Purging of deleterious burden in the endangered Iberian lynx.

SignificanceThe dynamics of deleterious variation under contrasting demographic scenarios remain poorly understood in spite of their relevance in evolutionary and conservation terms. Here we apply a genomic approach to study differences in the burden of deleterious alleles between the endangered Iberian lynx (Lynx pardinus) and the widespread Eurasian lynx (Lynx lynx). Our analysis unveils a significantly lower deleterious burden in the former species that should be ascribed to genetic purging, that is, to the increased opportunities of selection against recessive homozygotes due to the inbreeding caused by its smaller population size, as illustrated by our analytical predictions. This research provides theoretical and empirical evidence on the evolutionary relevance of genetic purging under certain demographic conditions.

Effectiveness of population-based recovery actions for threatened southern mountain caribou.

Habitat loss is affecting many species, including the southern mountain caribou (Rangifer tarandus caribou) population in western North America. Over the last half century, this threatened caribou population's range and abundance have dramatically contracted. An integrated population model was used to analyze 51 years (1973-2023) of demographic data from 40 southern mountain caribou subpopulations to assess the effectiveness of population-based recovery actions at increasing population growth. Reducing potential limiting factors on threatened caribou populations offered a rare opportunity to identify the causes of decline and assess methods of recovery. Southern mountain caribou abundance declined by 51% between 1991 and 2023, and 37% of subpopulations were functionally extirpated. Wolf reduction was the only recovery action that consistently increased population growth when applied in isolation, and combinations of wolf reductions with maternal penning or supplemental feeding provided rapid growth but were applied to only four subpopulations. As of 2023, recovery actions have increased the abundance of southern mountain caribou by 52%, compared to a simulation with no interventions. When predation pressure was reduced, rapid population growth was observed, even under contemporary climate change and high levels of habitat loss. Unless predation is reduced, caribou subpopulations will continue to be extirpated well before habitat conservation and restoration can become effective.

Evaluation of breeding strategies to reduce the inbreeding rate in the Friesian horse population: Looking back and moving forward.

In the past, small population sizes and unequal ancestor contributions have resulted in high inbreeding rates (ΔF) in the Friesian horse. Two decades ago, the studbook implemented a mating quota and started publishing individual kinships and reduced ΔF below 1% per generation. However, since then, the breeding population size has decreased and this raises the question whether current breeding strategies are sufficient to keep ΔF below desired rates. The aim of this study was to (1) reflect on past inbreeding trends and their main determinants, using pedigree analysis and (2) evaluate the effectiveness of the current and additional breeding strategies using stochastic simulations. We estimated the current ΔF (2013-2022) at 0.72% per generation. While the total contribution of the top 10 sires to the number of offspring per year has decreased from 75% in 1980 to 35% in 2022, this was mainly due to an increased number of approved studbook sires, and not due to more equalized contributions among sires. Of the simulated breeding strategies, selecting only breeding stallions with a below average mean kinship (i.e., "mean kinship selection") was most effective to decrease ΔF (from 0.66% to 0.33%). Increasing the number of breeding sires only had an effect when also a mating quota was applied. However, its effect remained limited. For example, a ~1.5 fold increase, combined with a mating quota of 80 offspring per sire per year, reduced ΔF from 0.55% to 0.51%. When increasing the number of breeding mares, a practically unfeasible large increase was needed for a meaningful reduction in ΔF (e.g. twice as many mares were needed to reduce ΔF from 0.66% to 0.56%). Stratified mating quotas, a novel approach in which we assigned each sire a mating quota (of 60, 80, 100 or 120 offspring per year) based on its mean kinship to recently born foals, resulted in a lower ΔF (0.43%) than a general mating quota of 90 offspring per sire per year (0.55%). Overall, while the current ΔF is below 1%, we recommend to implement additional strategies to further reduce ΔF below 0.5% in the Friesian horse population. For this breed and similar populations, we recommend to focus on breeding strategies based on kinship levels to effectively reduce ΔF.

