A roadmap to robust discriminant analysis of principal components.

Identification of population structure is a common goal for a variety of applications, including conservation, wildlife management, and medical genetics. The outcome of these analyses can have far reaching implications; therefore, it is important to ensure an understanding of the strengths and weaknesses of the methodologies used. Increasing in popularity, the discriminant analysis of principal components (DAPC) method incorporates combinations of genetic variables (alleles) into a model that differentiates individuals into genetic clusters. However, users may not have a full understanding of how to best parameterize the model. In this issue of Thia (Molecular Ecology Resources, 2022) looks under the hood of the DAPC. Using simulated data, he demonstrates the importance of careful parameter selection in developing a DAPC model, what the implications are for over-fitting the model, and finally, how best to evaluate the results of DAPC models. This work highlights the issues that can arise when over-parameterizing the DAPC model when gene flow is high among clusters and provides important guidelines to ensure researchers are making conclusions that are biologically relevant.

Temporal Monitoring of the Floreana Island Galapagos Giant Tortoise Captive Breeding Program.

Captive breeding programs benefit from genetic analyses that identify relatedness between individuals, assign parentage to offspring, and track levels of genetic diversity. Monitoring these parameters across breeding cycles is critical to the success of a captive breeding program as it allows conservation managers to iteratively evaluate and adjust program structure. However, in practice, genetic tracking of breeding outcomes is rarely conducted. Here, we examined the first three offspring cohorts (2017-2020) of the genetically informed captive breeding program for the Floreana Island Galapagos giant tortoise, Chelonoidis niger. This captive breeding program is unique as the Floreana tortoise has been extinct since the 1800s, but its genome persists, in part, in the form of living hybrids with the extant Volcano Wolf tortoise, C. becki. Breeding over the study period took place at the Galapagos National Park Directorate breeding facility in four corrals, each containing three females and two males. Using 17 microsatellite markers, we were able to assign parentage to 94 of the 98 offspring produced over the study period. We observe that despite the addition of more founders since the pilot breeding program, the effective population size remains low, and changes to the arrangements of breeding corrals may be necessary to encourage more equal reproductive output from the males. This study demonstrates the value of hybrids for species restoration and the importance of continually reassessing the outcomes of captive breeding.

Global genetic diversity status and trends: towards a suite of Essential Biodiversity Variables (EBVs) for genetic composition.

Biodiversity underlies ecosystem resilience, ecosystem function, sustainable economies, and human well-being. Understanding how biodiversity sustains ecosystems under anthropogenic stressors and global environmental change will require new ways of deriving and applying biodiversity data. A major challenge is that biodiversity data and knowledge are scattered, biased, collected with numerous methods, and stored in inconsistent ways. The Group on Earth Observations Biodiversity Observation Network (GEO BON) has developed the Essential Biodiversity Variables (EBVs) as fundamental metrics to help aggregate, harmonize, and interpret biodiversity observation data from diverse sources. Mapping and analyzing EBVs can help to evaluate how aspects of biodiversity are distributed geographically and how they change over time. EBVs are also intended to serve as inputs and validation to forecast the status and trends of biodiversity, and to support policy and decision making. Here, we assess the feasibility of implementing Genetic Composition EBVs (Genetic EBVs), which are metrics of within-species genetic variation. We review and bring together numerous areas of the field of genetics and evaluate how each contributes to global and regional genetic biodiversity monitoring with respect to theory, sampling logistics, metadata, archiving, data aggregation, modeling, and technological advances. We propose four Genetic EBVs: (i) Genetic Diversity; (ii) Genetic Differentiation; (iii) Inbreeding; and (iv) Effective Population Size (Ne ). We rank Genetic EBVs according to their relevance, sensitivity to change, generalizability, scalability, feasibility and data availability. We outline the workflow for generating genetic data underlying the Genetic EBVs, and review advances and needs in archiving genetic composition data and metadata. We discuss how Genetic EBVs can be operationalized by visualizing EBVs in space and time across species and by forecasting Genetic EBVs beyond current observations using various modeling approaches. Our review then explores challenges of aggregation, standardization, and costs of operationalizing the Genetic EBVs, as well as future directions and opportunities to maximize their uptake globally in research and policy. The collection, annotation, and availability of genetic data has made major advances in the past decade, each of which contributes to the practical and standardized framework for large-scale genetic observation reporting. Rapid advances in DNA sequencing technology present new opportunities, but also challenges for operationalizing Genetic EBVs for biodiversity monitoring regionally and globally. With these advances, genetic composition monitoring is starting to be integrated into global conservation policy, which can help support the foundation of all biodiversity and species' long-term persistence in the face of environmental change. We conclude with a summary of concrete steps for researchers and policy makers for advancing operationalization of Genetic EBVs. The technical and analytical foundations of Genetic EBVs are well developed, and conservation practitioners should anticipate their increasing application as efforts emerge to scale up genetic biodiversity monitoring regionally and globally.

