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.

Meta-Analysis Reveals Challenges and Gaps for Genome-to-Phenome Research Underpinning Plant Drought Response.

Severe drought conditions and extreme weather events are increasing worldwide with climate change, threatening the persistence of native plant communities and ecosystems. Many studies have investigated the genomic basis of plant responses to drought. However, the extent of this research throughout the plant kingdom is unclear, particularly among species critical for the sustainability of natural ecosystems. This study aimed to broaden our understanding of genome-to-phenome (G2P) connections in drought-stressed plants and identify focal taxa for future research. Bioinformatics pipelines were developed to mine and link information from databases and abstracts from 7730 publications. This approach identified 1634 genes involved in drought responses among 497 plant taxa. Most (83.30%) of these species have been classified for human use, and most G2P interactions have been described within model organisms or crop species. Our analysis identifies several gaps in G2P research literature and database connectivity, with 21% of abstracts being linked to gene and taxonomy data in NCBI. Abstract text mining was more successful at identifying potential G2P pathways, with 34% of abstracts containing gene, taxa, and phenotype information. Expanding G2P studies to include non-model plants, especially those that are adapted to drought stress, will help advance our understanding of drought responsive G2P pathways.

Expanding the conservation genomics toolbox: Incorporating structural variants to enhance genomic studies for species of conservation concern.

Structural variants (SVs) are large rearrangements (>50 bp) within the genome that impact gene function and the content and structure of chromosomes. As a result, SVs are a significant source of functional genomic variation, that is, variation at genomic regions underpinning phenotype differences, that can have large effects on individual and population fitness. While there are increasing opportunities to investigate functional genomic variation in threatened species via single nucleotide polymorphism (SNP) data sets, SVs remain understudied despite their potential influence on fitness traits of conservation interest. In this future-focused Opinion, we contend that characterizing SVs offers the conservation genomics community an exciting opportunity to complement SNP-based approaches to enhance species recovery. We also leverage the existing literature-predominantly in human health, agriculture and ecoevolutionary biology-to identify approaches for readily characterizing SVs and consider how integrating these into the conservation genomics toolbox may transform the way we manage some of the world's most threatened species.

Building strong relationships between conservation genetics and primary industry leads to mutually beneficial genomic advances.

Several reviews in the past decade have heralded the benefits of embracing high-throughput sequencing technologies to inform conservation policy and the management of threatened species, but few have offered practical advice on how to expedite the transition from conservation genetics to conservation genomics. Here, we argue that an effective and efficient way to navigate this transition is to capitalize on emerging synergies between conservation genetics and primary industry (e.g., agriculture, fisheries, forestry and horticulture). Here, we demonstrate how building strong relationships between conservation geneticists and primary industry scientists is leading to mutually-beneficial outcomes for both disciplines. Based on our collective experience as collaborative New Zealand-based scientists, we also provide insight for forging these cross-sector relationships.

Differential introgression and effective size of marker type influence phylogenetic inference of a recently divergent avian group (Phasianidae: Tympanuchus).

Life history strategies can influence the effective population size (Ne) of loci differently based on their mode of inheritance. Recognizing how this may affect the rate of lineage sorting among marker types is important for studies focused on resolving phylogenetic relationships among recently divergent taxa. In this study, we use gene tree, coalescent-based species tree, and isolation-with-migration analyses to explore the differences between marker types (autosomal, Z-linked, and mitochondrial) in resolving phylogenetic relationships among North American prairie grouse (Tympanuchus). We found that Z-linked loci were more likely to identify monophyletic relationships among prairie grouse species compared to autosomal and mtDNA loci in both species and gene tree analyses, with species tree analyses outperforming gene trees. These results were further supported with isolation-with-migration analyses, where Z-linked loci largely followed a strict isolation model while autosomal loci were more likely to fit a model with gene flow between species following population divergence. While accounting for differences in inheritance pattern (or Ne) for marker type, results suggest that additional factors, such as strong sexual selection and sex-biased introgression (i.e., male-biased postzygotic hybrid behavioral isolation or "unsexy son"), may further explain the decreased diversity levels and increased rate of lineage sorting observed with the Z-linked loci relative to autosomal and mtDNA loci. In fact, to our knowledge no hybrid male prairie grouse have been observed breeding in the wild, yet hybrid females along with backcross females are known to produce viable offspring. Overall, this study highlights that more work is needed to determine how complex models of gene flow (i.e., sex biased introgression) and differences in the effective size among marker types based on differing life history strategies influence divergence date estimation and species delimitation.

Relating gut microbiome composition and life history metrics for pronghorn (Antilocapra americana) in the Red Desert, Wyoming.