Give and take: Effects of genetic admixture on mutation load in endangered Florida panthers.

Genetic admixture is a biological event inherent to genetic rescue programs aimed at the long-term conservation of endangered wildlife. Although the success of such programs can be measured by the increase in genetic diversity and fitness of subsequent admixed individuals, predictions supporting admixture costs to fitness due to the introduction of novel deleterious alleles are necessary. Here, we analyzed nonsynonymous variation from conserved genes to quantify and compare levels of mutation load (i.e. proportion of deleterious alleles and genotypes carrying these alleles) among endangered Florida panthers and non-endangered Texas pumas. Specifically, we used canonical (i.e. non-admixed) Florida panthers, Texas pumas, and F1 (canonical Florida × Texas) panthers dating from a genetic rescue program and Everglades National Park panthers with Central American ancestry resulting from an earlier admixture event. We found neither genetic drift nor selection significantly reduced overall proportions of deleterious alleles in the severely bottlenecked canonical Florida panthers. Nevertheless, the deleterious alleles identified were distributed into a disproportionately high number of homozygous genotypes due to close inbreeding in this group. Conversely, admixed Florida panthers (either with Texas or Central American ancestry) presented reduced levels of homozygous genotypes carrying deleterious alleles but increased levels of heterozygous genotypes carrying these variants relative to canonical Florida panthers. Although admixture is likely to alleviate the load of standing deleterious variation present in homozygous genotypes, our results suggest that introduced novel deleterious alleles (temporarily present in heterozygous state) in genetically rescued populations could potentially be expressed in subsequent generations if their effective sizes remain small.

Capturing the dynamics of small populations: A retrospective assessment using long-term data for an island reintroduction.

The art of population modelling is to incorporate factors essential for capturing a population's dynamics while otherwise keeping the model as simple as possible. However, it is unclear how optimal model complexity should be assessed, and whether this optimal complexity has been affected by recent advances in modelling methodology. This issue is particularly relevant to small populations because they are subject to complex dynamics but inferences about those dynamics are often constrained by small sample sizes. We fitted Bayesian hierarchical models to long-term data on vital rates (survival and reproduction) for the toutouwai Petroica longipes population reintroduced to Tiritiri Matangi, a 220-ha New Zealand island, and quantified the performance of those models in terms of their likelihood of replicating the observed population dynamics. These dynamics consisted of overall growth from 33 (±0.3) to 160 (±6) birds from 1992-2018, including recoveries following five harvest events for further reintroductions to other sites. We initially included all factors found to affect vital rates, which included inbreeding, post-release effects (PRE), density-dependence, sex, age and random annual variation, then progressively removed these factors. We also compared performance of models where data analysis and simulations were done simultaneously to those produced with the traditional two-step approach, where vital rates are estimated first then fed into a separate simulation model. Parametric uncertainty and demographic stochasticity were incorporated in all projections. The essential factors for replicating the population's dynamics were density-dependence in juvenile survival and PRE, i.e. initial depression of survival and reproduction in translocated birds. Inclusion of other factors reduced the precision of projections, and therefore the likelihood of matching observed dynamics. However, this reduction was modest when the modelling was done in an integrated framework. In contrast, projections were much less precise when done with a two-step modelling approach, and the cost of additional parameters was much higher under the two-step approach. These results suggest that minimization of complexity may be less important than accounting for covariances in parameter estimates, which is facilitated by integrating data analysis and population projections using Bayesian methods.

Developmental instability and phenotypic evolution in a small and isolated bear population.

We explored fluctuating asymmetry (FA) and morphological integration (MI) in the skull of the small, highly inbred and divergent Apennine bear (Ursus arctos marsicanus), to explore its uniqueness and investigate any potential effects of inbreeding depression. We used 3D geometric morphometrics contrasting Apennine bears with other two large outbred bear populations from Scandinavia and Kamchatka as controls. Shape divergence and variability were explored by a principal component analysis on aligned coordinates of 39 landmarks. Procrustes ANOVA, morphological disparity and the global integration index were used to explore FA, shape variance and MI. By remarking Apennine bears as a highly divergent phenotype, we recorded the highest FA and deviation from self-similarity compared with the other two control populations. We conclude that Apennine bears are likely facing developmental instability as a consequence of inbreeding depression, whereas the divergent trait covariance pattern may represent a potential source of evolutionary novelties. We discuss the implications for the conservation and management of this imperiled taxon.