Ewe are what ewe wear: bigger horns, better ewes and the potential consequence of trophy hunting on female fitness in bighorn sheep.

In polygynous species, secondary sexual traits such as weapons or elaborate ornaments have evolved through intrasexual competition for mates. In some species, these traits are present in both sexes but are underdeveloped in the sex facing lower intrasexual competition for mates. It is often assumed that these underdeveloped sexually selected traits are a vestige of strong sexual selection on the other sex. Here, we challenge this assumption and investigate whether the expression of secondary sexual traits is associated with fitness in female bighorn sheep. Analyses of 45 years of data revealed that female horn length at 2 years, while accounting for mass and environmental variables, is associated with younger age at primiparity, younger age of first offspring weaned, greater reproductive lifespan and higher lifetime reproductive success. There was no association between horn length and fecundity. These findings highlight a potential conservation issue. In this population, trophy hunting selects against males with fast-growing horns. Intersexual genetic correlations imply that intense selective hunting of large-horned males before they can reproduce can decrease female horn size. Therefore, intense trophy hunting of males based on horn size could reduce female reproductive performance through the associations identified here, and ultimately reduce population growth and viability.

A new lineage of Galapagos giant tortoises identified from museum samples.

The Galapagos Archipelago is recognized as a natural laboratory for studying evolutionary processes. San Cristóbal was one of the first islands colonized by tortoises, which radiated from there across the archipelago to inhabit 10 islands. Here, we sequenced the mitochondrial control region from six historical giant tortoises from San Cristóbal (five long deceased individuals found in a cave and one found alive during an expedition in 1906) and discovered that the five from the cave are from a clade that is distinct among known Galapagos giant tortoises but closely related to the species from Española and Pinta Islands. The haplotype of the individual collected alive in 1906 is in the same clade as the haplotype in the contemporary population. To search for traces of a second lineage in the contemporary population on San Cristóbal, we closely examined the population by sequencing the mitochondrial control region for 129 individuals and genotyping 70 of these for both 21 microsatellite loci and >12,000 genome-wide single nucleotide polymorphisms [SNPs]. Only a single mitochondrial haplotype was found, with no evidence to suggest substructure based on the nuclear markers. Given the geographic and temporal proximity of the two deeply divergent mitochondrial lineages in the historical samples, they were likely sympatric, raising the possibility that the lineages coexisted. Without the museum samples, this important discovery of an additional lineage of Galapagos giant tortoise would not have been possible, underscoring the value of such collections and providing insights into the early evolution of this iconic radiation.

Linking genetic, morphological, and behavioural divergence between inland island and mainland deer mice.

The island syndrome hypothesis (ISH) stipulates that, as a result of local selection pressures and restricted gene flow, individuals from island populations should differ from individuals within mainland populations. Specifically, island populations are predicted to contain individuals that are larger, less aggressive, more sociable, and that invest more in their offspring. To date, tests of the ISH have mainly compared oceanic islands to continental sites, and rarely smaller spatial scales such as inland watersheds. Here, using a novel set of genome-wide SNP markers in wild deer mice (Peromyscus maniculatus) we conducted a genomic assessment of predictions underlying the ISH in an inland riverine island system: analysing island-mainland population structure, and quantifying heritability of phenotypes thought to underlie the ISH. We found clear genomic differentiation between the island and mainland populations and moderate to high marker-based heritability estimates for overall variation in traits previously found to differ in line with the ISH between mainland and island locations. FST outlier analyses highlighted 12 loci associated with differentiation between mainland and island populations. Together these results suggest that the island populations examined are on independent evolutionary trajectories, the traits considered have a genetic basis (rather than phenotypic variation being solely due to phenotypic plasticity). Coupled with the previous results showing significant phenotypic differentiation between the island and mainland groups in this system, this study suggests that the ISH can hold even on a small spatial scale.