Host microbial communities (hereafter, the 'microbiome') are recognized as an important aspect of host health and are gaining attention as a useful biomarker to understand the ecology and demographics of wildlife populations. Several studies indicate that the microbiome may contribute to the adaptive capacity of animals to changing environments associated with increasing habitat fragmentation and rapid climate change. To this end, we investigated the gut microbiome of pronghorn (Antilocapra americana), an iconic species in an environment that is undergoing both climatic and anthropogenic change. The bacterial composition of the pronghorn gut microbiome has yet to be described in the literature, and thus our study provides important baseline information about this species. We used 16S rRNA amplicon sequencing of fecal samples to characterize the gut microbiome of pronghorn-a facultative sagebrush (Artemisia spp.) specialist in many regions where they occur in western North America. We collected fecal pellets from 159 captured female pronghorn from four herds in the Red Desert of Wyoming during winters of 2013 and 2014. We found small, but significant differences in diversity of the gut microbiome relative to study area, capture period, and body fat measurements. In addition, we found a difference in gut microbiome composition in pronghorn across two regions separated by Interstate 80. Results indicated that the fecal microbiome may be a potential biomarker for the spatial ecology of free-ranging ungulates. The core gut microbiome of these animals-including bacteria in the phyla Firmicutes (now Bacillota) and Bacteroidota-remained relatively stable across populations and biological metrics. These findings provide a baseline for the gut microbiome of pronghorn that could potentially be used as a target in monitoring health and population structure of pronghorn relative to habitat fragmentation, climate change, and management practices.

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.

Comparing genome-based estimates of relatedness for use in pedigree-based conservation management.

Researchers have long debated which estimator of relatedness best captures the degree of relationship between two individuals. In the genomics era, this debate continues, with relatedness estimates being sensitive to the methods used to generate markers, marker quality, and levels of diversity in sampled individuals. Here, we compare six commonly used genome-based relatedness estimators (kinship genetic distance [KGD], Wang maximum likelihood [TrioML], Queller and Goodnight [Rxy ], Kinship INference for Genome-wide association studies [KING-robust), and pairwise relatedness [RAB ], allele-sharing coancestry [AS]) across five species bred in captivity-including three birds and two mammals-with varying degrees of reliable pedigree data, using reduced-representation and whole genome resequencing data. Genome-based relatedness estimates varied widely across estimators, sequencing methods, and species, yet the most consistent results for known first order relationships were found using Rxy , RAB , and AS. However, AS was found to be less consistently correlated with known pedigree relatedness than either Rxy or RAB . Our combined results indicate there is not a single genome-based estimator that is ideal across different species and data types. To determine the most appropriate genome-based relatedness estimator for each new data set, we recommend assessing the relative: (1) correlation of candidate estimators with known relationships in the pedigree and (2) precision of candidate estimators with known first-order relationships. These recommendations are broadly applicable to conservation breeding programmes, particularly where genome-based estimates of relatedness can complement and complete poorly pedigreed populations. Given a growing interest in the application of wild pedigrees, our results are also applicable to in situ wildlife management.

G2PMineR: A Genome to Phenome Literature Review Approach.

There is a gap in the conceptual framework linking genes to phenotypes (G2P) for non-model organisms, as most non-model organisms do not yet have genomic resources readily available. To address this, researchers often perform literature reviews to understand G2P linkages by curating a list of likely gene candidates, hinging upon other studies already conducted in closely related systems. Sifting through hundreds to thousands of articles is a cumbersome task that slows down the scientific process and may introduce bias into a study. To fill this gap, we created G2PMineR, a free and open source literature mining tool developed specifically for G2P research. This R package uses automation to make the G2P review process efficient and unbiased, while also generating hypothesized associations between genes and phenotypes within a taxonomical framework. We applied the package to a literature review for drought-tolerance in plants. The analysis provides biologically meaningful results within the known framework of drought tolerance in plants. Overall, the package is useful for conducting literature reviews for genome to phenome projects, and also has broad appeal to scientists investigating a wide range of study systems as it can conduct analyses under the auspices of three different kingdoms (Plantae, Animalia, and Fungi).

A draft genome provides hypotheses on drought tolerance in a keystone plant species in Western North America threatened by climate change.

Climate change presents distinct ecological and physiological challenges to plants as extreme climate events become more common. Understanding how species have adapted to drought, especially ecologically important nonmodel organisms, will be crucial to elucidate potential biological pathways for drought adaptation and inform conservation strategies. To aid in genome-to-phenome research, a draft genome was assembled for a diploid individual of Artemisia tridentata subsp. tridentata, a threatened keystone shrub in western North America. While this taxon has few genetic resources available and genetic/genomics work has proven difficult due to genetic heterozygosity in the past, a draft genome was successfully assembled. Aquaporin (AQP) genes and their promoter sequences were mined from the draft genome to predict mechanisms regulating gene expression and generate hypotheses on key genes underpinning drought response. Fifty-one AQP genes were fully assembled within the draft genome. Promoter and phylogenetic analyses revealed putative duplicates of A. tridentata subsp. tridentata AQPs which have experienced differentiation in promoter elements, potentially supporting novel biological pathways. Comparison with nondrought-tolerant congener supports enrichments of AQP genes in this taxon during adaptation to drought stress. Differentiation of promoter elements revealed that paralogues of some genes have evolved to function in different pathways, highlighting these genes as potential candidates for future research and providing critical hypotheses for future genome-to-phenome work.

