can be a useful tool for the management of threatened species, but such programs often begin with a bottleneck which may lower reproductive fitness by increasing inbreeding and allowing deleterious alleles to increase in frequency by reducing effective population size. Dryococelus australis, the Lord Howe Island stick insect, is a critically endangered insect that has been bred at the Melbourne Zoo in Melbourne, Australia, since shortly after the species was rediscovered in 2001. We analysed a long-term dataset of phenotypic measurements from this captive breeding program to determine whether reproductive fitness has declined in captivity. We found a clear signal of decline in direct (egg hatching success) and indirect (egg and nymph size) fitness indicators over time. We compared a captive line descended from the original program founders to a new hybrid line that has recent wild ancestry. The hybrid line showed an immediate improvement in egg hatch rate, suggesting the potential for genetic rescue. Egg and nymph size did not show as dramatic a change, although this line had only existed for just over a single generation at the time of data collection. These results argue strongly for the regular sourcing of new genetic material from the wild D. australis population to counteract fitness declines in captivity. Aside from benefiting the captive population, this would also improve the prospects of establishing new wild populations in the future, using individuals sourced from captivity.

Transitions to captivity are usually population bottlenecks and so may contribute to the fitness decline of captive-bred species by genetic drift and increased inbreeding. Purging can remove deleterious alleles from populations during declines and bottlenecks but the strength of this effect across different scenarios is unknown. The Lord Howe Island stick insect, Dryococelus australis , has been bred in captivity since 2003 and passed through an extreme bottleneck: only two mating pairs with only one new addition since then. We document extremely low heterozygosity and high inbreeding in the wild, suggestive of low population size and/or a recent colonisation bottleneck. We then test the ability of natural selection to purge deleterious alleles following an extreme population bottleneck by comparing patterns of genetic diversity in wild and captive-bred D. australis . Captive-bred individuals had lower heterozygosity and a greater number of long runs-of-homozygosity compared to wild individuals, implying strong inbreeding in captivity which would facilitate purging. Highly deleterious alleles were preferentially depleted in captivity but all other alleles, coding and non-coding, had the same mean frequency change in captivity compared to the wild. The more deleterious an allele was predicted to be, the more likely it was found outside of runs-of-homozygosity. These results are consistent with inbreeding purging these deleterious alleles. We show that purging can operate on highly deleterious alleles via inbreeding, even after an extreme bottleneck. This may contribute to the persistence of captive populations, although strong drift will also limit their adaptive potential in the future, in captivity and once reintroduced into the wild. ### Competing Interest Statement The authors have declared no competing interest.

Conservation breeding programs are an increasingly important tool to help supplement declining wild populations, but captive-bred animals generally do not survive well post-release. Early life in captivity has the potential to influence growth and development, with impacts carrying over to affect survival. Understanding carry-over effects of captivity and consequences on survival is critically important for conservation efforts globally but remains poorly understood. We examined the relationship between early-life environment, physical condition, and juvenile survival of wild and captive-bred critically endangered orange-bellied parrots (Neophema chrysogaster). Using nestling growth models, we calculated a body condition index for 1,039 wild and captive-bred orange-bellied parrots hatched over six breeding seasons. Nestling body condition varied with year, provenance, and brood position. Wild nestlings had consistently higher body condition than captive-bred nestlings, and first-hatched nestlings were typically heavier than later hatched siblings. We then investigated first-year survival for 298 wild-born and captive-bred released parrots in the wild. Overall, first-year survival was 27.5%, and individual body condition was more influential than provenance in predicting survival. Our findings could be used to aid the selection of individuals for release that have the best prospects of surviving in the wild. This study addresses important questions about the post-release fitness of captive-bred animals, and our metric of assessing physical condition provides a straightforward tool for other conservation breeding programs to adapt management techniques to improve survival outcomes.