The crucial role of genome-wide genetic variation in conservation.

The unprecedented rate of extinction calls for efficient use of genetics to help conserve biodiversity. Several recent genomic and simulation-based studies have argued that the field of conservation biology has placed too much focus on conserving genome-wide genetic variation, and that the field should instead focus on managing the subset of functional genetic variation that is thought to affect fitness. Here, we critically evaluate the feasibility and likely benefits of this approach in conservation. We find that population genetics theory and empirical results show that conserving genome-wide genetic variation is generally the best approach to prevent inbreeding depression and loss of adaptive potential from driving populations toward extinction. Focusing conservation efforts on presumably functional genetic variation will only be feasible occasionally, often misleading, and counterproductive when prioritized over genome-wide genetic variation. Given the increasing rate of habitat loss and other environmental changes, failure to recognize the detrimental effects of lost genome-wide genetic variation on long-term population viability will only worsen the biodiversity crisis.

Ancient hybridization patterns between bighorn and thinhorn sheep.

Whole-genome sequencing has advanced the study of species evolution, including the detection of genealogical discordant events such as ancient hybridization and incomplete lineage sorting (ILS). The evolutionary history of bighorn (Ovis canadensis) and thinhorn (Ovis dalli) sheep present an ideal system to investigate evolutionary discordance due to their recent and rapid radiation and putative secondary contact between bighorn and thinhorn sheep subspecies, specifically the dark pelage Stone sheep (O. dalli stonei) and predominately white Dall sheep (O. dalli dalli), during the last ice age. Here, we used multiple genomes of bighorn and thinhorn sheep, together with snow (O. nivicola) and the domestic sheep (O. aries) as outgroups, to assess their phylogenomic history, potential introgression patterns and their adaptive consequences. Among the Pachyceriforms (snow, bighorn and thinhorn sheep) a consistent monophyletic species tree was retrieved; however, many genealogical discordance patterns were observed. Alternative phylogenies frequently placed Stone and bighorn as sister clades. This relationship occurred more often and was less divergent than that between Dall and bighorn. We also observed many blocks containing introgression signal between Stone and bighorn genomes in which coat colour genes were present. Introgression signals observed between Dall and bighorn were more random and less frequent, and therefore probably due to ILS or intermediary secondary contact. These results strongly suggest that Stone sheep originated from a complex series of events, characterized by multiple, ancient periods of secondary contact with bighorn sheep.

Opportunities and challenges of macrogenetic studies.

The rapidly emerging field of macrogenetics focuses on analysing publicly accessible genetic datasets from thousands of species to explore large-scale patterns and predictors of intraspecific genetic variation. Facilitated by advances in evolutionary biology, technology, data infrastructure, statistics and open science, macrogenetics addresses core evolutionary hypotheses (such as disentangling environmental and life-history effects on genetic variation) with a global focus. Yet, there are important, often overlooked, limitations to this approach and best practices need to be considered and adopted if macrogenetics is to continue its exciting trajectory and reach its full potential in fields such as biodiversity monitoring and conservation. Here, we review the history of this rapidly growing field, highlight knowledge gaps and future directions, and provide guidelines for further research.

Macrogenetic studies must not ignore limitations of genetic markers and scale.

Millette et al. (Ecology Letters, 2020, 23:55-67) reported no consistent worldwide anthropogenic effects on animal genetic diversity using repurposed mitochondrial DNA sequences. We reexamine data from this study, describe genetic marker and scale limitations which might lead to misinterpretations with conservation implications, and provide advice to improve future macrogenetic studies.

