Abstract Near-extinction events impose severe genomic bottlenecks that can have lasting fitness consequences, yet the specific mechanisms involved remain poorly understood. ‘Alalā, the last remaining Hawaiian crow, narrowly avoided extinction when a conservation breeding program was initiated from just eight genetic founders. Although the conservation breeding program has since recovered to ∼120 birds, it remains plagued by egg hatching failure rates >50%. To investigate the impacts of this bottleneck on hatching failure and other fitness components, we generated a chromosome-level reference genome and resequenced 175 individuals, including 78 deceased embryos. Although long runs of homozygosity (ROH) >1Mb are abundant in ‘Alalā (mean F ROH =0.32), associations between F ROH and measures of survival and reproduction, including egg failure, were weak or nonexistent. Instead, we identify two recessive lethal haplotypes that together account for nearly all of the excess hatching failure and have persisted in the population at high frequency (15-25%), hinting at impaired purifying selection. Eco-evolutionary simulation models demonstrate that these findings are expected for a species that has endured a severe population bottleneck and exhibits modest levels of pre-bottleneck heterozygosity. Our findings suggest that elevated recessive allele frequencies may be a broadly important consequence of population bottlenecks.

Genetic and genomic data are increasingly used to aid conservation management of endangered species by providing insights into evolutionary histories, factors associated with extinction risks, and potential for future adaptation. For the 'Alalā, or Hawaiian crow (Corvus hawaiiensis), genetic concerns include negative correlations between inbreeding and hatching success. However, it is unclear if low genetic diversity and inbreeding depression are consequences of a historical population bottleneck, or if 'Alalā had historically low genetic diversity that predated human influence, perhaps as a result of earlier declines or founding events. In this study, we applied a hybridization-based sequence capture to generate a genome-wide single nucleotide polymorphism (SNP) dataset for comparing historical specimens collected in the 1890s, when 'Alalā were more numerous, to samples taken between 1973 and 1998, when 'Alalā population densities were near the lowest documented levels in the wild, prior to all individuals being collected for captive rearing. We found low genome-wide diversity in both sample groups, however, the modern sample group (1973 to 1998 cohort) exhibited relatively fewer polymorphic alleles, a lower proportion of polymorphic loci, and lower observed heterozygosity, consistent with a population decline and potential bottleneck effects. These results combined with a current low population size highlight the importance of continued efforts by conservation managers to mitigate inbreeding and maintain founder representation to preserve what genetic diversity remains.

Culex quinquefasciatus mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people and, separately, poses a threat to the conservation of insular birds. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations. Yet, research into Cx. quinquefasciatus has been lagging compared to other disease vectors. Here, we use this tool to disrupt a set of five pigmentation genes in Cx. quinquefasciatus that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of Cx. quinquefasciatus that are relevant to potential future public health and bird conservation applications. We generate a double-mutant line, demonstrating the possibility of sequentially combining multiple such mutations in a single individual. Lastly, we target two loci, doublesex in the sex-determination pathway and proboscipedia, a hox gene, demonstrating the flexibility of these methods applied to novel targets. Our work provides a platform of seven validated loci that could be used for targeted mutagenesis in Cx. quinquefasciatus and the future development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily scored transgenesis marker.

Understanding how inbreeding affects endangered species in conservation breeding programs is essential for their recovery. The Hawaiian Crow ('Alalā) (Corvus hawaiiensis) is one of the world's most endangered birds. It went extinct in the wild in 2002, and, until recent release efforts starting in 2016, nearly all of the population remained under human care for conservation breeding. Using pedigree inbreeding coefficients (F), we evaluated the effects of inbreeding on Hawaiian Crow offspring survival and reproductive success. We used regression tree analysis to identify the level of inbreeding (i.e., inbreeding threshold) that explains a substantial decrease in 'Alalā offspring survival to recruitment. Similar to a previous study of inbreeding in 'Alalā, we found that inbreeding had a negative impact on offspring survival but that parental (vs. artificial) egg incubation improved offspring survival to recruitment. Furthermore, we found that inbreeding did not substantially affect offspring reproductive success, based on the assumption that offspring that survive to adulthood breed with distantly related mates. Our novel application of regression tree analysis showed that offspring with inbreeding levels exceeding F = 0.098 were 69% less likely to survive to recruitment than more outbred offspring, providing a specific threshold value for ongoing population management. Our results emphasize the importance of assessing inbreeding depression across all life history stages, confirm the importance of prioritizing parental over artificial egg incubation in avian conservation breeding programs, and demonstrate the utility of regression tree analysis as a tool for identifying inbreeding thresholds, if present, in any pedigree-managed population.