In recent years, the potential to locate endangered animals using scent trained detection dogs (conservation detection dogs) has gained attention. Among vertebrates, conservation detection dogs have demonstrated a remarkable capacity to detect the scent of endangered mammals, reptiles, and birds, but their use in detecting amphibians is only beginning to be explored. A lack of work in this area is surprising given that amphibians are declining faster than any other vertebrate taxa. Moreover, amphibians are generally small, highly cryptic and breed in complex habitats, making them difficult to locate for the purpose of monitoring or establishing conservation breeding programs. This study aimed to provide a preliminary investigation into whether conservation detection dogs can be imprinted on the scent of the critically endangered Baw Baw frog ( Philoria frosti ) under captive conditions, and then effectively trained to locate wild frogs in their complex natural habitat. Two conservation detection dogs were trained to identify and locate P. frosti scent under controlled conditions before assessing their ability to locate wild P. frosti . Both conservation detection dogs were effective at locating P. frosti scent under controlled conditions, and also demonstrated an ability to detect live frogs under natural conditions. From an applied perspective, our findings provide new evidence that conservation detection dogs are capable of learning to detect the scent of small, cryptic anuran species. They also indicate that detection dogs are capable of locating frogs in highly complex forest habitat, confirming their untapped potential to aid in the management of imperiled species that have previously proven difficult to detect, monitor, and protect. We discuss the limitations of our approach and provide recommendations to help direct future amphibian conservation detection dog research.

We present a chromosome-scale genome assembly for Dryococelus australis, a critically endangered Australian phasmid. The assembly, constructed with Pacific Biosciences continuous long reads and chromatin conformation capture (Omni-C) data, is 3.42 Gb in length with a scaffold N50 of 262.27 Mb and L50 of 5. Over 99% of the assembly is contained in 17 major scaffolds, which corresponds to the species' karyotype. The assembly contains 96.3% of insect Benchmarking Unique Single Copy Ortholog genes in single copy. A custom repeat library identified 63.29% of the genome covered by repetitive elements; most were not identifiable based on similarity to sequences in existing databases. A total of 33,793 putative protein-coding genes were annotated. Despite the high contiguity and single-copy Benchmarking Unique Single Copy Ortholog content of the assembly, over 1 Gb of the flow-cytometry-estimated genome size is not represented, likely due to the large and repetitive nature of the genome. We identified the X chromosome with a coverage-based analysis and searched for homologs of genes known to be X-linked across the genus Timema. We found 59% of these genes on the putative X chromosome, indicating strong conservation of X-chromosomal content across 120 million years of phasmid evolution.

Small, isolated populations risk extinction through inbreeding depression, chance loss of beneficial variation, and reduced adaptability to changing environments. Genetic rescue via gene flow from genetically diverse sources is the most effective way to improve fitness of such populations. However, when the only potential source of immigrants is a different subspecies that diverged long ago and occupies a different environment, genetic rescue may lead to reduced fitness of admixed offspring through outbreeding depression or maladaptation. Test cases are needed to evaluate how to manage such potentially risky rescues to deliver enhanced population fitness. The helmeted honeyeater Lichenostomus melanops cassidix is a critically endangered subspecies of the yellow-tufted honeyeater. The sole remaining natural wild population experiences strong inbreeding depression for lifetime reproductive fitness. Captive genetic rescue trials are underway using a neighbouring subspecies, gippslandicus, which diverged from cassidix thousands of years ago and differs in morphology, mobility and preferred habitat. We evaluated short-term reproductive fitness for captive cassidix-cassidix pairs, first- and second-generation intersubspecific crosses and backcrosses to cassidix, while accounting for breeding season, sex, age at breeding, and wild/captive origin of each bird. Most admixed pair-types more readily engaged in breeding, raised more nestlings per nest, and had less male-biased chick sex-ratios than did cassidix-cassidix pairs, with negligible evidence of outbreeding depression. Continuing monitoring of fitness after releases into the wild is recommended, to ensure local adaptation is retained. With potentially riskier rescue increasingly becoming the only option for many populations, our study provides an encouraging test case.

Identifying sex-linked markers in genomic datasets is important because their presence in supposedly neutral autosomal datasets can result in incorrect estimates of genetic diversity, population structure and parentage. However, detecting sex-linked loci can be challenging, and available scripts neglect some categories of sex-linked variation. Here, we present new R functions to (1) identify and separate sex-linked loci in ZW and XY sex determination systems and (2) infer the genetic sex of individuals based on these loci. We tested these functions on genomic data for two bird and one mammal species and compared the biological inferences made before and after removing sex-linked loci using our function. We found that our function identified autosomal loci with ≥98.8% accuracy and sex-linked loci with an average accuracy of 87.8%. We showed that standard filters, such as low read depth and call rate, failed to remove up to 54.7% of sex-linked loci. This led to (i) overestimation of population FIS by up to 24%, and the number of private alleles by up to 8%; (ii) wrongly inferring significant sex differences in heterozygosity; (iii) obscuring genetic population structure and (iv) inferring ~11% fewer correct parentages. We discuss how failure to remove sex-linked markers can lead to incorrect biological inferences (e.g. sex-biased dispersal and cryptic population structure) and misleading management recommendations. For reduced-representation datasets with at least 15 known-sex individuals of each sex, our functions offer convenient resources to remove sex-linked loci and to sex the remaining individuals (freely available at https://github.com/drobledoruiz/conservation_genomics).