Historical Introgression from Wild Relatives Enhanced Climatic Adaptation and Resistance to Pneumonia in Sheep.

How animals, particularly livestock, adapt to various climates and environments over short evolutionary time is of fundamental biological interest. Further, understanding the genetic mechanisms of adaptation in indigenous livestock populations is important for designing appropriate breeding programs to cope with the impacts of changing climate. Here, we conducted a comprehensive genomic analysis of diversity, interspecies introgression, and climate-mediated selective signatures in a global sample of sheep and their wild relatives. By examining 600K and 50K genome-wide single nucleotide polymorphism data from 3,447 samples representing 111 domestic sheep populations and 403 samples from all their seven wild relatives (argali, Asiatic mouflon, European mouflon, urial, snow sheep, bighorn, and thinhorn sheep), coupled with 88 whole-genome sequences, we detected clear signals of common introgression from wild relatives into sympatric domestic populations, thereby increasing their genomic diversities. The introgressions provided beneficial genetic variants in native populations, which were significantly associated with local climatic adaptation. We observed common introgression signals of alleles in olfactory-related genes (e.g., ADCY3 and TRPV1) and the PADI gene family including in particular PADI2, which is associated with antibacterial innate immunity. Further analyses of whole-genome sequences showed that the introgressed alleles in a specific region of PADI2 (chr2: 248,302,667-248,306,614) correlate with resistance to pneumonia. We conclude that wild introgression enhanced climatic adaptation and resistance to pneumonia in sheep. This has enabled them to adapt to varying climatic and environmental conditions after domestication.

The influence of a priori grouping on inference of genetic clusters: simulation study and literature review of the DAPC method.

Inference of genetic clusters is a key aim of population genetics, sparking development of numerous analytical methods. Within these, there is a conceptual divide between finding de novo structure versus assessment of a priori groups. Recently developed, Discriminant Analysis of Principal Components (DAPC), combines discriminant analysis (DA) with principal component (PC) analysis. When applying DAPC, the groups used in the DA (specified a priori or described de novo) need to be carefully assessed. While DAPC has rapidly become a core technique, the sensitivity of the method to misspecification of groups and how it is being empirically applied, are unknown. To address this, we conducted a simulation study examining the influence of a priori versus de novo group designations, and a literature review of how DAPC is being applied. We found that with a priori groupings, distance between genetic clusters reflected underlying FST. However, when migration rates were high and groups were described de novo there was considerable inaccuracy, both in terms of the number of genetic clusters suggested and placement of individuals into those clusters. Nearly all (90.1%) of 224 studies surveyed used DAPC to find de novo clusters, and for the majority (62.5%) the stated goal matched the results. However, most studies (52.3%) omit key run parameters, preventing repeatability and transparency. Therefore, we present recommendations for standard reporting of parameters used in DAPC analyses. The influence of groupings in genetic clustering is not unique to DAPC, and researchers need to consider their goal and which methods will be most appropriate.

Acute effects of static stretching on Wingate testing in men.

BACKGROUND: Previous research has demonstrated a reduction in peak power via isokinetic dynamometry and power testing. The purpose of this study was to determine the effects of static stretching (SS) on the Wingate test (WAnT). METHODS: Thirteen recreationally active males (mean age: 22.5±1.9 years, height: 172.5±8.4 cm, body mass: 83.4±24.6 kg) were tested for peak (PP), average (AP), and minimum (MP) power output using the WAnT. Two WAnT trials were completed by each subject on a Monark 894-E ergometer that was interfaced with a desktop computer. Participants completed a 5-minute warm-up on the cycle ergometer and then performed the pre-WAnT. Upon completion, the stretching protocol four static stretches were performed: one was participant self-administered and the remaining three by the investigator. Each stretch was held for 30 seconds and was repeated 4 times on each leg and repeated after 4 minutes of rest. RESULTS: Significant differences (P<0.05) were found for PP only for the pre- versus post-WAnT (802.6±207.6 vs. 768.8±199.7 W). CONCLUSIONS: Static stretching before performance of the WAnT demonstrated a significant decrease in peak power output. Based upon these results, it appears that SS decreases peak power output when performing an anaerobic capacity test on a cycle ergometer.