The avifauna endemic to islands is particularly susceptible to population declines and extinctions resulting from the introduction of non-native pathogens. Three pathogens of concern are the avian malaria parasites, the avian poxviruses, and West Nile virus—each of which can be transmitted by Culex quinquefasciatus, a highly adaptive and invasive mosquito. Culex quinquefasciatus has dramatically expanded its range in recent centuries and is now established throughout much of the tropics and sub-tropics, including on many islands that are remote from mainland landmasses and where this geographic separation historically protected island species from mosquito-borne diseases. The potential for ecological disruption by Cx. quinquefasciatus has been particularly striking in the Hawaiian Islands, where the introduction and transmission of avian malaria and avian poxvirus led to the extinction of several endemic bird species, with many more at risk. With Cx. quinquefasciatus now present in many insular communities and global trade and tourism increasing links between these areas, both to each other and to mainlands, there is growing concern that patterns of avian decline in Hawai‘i may be played out in other insular ecosystems. The implementation of traditional methods for Cx. quinquefasciatus control, including larval source management, is often impractical at large scale and when breeding sites are numerous and difficult to locate—typical issues associated with invasive species removal. One alternative approach would be the utilisation of genetic control methods, several of which have been successfully developed in other mosquitos such as Aedes aegypti and the malaria vector Anopheles gambiae. However, the development of similar tools for Cx. quinquefasciatus has been comparatively limited. Here we review the threat that Cx. quinquefasciatus poses as a vector of avian pathogens to island avifauna and discuss specific examples of at-risk bird populations on the islands of Hawai‘i, New Zealand and Galápagos. We also review the major options for the deployment of genetic control tools against Cx. quinquefasciatus, and discuss the current state of the field with a focus on radiation-based sterilisation, transgenic methods, and transinfections using the bacterial endosymbiont Wolbachia.

We must ensure that trials are scientifically, politically, and socially robust, publicly accountable, and widely transparent

Background Extensive phenotypic plasticity in oysters makes them difficult to identify based on morphology alone, but their identities can be resolved by applying genetic and genomic technologies. In this study, we collected unknown oyster specimens from Hawaiian waters for genetic identification. Methods We sequenced two partial gene fragments, mitochondrial 16S ribosomal RNA ( 16S ) and cytochrome c oxidase subunit I ( COI ), in 48 samples: 27 unidentified oyster specimens collected from two locations on O‘ahu, 13 known specimens from a hatchery in Hilo, Hawai‘i Island, and 8 known specimens from Hilo Bay, Hawai‘i Island. Results Molecular data identified approximately 85% of unknown samples as belonging to the Ostrea stentina/aupouria/equestris species complex, a globally distributed group with a history of uncertain and controversial taxonomic status. The remaining unknown samples were the native Dendostrea sandvichensis (G. B. Sowerby II, 1871), and nonnative Crassostrea gigas (Thunberg, 1793), the latter of which is a commercial species that was introduced to Hawai‘I from multiple sources during the 20th century. Phylogenetic analysis placed Hawai‘i Ostrea alongside samples from China, Japan, and New Zealand, grouping them within the recently classified western Pacific O. equestris . Until now, four extant species of true oyster have been documented in Hawai‘i. This study expands the known range of O. equestris by providing the first verification of its occurrence in Hawai‘i.

Despite the advent of several novel, synthetic gene drive mechanisms and their potential to one-day control a number of devastating diseases, among other applications, practical use of these systems remains contentious and risky. In particular, there is little in the way of empirical evidence of the long-term robustness of these synthetic systems against mutational breakdown. Rather, most existing systems are either known or predicted to be susceptible to rapid inactivation, though methodological designs continue to be refined. Here we evaluate a currently existing synthetic, underdominance-based gene drive system 200+ generations after it was first established in a laboratory colony of Drosophila melanogaster. Not only do we find that the system is still functioning as designed, we also show evidence that disruptions to the genetic construct are highly likely to be removed by natural selection, contributing to the system's robust, long-term stability. This stability appears to be a result of a fundamental relationship between ribosomal proteins (a novel target of the system) and natural cellular defenses that protect against cancer development. As far as we are aware, this is the longest continually functioning synthetic gene drive system thus verified, making it highly appropriate for additional research into its eventual suitability for field trials. Due to inherent properties of this gene drive, it is also likely to be adaptable for use in many different species. The insect lines established and used to test this system have been deposited at a Drosophila stock center, and are available to labs for further, independent testing.