Captive breeding programs are an increasingly important tool for species' conservation efforts, but not all species reproduce well in captivity. Identifying factors that affect the reproductive success of captive populations is crucial to improving the performance and management of conservation-breeding programs, both by providing individuals for release and informing decision making. We examined breeding records collected from the long-running conservation-breeding program for the critically endangered Orange-bellied Parrot Neophema chrysogaster over an 11-year period. We examined egg hatching rate, nestling survival rate, and offspring sex ratio in response to a wide range of variables related to characteristics of individual birds, breeding events, and the captive environment. The hatch rate of eggs was higher in first clutches compared to second clutches and was lower than the wild population. The survival rate of nestlings through to fledging was variable between years but became higher and more consistent over the last five years of the study period. Variation in brood sex ratio was not related to any of the potential explanatory variables that we examined. This is one of the first studies to examine reproductive data in a long-running conservation-breeding program and shows that many common metrics do not explain reproductive variation. Our approach provides a framework for managers to investigate factors affecting reproductive success in conservation breeding programs more broadly.

The decision to intervene in endangered species management is often complicated. Migratory species exemplify this difficulty because they experience diverse threats at different times and places that can act cumulatively and synergistically on their populations. We use population viability analysis (PVA) to compare potential conservation interventions on the critically endangered, migratory Orange-bellied Parrot Neophema chrysogaster . This species suffers high juvenile mortality, but it is not clear why this is so. Given uncertainty about the best recovery strategy, we compare PVA scenarios that simulate various ways of utilizing captive-bred parrots to support the wild population in the context of unresolved threatening processes. Increasing the number of juveniles entering the population each year had the greatest benefit for population growth rate and size. Directly lowering juvenile mortality rates is difficult given uncertainty about the drivers of mortality in the wild. In lieu of this, releasing 100 juveniles from captivity to the wild population each autumn (either as a stand-alone action, or in combination with other interventions) was the most feasible and straightforward intervention of the options we tested. However, our PVAs also show that unless substantial and sustainable reductions can be made to juvenile mortality rates, Orange-bellied Parrots will remain dependent on intensive conservation management. This study highlights the utility of PVAs for answering practical questions about how to implement species conservation. PVAs provide a way to incorporate the best available information in a replicable modelling framework, and to identify impacts of parameter uncertainty on demographic trends.

Conservation breeding management aims to reduce inbreeding and maximize the retention of genetic diversity in endangered populations. However, breeding management of wild populations is still rare, and there is a need for approaches that provide data-driven evidence of the likelihood of success of alternative in situ strategies. Here, we provide an analytical framework that uses in silico simulations to evaluate, for real wild populations, (i) the degree of population-level inbreeding avoidance, (ii) the genetic quality of mating pairs, and (iii) the potential genetic benefits of implementing two breeding management strategies. The proposed strategies aim to improve the genetic quality of breeding pairs by splitting detrimental pairs and allowing the members to re-pair in different ways. We apply the framework to the wild population of the Critically Endangered helmeted honeyeater by combining genomic data and field observations to estimate the inbreeding (i.e., pair-kinship) and genetic quality (i.e., Mate Suitability Index) of all mating pairs for seven consecutive breeding seasons. We found no evidence of population-level inbreeding avoidance and that ~91.6% of breeding pairs were detrimental to the genetic health of the population. Furthermore, the framework revealed that neither proposed management strategy would significantly improve the genetic quality or reduce inbreeding of the mating pairs in this population. Our results demonstrate the usefulness of our analytical framework for testing the efficacy of different in situ breeding management strategies and for making evidence-based management decisions.