The genetic basis of animal behavioural diversity in natural populations.

Individual differences in animal behaviour influence ecological and evolutionary processes. Much behavioural variation has a heritable component, suggesting that genetics may play a role in its development. Yet, the study of the mechanistic description linking genes to behaviour in nature remains in its infancy, and such research is considered a challenge in contemporary biology. Here, we performed a literature review and meta-analysis to assess trends in analytical approaches used to investigate the relationship between genes and behaviour in natural systems, specifically candidate gene approaches, quantitative trait locus (QTL) mapping, and genome-wide association studies (GWAS). We aimed to determine the efficacy and success of each approach, while also describing which behaviours and species were examined by researchers most often. We found that the majority of QTL mapping and GWAS results revealed a significant or suggestive effect (Zr = 0.3 [95% CI: 0.25:0.35] and Zr = 0.39 [0.33:0.46], respectively) between the trait of interest and genetic marker(s) tested, while over half of candidate gene accounts (Zr = 0.16 [0.11:0.21]) did not find a significant association. Approximately a third of all study estimates investigated animal personality traits; though, reproductive and migratory behaviours were also well-represented. Our findings show that despite widespread accessibility of molecular approaches given current sequencing technologies, efforts to elucidate the genetic basis of behaviour in free-ranging systems has been limited to relatively few species. We discuss challenges encountered by researchers, and recommend integration of novel genomic methods with longitudinal studies to usher in the next wave of behavioural genomic research.

Confidently identifying the correct K value using the ΔK method: When does K = 2?

Populations delineated based on genetic data are commonly used for wildlife conservation and management. Many studies use the program structure combined with the ΔK method to identify the most probable number of populations (K). We recently found K = 2 was identified more often when studies used ΔK compared to studies that did not. We suggested two reasons for this: hierarchical population structure leads to underestimation, or the ΔK method does not evaluate K = 1 causing an overestimation. The present contribution aims to develop a better understanding of the limits of the method using one, two and three population simulations across migration scenarios. From these simulations we identified the "best K" using model likelihood and ΔK. Our findings show that mean probability plots and ΔK are unable to resolve the correct number of populations once migration rate exceeds 0.005. We also found a strong bias towards selecting K = 2 using the ΔK method. We used these data to identify the range of values where the ΔK statistic identifies a value of K that is not well supported. Finally, using the simulations and a review of empirical data, we found that the magnitude of ΔK corresponds to the level of divergence between populations. Based on our findings, we suggest researchers should use the ΔK method cautiously; they need to report all relevant data, including the magnitude of ΔK, and an estimate of connectivity for the research community to assess whether meaningful genetic structure exists within the context of management and conservation.

Metabolic Syndrome and Physical Activity Levels in College Students.

Background: It is estimated that >30% of young adults attending college are overweight or obese and do not participate in enough physical activity (PA) to maintain a healthy body mass. Many of the known risk factors that are often associated with obesity also place an individual at risk for developing metabolic syndrome (MetS). The purpose of this study was to examine the prevalence of MetS and the magnitude and rate of PA levels in college students. Methods: Seventy-six college-aged students participated in the study. The following information was obtained from each participant: body anthropometrics, fasting glucose and lipoproteins, and accelerometer-measured activity levels. Participants wore, at the waist, the wireless activity monitor (wGT3X-BT; ActiGraph, Pensacola, FL) for seven consecutive days to monitor PA levels. MetS was determined if the participant met three of the five criteria utilizing the National Cholesterol Education Program guidelines. Results: More than half of the participants had at least one criterion, with the overall prevalence of MetS being 9.2%. MetS participants were more sedentary (84.8% vs. 91.0%, P < 0.001) and engaged in less light activities (8.9% vs. 6.0%, P < 0.001). Conclusions: Current activity levels in college students do not meet the established guidelines for total steps per day, elevating the risk of acquiring metabolic disorders.