Genome-level data can provide researchers with unprecedented precision to examine the causes and genetic consequences of population declines, which can inform conservation management. Here, we present a high-quality, long-read, de novo genome assembly for one of the world’s most endangered bird species, the ʻAlalā (Corvus hawaiiensis; Hawaiian crow). As the only remaining native crow species in Hawaiʻi, the ʻAlalā survived solely in a captive-breeding program from 2002 until 2016, at which point a long-term reintroduction program was initiated. The high-quality genome assembly was generated to lay the foundation for both comparative genomics studies and the development of population-level genomic tools that will aid conservation and recovery efforts. We illustrate how the quality of this assembly places it amongst the very best avian genomes assembled to date, comparable to intensively studied model systems. We describe the genome architecture in terms of repetitive elements and runs of homozygosity, and we show that compared with more outbred species, the ʻAlalā genome is substantially more homozygous. We also provide annotations for a subset of immunity genes that are likely to be important in conservation management, and we discuss how this genome is currently being used as a roadmap for downstream conservation applications.

There has been recent interest in the mutational stability of different gene-drive mechanisms. Here we test an underdominance system 200 generations after it was first established. Not only do we find that the system is still functioning as expected, we also find evidence that disruptions to the genetic construct are likely to be removed by natural selection, contributing to the system's long term stability. This appears to be a result of a fundamental relationship between ribosomal proteins and cellular defences which protect against cancer development. As far as the authors are aware, this is the longest continually functioning gene-drive system so far verified. The Drosophila lines used to test this system have been deposited at a Drosophila stock center and are available to labs for independent testing of various aspects of the system.

Despite the advent of several novel, synthetic gene drive mechanisms and their potential to one-day control a number of devastating diseases, among other applications, practical use of these systems remains contentious and risky. In particular, there is little in the way of empirical evidence of the long-term robustness of these synthetic systems against mutational breakdown. Rather, most existing systems are either known or predicted to be susceptible to rapid inactivation, though methodological designs continue to be refined. Here we evaluate a currently existing synthetic, underdominance-based gene drive system 200+ generations after it was first established in a laboratory colony of Drosophila melanogaster. Not only do we find that the system is still functioning as designed, we also show evidence that disruptions to the genetic construct are highly likely to be removed by natural selection, contributing to the system's robust, long-term stability. This stability appears to be a result of a fundamental relationship between ribosomal proteins (a novel target of the system) and natural cellular defenses that protect against cancer development. As far as we are aware, this is the longest continually functioning synthetic gene drive system thus verified, making it highly appropriate for additional research into its eventual suitability for field trials. Due to inherent properties of this gene drive, it is also likely to be adaptable for use in many different species. The insect lines established and used to test this system have been deposited at a Drosophila stock center, and are available to labs for further, independent testing.

Genetic rescue can reduce inbreeding depression and increase fitness of small populations, even when the donor populations are highly inbred. In a recent experiment involving two inbred island populations of the New Zealand South Island robin, Petroica australis, reciprocal translocations improved microsatellite diversity and individual fitness. While microsatellite loci may reflect patterns of genome-wide diversity, they generally do not indicate the specific genetic regions responsible for increased fitness. We tested the effectiveness of this reciprocal translocation for rescuing diversity of two immunogenetic regions: Toll-like receptor (TLR) and major histocompatibility complex (MHC) genes. We found that the relatively small number of migrants (seven and ten per island) effectively brought the characteristic TLR gene diversity of each source population into the recipient population. However, when migrants transmitted TLR alleles that were already present at high frequency in the recipient population, it was possible for offspring of mixed heritage to have decreased gene diversity compared to recipient population diversity prior to translocation. In contrast to TLRs, we did not observe substantial changes in MHC allelic diversity following translocation, with limited evidence of a decrease in differentiation, perhaps because most MHC alleles were observed at both sites prior to the translocation. Overall, we conclude that small numbers of migrants may successfully restore the diversity of immunogenetic loci with few alleles, but that translocating larger numbers of animals would provide additional opportunity for the genetic rescue of highly polymorphic immunity regions, such as the MHC, even when the source population is inbred.