Radio-tracking tagged wildlife remains a critical research technique for understanding the movements, behaviours and survival of many species. However, traditional hand-held tracking techniques on the ground are labour intensive and time consuming. Therefore, researchers are increasingly seeking new technologies to address these challenges, including drone radio-tracking receivers. Following the implementation of drone radio-tracking techniques for five different threatened species projects within different habitat and landscape types, we identified the need to quantity the relative spatial extent of surveys using both drone and hand-held techniques for each project. This was undertaken using viewshed analyses. These analyses demonstrated that survey coverage with drone-based radio-tracking was substantially greater than that of hand-held radio-tracking for all species and landscapes examined. Within mountainous landscapes, drone radio-tracking covered up to four times the area of hand-held tracking, whereas in flat to undulating landscapes, drone surveys covered up to 11.3 times the area that could be surveyed using hand-held techniques from the same locations on the ground. The viewshed analyses were also found to be a valuable visualisation tool for identifying areas for targeted surveys to reduce the risk of 'losing' tagged animals, which has traditionally been one of the biggest radio-tracking challenges.

Background The helmeted honeyeater (Lichenostomus melanops cassidix) is a Critically Endangered bird endemic to Victoria, Australia. To aid its conservation, the population is the subject of genetic rescue. To understand, monitor, and modulate the effects of genetic rescue on the helmeted honeyeater genome, a chromosome-length genome and a high-density linkage map are required. Results We used a combination of Illumina, Oxford Nanopore, and Hi-C sequencing technologies to assemble a chromosome-length genome of the helmeted honeyeater, comprising 906 scaffolds, with length of 1.1 Gb and scaffold N50 of 63.8 Mb. Annotation comprised 57,181 gene models. Using a pedigree of 257 birds and 53,111 single-nucleotide polymorphisms, we obtained high-density linkage and recombination maps for 25 autosomes and Z chromosome. The total sex-averaged linkage map was 1,347 cM long, with the male map being 6.7% longer than the female map. Recombination maps revealed sexually dimorphic recombination rates (overall higher in males), with average recombination rate of 1.8 cM/Mb. Comparative analyses revealed high synteny of the helmeted honeyeater genome with that of 3 passerine species (e.g., 32 Hi-C scaffolds mapped to 30 zebra finch autosomes and Z chromosome). The genome assembly and linkage map suggest that the helmeted honeyeater exhibits a fission of chromosome 1A into 2 chromosomes relative to zebra finch. PSMC analysis showed a similar to 15-fold decline in effective population size to similar to 60,000 from mid- to late Pleistocene. Conclusions The annotated chromosome-length genome and high-density linkage map provide rich resources for evolutionary studies and will be fundamental in guiding conservation efforts for the helmeted honeyeater.

Abstract Emergency conservation interventions will be increasingly necessary to prevent extinctions or severe population bottlenecks as extreme events become more frequent. We detail the emergency extraction of the endangered Eastern Bristlebird (Dasyornis brachpterus) during the unprecedented 2019–2020 Australian Black Summer bushfires, an intervention that led to the rapid establishment of a temporary ex situ insurance population sourced from an area under immediate threat from bushfire (Croajingolong National Park, Victoria). The intervention was triggered, coordinated, and implemented within a 4‐week period, with re‐release to the wild within 2 months. We present this case study within a framework for emergency conservation interventions, based on the emergency management phases of preparation, response, and recovery, with the addition of an evaluation phase. The preparation phase involved compiling existing knowledge and capacity to facilitate the operation. The response phase consisted of (a) initiation and planning of the intervention (coordination) and (b) implementation, that is, the translocation of 15 birds from an area under threat of bushfire to a captive institution (>500 km). The recovery phase saw the insurance population re‐released to unburnt habitat after the bushfire had ceased. The evaluation phase incorporated lessons learnt from the other three phases as part of an adaptive management approach. We reflect on the Eastern Bristlebird emergency conservation intervention to explore how we can better prepare for, respond to, and recover from the large range of emergencies faced by biodiversity around the world.

Abstract Threatened species managers are often required to make swift decisions in the face of considerable uncertainty. We tested a structured approach for evaluating conservation options for the critically endangered orange‐bellied parrot. The Orange‐bellied Parrot Recovery Team provides advice to government agencies to inform the allocation of a limited number of captive‐bred birds to different release scenarios. Using a structured expert elicitation approach, we determined that scenarios where more fledglings were released were more cost‐effective compared to other options. Following this finding in 2019, the recovery team adjusted plans and allocated additional birds to fledgling release, a response that contributed to an increase in wild birds migrating in 2020 and 2021. The challenges facing orange‐bellied parrot conservation, including limitations in animals, time and resources, and high uncertainty, are common for threatened species recovery programs. Here we show that a structured process can help managers grapple with these complex trade‐offs to make timely decisions.