Genetically informed captive breeding of hybrids of an extinct species of Galapagos tortoise.

Hybridization poses a major challenge for species conservation because it threatens both genetic integrity and adaptive potential. Yet, hybridization can occasionally offer unprecedented opportunity for species recovery if the genome of an extinct taxon is present among living hybrids such that selective breeding could recapture it. We explored the design elements for establishing a captive-breeding program for Galapagos tortoises (Chelonoidis spp.) built around individuals with admixed ancestry involving an extinct species. The target individuals were hybrids between the extinct species from Floreana Island, C. niger, and an extant species, C. becki, which were recently found in the endemic range of C. becki, from Wolf Volcano on Isabela Island. We combined genotypic data from 35 tortoises with high ancestry from C. niger with forward-in-time simulations to explore captive breeding strategies that maximized overall genetic diversity and ancestry from C. niger while accommodating resource constraints, species biology, and the urgency to return tortoises to Floreana Island for facilitating ecosystem restoration. Overall genetic diversity was maximized when in the simulation tortoises were organized in relatively small breeding groups. Substantial amounts of the C. niger genome were captured despite limited resources available for selectively breeding tortoises in captivity. Genetic diversity was maximized when captive-bred offspring were released to the wild rather than being used as additional breeders. Our results provide genetic-based and practical guidance on the inclusion of hybrids with genomic representation from extinct taxa into species restoration programs and informs the ongoing debate on the value of hybrids in biodiversity conservation.

Reproducción en Cautiverio Informada Genéticamente de Híbridos de una Especie Extinta de Tortuga de las Galápagos Resumen La hibridación representa un obstáculo importante para la conservación de especies ya que amenaza tanto a la integridad genética como al potencial adaptativo. Aun así, la hibridación ocasionalmente puede ofrecer una oportunidad sin precedentes para la recuperación de una especie si el genoma de un taxón extinto está presente entre los híbridos vivientes de tal manera que la reproducción selectiva pudiera recuperarlo. Exploramos los elementos de diseño para el establecimiento de un programa de reproducción en cautiverio de la tortuga de las Galápagos (Chelonoidis spp.) construido en torno a los individuos con linajes mixtos que incluyeran una especie extinta. Los individuos fueron los híbridos de la especie extinta en la Isla Floreana, C. niger, y la especie viviente C. becki, encontrados recientemente en la distribución geográfica endémica de la segunda especie en el Volcán Wolf (Isla Isabela). Combinamos los datos genotípicos de 35 tortugas con un linaje cargado de C. niger usando simulaciones futuras de la descendencia generada por el programa para explorar las estrategias de reproducción en cautiverio que maximizaran en general la diversidad genética y el linaje de C. niger a la vez que se ajustaba a las restricciones de recursos, la biología de la especie y la urgencia por regresar las tortugas a la Isla Floreana para facilitar la restauración del ecosistema. En general, la diversidad genética se maximizó cuando en la simulación las tortugas estuvieron organizadas en grupos de reproducción relativamente pequeños y cuando cantidades sustanciales del genoma de C. niger fueron capturados con base en los recursos disponibles para reproducir selectivamente a las tortugas en cautiverio. La diversidad genética se vio especialmente maximizada cuando las crías reproducidas en cautiverio fueron liberadas en lugar de ser utilizadas como reproductoras adicionales. Nuestros resultados proporcionan una guía práctica y basada en la genética para la inclusión de híbridos con representación genómica de un taxón extinto en los programas de restauración de especies. Cuando incorporamos a los híbridos con diversidad genética que previamente se creía perdida en los programas con el propósito de la reintroducción de especies, nuestro estudio informa al debate continuo sobre el valor de los híbridos para la conservación de la biodiversidad.

Functional Movement Screen scores in a group of division IA athletes.