For populations that experienced historical population bottlenecks, subsequent contemporary bottlenecks may continue to degrade genetic diversity at loci that are still variable. However, it is currently unclear how different types of contemporary bottlenecks may affect diversity at functional versus neutral loci. In this study, we examine genetic diversity of microsatellite, major histocompatibility complex (MHC), and toll-like receptor (TLR) loci of three New Zealand South Island saddleback (Philesturnus carunculatus) populations: Big Island, Kaimohu Island and Motuara Island. One population, Motuara, experienced two sets of contemporary bottlenecks (translocation event followed by a disease-outbreak and population crash) and was sampled in three different years. For all populations and sampling periods, we compare patterns of diversity across the three sets of loci to infer the effects of contemporary bottlenecks. We found that translocation-induced bottlenecks resulted in greater losses of microsatellite than MHC diversity, whereas temporal (encompassing a potential disease-induced bottleneck) fluctuations in diversity were greater for MHC markers than microsatellite loci. Both translocation- and disease-induced contemporary bottlenecks affected TLR diversity to a greater extent than diversity of either microsatellites or MHC. We found significant temporal changes in the frequencies of two TLR1LA alleles on Motuara Island (coincident with the population crash), but no further subsequent change. These alleles differ by an amino acid variant (associated with the leucine-rich repeat region of TLR1LA). Since TLR1LA may initiate an innate immune response in the presence of haemosporidia parasites, it is possible that a selection event has contributed to the observed patterns at TLR1LA. Our results indicate that patterns of genetic diversity loss at microsatellite, MHC and TLR loci are not equally affected by contemporary population bottlenecks, and may be influenced by bottleneck type.

Studies of wildlife populations have identified associations between disease resistance and diversity at genes of the major histocompatibility complex (MHC), which are involved with adaptive immunity.We compare MHC class II B (MHCIIB) and microsatellite genetic diversity in a population of New Zealand passerine birds, North Island saddlebacks on Mokoia Island, that was also tested for avian malaria.Prevalence of infection was low, and we found no conclusive evidence that infection status was linked to particular MHCIIB alleles.Individual-level MHCIIB nucleotide diversity, and the number of alleles per bird were both lower in infected compared with uninfected samples; however, neither trend was statistically significant.Genetic structure was observed in the MHCIIB dataset, suggesting that selective forces are shaping MHCIIB diversity in this population.Future research would benefit from long-term monitoring of allele frequencies and larger sample sizes where possible.Additionally, including disease intensity data (i.e. the degree of infection in an individual) alongside prevalence data (i.e.presence/absence of infection in an individual) may enhance the ability to detect phenotype-genotype associations in future research.

Most empirical evidence suggests that balancing selection does not counter the effects of genetic drift in shaping postbottleneck major histocompatibility complex ( MHC ) genetic diversity when population declines are severe or prolonged. However, few studies have been able to include data from historical specimens, or to compare populations/species with different bottleneck histories. In this study, we examined MHC class II B and microsatellite diversity in four N ew Z ealand passerine (songbird) species that experienced moderate to very severe declines. We compared diversity from historical samples (collected c . 1884–1938) to present‐day populations. Using a Bayesian framework, we found that the change in genetic diversity from historical to contemporary samples was affected by three main factors: (i) whether the data were based on MHC or microsatellite markers, (ii) species (as a surrogate for bottleneck severity) and (iii) whether the comparison between historical and contemporary samples was made using historical samples originating from the mainland, or using historical samples originating from islands. The greatest losses in genetic diversity occurred for the most severely bottlenecked species, particularly between historical mainland and contemporary samples. Additionally, where loss of diversity occurred, the change was greater for MHC genes compared to microsatellite loci.