Captive breeding programmes are key to many threatened species reintroduction strategies but could potentially be associated with adaptations to captivity that are maladaptive in their natural habitat. Despite the importance of sensory ecology to biological fitness, few studies explore sensory system adaptations to captivity. Captive environments are devoid of predators and provide ready access to food sources and potential mates, thus reducing the need for individuals to use signals and cues to identify and locate resources or detect potential threats. With reduced complexity of the signalling environment, relaxation of selective pressures may favour reduced investment in sensory organs in captivity. We test this prediction in an iconic critically endangered invertebrate, the Lord Howe Island stick insect Dryococelus australis, which was extirpated from the island in the 1920s/30s and rediscovered on a nearby volcanic stack, Ball's Pyramid, in 2001. Using historical specimens from these populations and specimens from the 8-10th and 14th generations of a long-term conservation captive breeding programme, we examine differences in behaviourally relevant morphological traits of the compound eyes (visual organs) and antennae (olfactory organs). We find that captivity is associated with smaller compound eye size, smaller eye ommatidia and reduced density of antennal odour receptors. These morphological changes are indicative of reduced sensitivity to visual and olfactory signals and cues, and therefore are likely to have fitness implications when reintroducing a captive population into the wild. Synthesis and applications. We observe differences in sensory organ morphology between wild and captive-bred populations of the critically endangered Lord Howe Island stick insect. Our results emphasise the importance of incorporating evolutionary biology and sensory ecology into conservation programme design: to minimise the potential for captive breeding environments to compromise sensory systems that support appropriate behaviours upon reintroduction of populations into a natural habitat.

In species with biparental care, diel patterns in male and female incubation arise due to the differential costs and benefits of incubation. This pattern should be less obvious or absent in species where both sexes stand to benefit significantly from investing in incubation (i.e. in monogamous species with high paternity certainty), in temperate climates and in sexually monomorphic species where sexes probably experience similar physiological costs and risks of detection by predators. Despite no obvious size or colour differences between male and female Hooded Dotterel Thinornis cucullatus, males incubated significantly more at night (59% of the time), and less during the day (43% of the time). We show that despite Hooded Dotterel being sexually monomorphic, long-lived with probably high paternity certainty, incubation is not shared equitably, with a bias towards male nocturnal incubation.

As the global biodiversity crisis deepens, with increasing habitat fragmentation and a changing climate, innovative options for conserving species are being explored. One such conservation action is genetic rescue: introduction of new alleles to promote population fitness. However, for critically endangered species where only one viable population remains, options for introducing new alleles are limited. Interspecies hybridization offers a potential solution but requires resolution of evolutionary relationships, a sound understanding of species biology, social license, and permissive legislative frameworks. Here, we show how phylogenetics and species biology can inform genetic rescue options for the orange‐bellied parrot (OBP; Neophema chrysogaster ), a critically endangered Australian bird with one small remaining wild population. Our phylogenetic analysis of mitochondrial genomes and nuclear loci for all congeneric species provided strong support for OBPs being the sister species to a group comprising elegant, rock, and blue‐winged parrots. Accounting for species distribution, behavior, and ecology, a captive trial of interspecific hybridization with the blue‐winged parrot is recommended, including assessment of the fitness of hybrid individuals. Introduction of new alleles into the OBP genome would achieve the conservation goal of improving genetic diversity in a critically endangered species. Concurrently, legislative issues will need to be resolved.

Living in a captive environment may compromise phenotypic traits critical to survival in the wild. Captive animals that differ from the ideal wild phenotype may have impaired fitness after release, especially if there is extreme phenotypic selection during some life history stages. Wing shape of migratory birds is crucial to migration efficiency, and changes to wing shape in captivity may severely affect survival after release. We investigate wing shape of migratory Orange-bellied Parrots Neophema chrysogaster in captivity and the wild. The first two flight feathers were shorter in captive birds, and the fifth and sixth feathers were longer than wild conspecifics. These differences altered wing shape, producing a more proximal tip and a more convex trailing edge than the wild phenotype, which likely alters flight performance. This wing shape contravenes expectations from general patterns of wing shape in migratory birds. Wing shape in the captive birds sampled was independent of inbreeding, generations in captivity and ancestry. Captive environments may affect feather development or impose some selective pressure on wing shape. Alternatively, release from intense phenotypic selection during migration may allow parrots with wing shapes poorly adapted to migration to survive and breed in captivity. Altered wing shape may contribute to low observed survival of parrots released to the wild.