BACKGROUND: To determine mean values of the Functional Movement Screen scores in Division IA collegiate athletes. METHODS: One hundred sixty-one Division I collegiate athletes were recruited to participate in the study. The Functional Movement Screen was performed during pre-participation screenings prior to the beginning of the season. All athletes performed all seven tests and clearing tests as part of their participation. All the athletes were injury free at the time of testing. A χ2 analysis was used to evaluate whether significant differences in the individual scores existed and t-test comparisons were conducted for the composite scores of the FMS and to examine if there were significant differences between genders. RESULTS: No significant difference was found between genders. The overall mean FMS score amongst players was 16.2±2.6. Female athletes scored highest on the active straight leg raise (2.64±0.54) and shoulder mobility (2.52±0.72) while their lowest scoring trials were trunk stability (1.95±0.25) and IL (2.25±0.59). Male athletes scored highest on the push-pp test (2.78±0.49) and active straight leg raise (2.47±0.62) and the lowest on trunk stability (1.98±0.13). CONCLUSIONS: This study provides mean value scores for FMS scores when testing Division IA collegiate athletes. The FMS may be useful for recognizing deficiencies in movements.

Population genomics through time provides insights into the consequences of decline and rapid demographic recovery through head-starting in a Galapagos giant tortoise.

Population genetic theory related to the consequences of rapid population decline is well-developed, but there are very few empirical studies where sampling was conducted before and after a known bottleneck event. Such knowledge is of particular importance for species restoration, given links between genetic diversity and the probability of long-term persistence. To directly evaluate the relationship between current genetic diversity and past demographic events, we collected genome-wide single nucleotide polymorphism data from prebottleneck historical (c.1906) and postbottleneck contemporary (c.2014) samples of Pinzón giant tortoises (Chelonoidis duncanensis; n = 25 and 149 individuals, respectively) endemic to a single island in the Galapagos. Pinzón giant tortoises had a historically large population size that was reduced to just 150-200 individuals in the mid 20th century. Since then, Pinzón's tortoise population has recovered through an ex situ head-start programme in which eggs or pre-emergent individuals were collected from natural nests on the island, reared ex situ in captivity until they were 4-5 years old and subsequently repatriated. We found that the extent and distribution of genetic variation in the historical and contemporary samples were very similar, with the latter group not exhibiting the characteristic genetic patterns of recent population decline. No population structure was detected either spatially or temporally. We estimated an effective population size (N e) of 58 (95% CI = 50-69) for the postbottleneck population; no prebottleneck N e point estimate was attainable (95% CI = 39-infinity) likely due to the sample size being lower than the true N e. Overall, the historical sample provided a valuable benchmark for evaluating the head-start captive breeding programme, revealing high retention of genetic variation and no skew in representation despite the documented bottleneck event. Moreover, this work demonstrates the effectiveness of head-starting in rescuing the Pinzón giant tortoise from almost certain extinction.

Theory, practice, and conservation in the age of genomics: The Galápagos giant tortoise as a case study.

High-throughput DNA sequencing allows efficient discovery of thousands of single nucleotide polymorphisms (SNPs) in nonmodel species. Population genetic theory predicts that this large number of independent markers should provide detailed insights into population structure, even when only a few individuals are sampled. Still, sampling design can have a strong impact on such inferences. Here, we use simulations and empirical SNP data to investigate the impacts of sampling design on estimating genetic differentiation among populations that represent three species of Galápagos giant tortoises (Chelonoidis spp.). Though microsatellite and mitochondrial DNA analyses have supported the distinctiveness of these species, a recent study called into question how well these markers matched with data from genomic SNPs, thereby questioning decades of studies in nonmodel organisms. Using >20,000 genomewide SNPs from 30 individuals from three Galápagos giant tortoise species, we find distinct structure that matches the relationships described by the traditional genetic markers. Furthermore, we confirm that accurate estimates of genetic differentiation in highly structured natural populations can be obtained using thousands of SNPs and 2-5 individuals, or hundreds of SNPs and 10 individuals, but only if the units of analysis are delineated in a way that is consistent with evolutionary history. We show that the lack of structure in the recent SNP-based study was likely due to unnatural grouping of individuals and erroneous genotype filtering. Our study demonstrates that genomic data enable patterns of genetic differentiation among populations to be elucidated even with few samples per population, and underscores the importance of sampling design. These results have specific implications for studies of population structure in endangered species and subsequent management decisions.