PREMISE OF STUDY:Molecular population genetics is a powerful tool to infer how species responded to past environmental change. In the northern hemisphere, interest is increasing in how species responded to changes in ice coverage and temperature during the last glaciation maximum (LGM, between 18000-21000 yr ago) with a common assumption that glacial refugia were located at the southern edge of a species range.METHODS:We reconstructed the glacial and postglacial phylogeography of Sabatia kennedyana, a member of the Atlantic Coastal Plains Flora with a current distribution from Nova Scotia (NS) to South Carolina, using both cpDNA and nuclear markers. We also examined clinal variation in morphological traits, in particular relative investment in asexual vs sexual growth.KEY RESULTS:We find strong evidence that the species did not reside in southern glacial refugia, but rather in primary glacial refugia off the exposed continental shelf extending from Cape Cod and that this area was responsible for the founding of modern populations across the range from Nova Scotia (NS) to the United States. Additionally, based on the finding of higher cpDNA diversity and older cpDNA lineages in NS, we propose that multiple founder events occurred in NS, while only a single lineage gave rise to current populations in the United States.CONCLUSIONS:By understanding how S. kennedyana responded to past shifts in climate and by identifying areas of high genetic diversity in the northern range edge, we discuss the potential response of the species to future climate change scenarios.

BACKGROUND:The New Zealand native frogs, family Leiopelmatidae, are among the most archaic in the world. Leiopelma hochstetteri (Hochstetter's frog) is a small, semi-aquatic frog with numerous, fragmented populations scattered across New Zealand's North Island. We characterized a major histocompatibility complex (MHC) class II B gene (DAB) in L. hochstetteri from a spleen transcriptome, and then compared its diversity to neutral microsatellite markers to assess the adaptive genetic diversity of five populations ("evolutionarily significant units", ESUs).RESULTS:L. hochstetteri possessed very high MHC diversity, with 74 DAB alleles characterized. Extremely high differentiation was observed at the DAB locus, with only two alleles shared between populations, a pattern that was not reflected in the microsatellites. Clustering analysis on putative peptide binding residues of the DAB alleles indicated four functional supertypes, all of which were represented in 4 of 5 populations, albeit at different frequencies. Otawa was an exception to these observations, with only two DAB alleles present.CONCLUSIONS:This study of MHC diversity highlights extreme population differentiation at this functional locus. Supertype differentiation was high among populations, suggesting spatial and/or temporal variation in selection pressures. Low DAB diversity in Otawa may limit this population's adaptive potential to future pathogenic challenges.

The major histocompatibility complex (MHC) is integral to the vertebrate adaptive immune system. Characterizing diversity at functional MHC genes is invaluable for elucidating patterns of adaptive variation in wild populations, and is particularly interesting in species of conservation concern, which may suffer from reduced genetic diversity and compromised disease resilience. Here, we use next generation sequencing to investigate MHC class II B (MHCIIB) diversity in two sister taxa of New Zealand birds: South Island saddleback (SIS), Philesturnus carunculatus, and North Island saddleback (NIS), Philesturnus rufusater. These two species represent a passerine family outside the more extensively studied Passerida infraorder, and both have experienced historic bottlenecks. We examined exon 2 sequence data from populations that represent the majority of genetic diversity remaining in each species. A high level of locus co-amplification was detected, with from 1 to 4 and 3 to 12 putative alleles per individual for South and North Island birds, respectively. We found strong evidence for historic balancing selection in peptide-binding regions of putative alleles, and we identified a cluster combining non-classical loci and pseudogene sequences from both species, although no sequences were shared between the species. Fewer total alleles and fewer alleles per bird in SIS may be a consequence of their more severe bottleneck history; however, overall nucleotide diversity was similar between the species. Our characterization of MHCIIB diversity in two closely related species of New Zealand saddlebacks provides an important step in understanding the mechanisms shaping MHC diversity in wild, bottlenecked populations.

Molecular ecologists must be vigilant in detecting and accounting for genotyping error, yet potential errors stemming from dye-induced mobility shift (dye shift) may be frequently neglected and largely unknown to researchers who employ 3-primer systems with automated genotyping. When left uncorrected, dye shift can lead to mis-scoring alleles and even to falsely calling new alleles if different dyes are used to genotype the same locus in subsequent reactions. When we used four different fluorophore labels from a standard dye set to genotype the same set of loci, differences in the resulting size estimates for a single allele ranged from 2.07 bp to 3.68 bp. The strongest effects were associated with the fluorophore PET, and relative degree of dye shift was inversely related to locus size. We found little evidence in the literature that dye shift is regularly accounted for in 3-primer studies, despite knowledge of this phenomenon existing for over a decade. However, we did find some references to erroneous standard correction factors for the same set of dyes that we tested. We thus reiterate the need for strict quality control when attempting to reduce possible sources of genotyping error, and in cases where different dyes are applied to a single locus, perhaps mistakenly, we strongly discourage researchers from assuming generic correction patterns.