Fragmentation of Apolipoprotein E4 is Required for Differential Expression of Inflammation and Activation Related Genes in Microglia Cells.

The apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD) and accumulating evidence suggests that fragmentation with a toxic-gain of function may be a key molecular step associated with this risk. Recently, we demonstrated strong immunoreactivity of a 151 amino-terminal fragment of apoE4 (E4-fragment) within the nucleus of microglia in the human AD brain. In vitro, this fragment led to toxicity and activation of inflammatory processes in BV2 microglia cells. Additionally, a transcriptome analysis following exogenous treatment of BV2 microglia cells with this E4 fragment led to a > 2-fold up regulation of 1,608 genes, with many genes playing a role in inflammation and microglia activation. To extend these findings, we here report a similar transcriptome analysis in BV2 microglia cells following treatment with full-length ApoE4 (FL-ApoE4). The results indicated that full-length ApoE4 had a very small effect on gene expression compared to the fragment. Only 48 differentially expressed genes (DEGs) were identified (p < 0.05, and greater than 2-fold change). A gene ontology analysis of these DEGs indicated that they are not involved in inflammatory and activation processes, in contrast to the genes up regulated by the E4-fragment. In addition, genes that showed a negative fold-change upon FL-E4 treatment typically showed a strong positive fold-change upon treatment with the fragment (Pearson's r = -0.7). Taken together, these results support the hypothesis that a key step in the conversion of microglia to an activated phenotype is proteolytic cleavage of FL-ApoE4. Therefore, the neutralization of this amino-terminal fragment of ApoE4, specifically, may serve as an important therapeutic strategy in the treatment of AD.

A comparison of pedigree, genetic and genomic estimates of relatedness for informing pairing decisions in two critically endangered birds: Implications for conservation breeding programmes worldwide.

Conservation management strategies for many highly threatened species include conservation breeding to prevent extinction and enhance recovery. Pairing decisions for these conservation breeding programmes can be informed by pedigree data to minimize relatedness between individuals in an effort to avoid inbreeding, maximize diversity and maintain evolutionary potential. However, conservation breeding programmes struggle to use this approach when pedigrees are shallow or incomplete. While genetic data (i.e., microsatellites) can be used to estimate relatedness to inform pairing decisions, emerging evidence indicates this approach may lack precision in genetically depauperate species, and more effective estimates will likely be obtained from genomic data (i.e., thousands of genome-wide single nucleotide polymorphisms, or SNPs). Here, we compare relatedness estimates and subsequent pairing decisions using pedigrees, microsatellites and SNPs from whole-genome resequencing approaches in two critically endangered birds endemic to New Zealand: kaki/black stilt (Himantopus novaezelandiae) and kakariki karaka/orange-fronted parakeet (Cyanoramphus malherbi). Our findings indicate that SNPs provide more precise estimates of relatedness than microsatellites when assessing empirical parent-offspring and full sibling relationships. Further, our results show that relatedness estimates and subsequent pairing recommendations using PMx are most similar between pedigree- and SNP-based approaches. These combined results indicate that in lieu of robust pedigrees, SNPs are an effective tool for informing pairing decisions, which has important implications for many poorly pedigreed conservation breeding programmes worldwide.

Reference Genomes from Distantly Related Species Can Be Used for Discovery of Single Nucleotide Polymorphisms to Inform Conservation Management.

Threatened species recovery programmes benefit from incorporating genomic data into conservation management strategies to enhance species recovery. However, a lack of readily available genomic resources, including conspecific reference genomes, often limits the inclusion of genomic data. Here, we investigate the utility of closely related high-quality reference genomes for single nucleotide polymorphism (SNP) discovery using the critically endangered kaki/black stilt (Himantopus novaezelandiae) and four Charadriiform reference genomes as proof of concept. We compare diversity estimates (i.e., nucleotide diversity, individual heterozygosity, and relatedness) based on kaki SNPs discovered from genotyping-by-sequencing and whole genome resequencing reads mapped to conordinal (killdeer, Charadrius vociferus), confamilial (pied avocet, Recurvirostra avosetta), congeneric (pied stilt, Himantopus himantopus) and conspecific reference genomes. Results indicate that diversity estimates calculated from SNPs discovered using closely related reference genomes correlate significantly with estimates calculated from SNPs discovered using a conspecific genome. Congeneric and confamilial references provide higher correlations and more similar measures of nucleotide diversity, individual heterozygosity, and relatedness. While conspecific genomes may be necessary to address other questions in conservation, SNP discovery using high-quality reference genomes of closely related species is a cost-effective approach for estimating diversity measures in threatened species.