Conservation resource allocation, small population resiliency, and the fallacy of conservation triage.

Some conservation prioritization methods are based on the assumption that conservation needs overwhelm current resources and not all species can be conserved; therefore, a conservation triage scheme (i.e., when the system is overwhelmed, species should be divided into three groups based on likelihood of survival, and efforts should be focused on those species in the group with the best survival prospects and reduced or denied to those in the group with no survival prospects and to those in the group not needing special efforts for their conservation) is necessary to guide resource allocation. We argue that this decision-making strategy is not appropriate because resources are not as limited as often assumed, and it is not evident that there are species that cannot be conserved. Small population size alone, for example, does not doom a species to extinction; plants, reptiles, birds, and mammals offer examples. Although resources dedicated to conserving all threatened species are insufficient at present, the world's economic resources are vast, and greater resources could be dedicated toward species conservation. The political framework for species conservation has improved, with initiatives such as the UN Sustainable Development Goals and other international agreements, funding mechanisms such as The Global Environment Facility, and the rise of many nongovernmental organizations with nimble, rapid-response small grants programs. For a prioritization system to allow no extinctions, zero extinctions must be an explicit goal of the system. Extinction is not inevitable, and should not be acceptable. A goal of no human-induced extinctions is imperative given the irreversibility of species loss.

Asignación de Recursos para la Conservación, Resiliencia de Poblaciones Pequeñas y la Falacia del Triaje de Conservación Resumen Algunos métodos de priorización de la conservación están basados en el supuesto de que las necesidades de la conservación superan a los actuales recursos y que no todas las especies pueden ser conservadas; por lo tanto, se necesita un esquema de triaje (esto es, cuando el sistema está abrumado, las especies deben dividirse en tres grupos con base en su probabilidad de supervivencia y los esfuerzos deben enfocarse en aquellas especies dentro del grupo con las mejores probabilidades de supervivencia y a aquellas en el grupo sin probabilidades de supervivencia o aquellas en el grupo que no necesita esfuerzos especializados para su conservación se les deben reducir o negar los esfuerzos de conservación) para dirigir la asignación de recursos. Discutimos que esta estrategia para la toma de decisiones no es apropiada porque los recursos no están tan limitados como se asume con frecuencia y tampoco es evidente que existan especies que no puedan ser conservadas. Por ejemplo, tan sólo un tamaño poblacional pequeño no es suficiente para condenar a una especie a la extinción; contamos con ejemplos en plantas, reptiles, aves y mamíferos. Aunque actualmente todos los recursos dedicados a la conservación de todas las especies amenazadas son insuficientes, los recursos económicos mundiales son vastos y se podrían dedicar mayores recursos a la conservación de especies. El marco de trabajo político para la conservación de especies ha mejorado, con iniciativas como los Objetivos de Desarrollo Sustentable de la ONU y otros acuerdos internacionales, el financiamiento de mecanismos como el Fondo para el Medio Ambiente Mundial, y el surgimiento de muchas organizaciones no gubernamentales mediante programas de subsidios pequeños hábiles y de respuesta rápida. Para que un sistema de priorización no permita las extinciones, las cero extinciones deben ser un objetivo explícito del sistema. La extinción no es inevitable y no debería ser aceptable. El objetivo de cero extinciones inducidas por humanos es imperativo dada la irreversibilidad de la pérdida de especies.

Using long-term data for a reintroduced population to empirically estimate future consequences of inbreeding.

Inbreeding depression is an important long-term threat to reintroduced populations. However, the strength of inbreeding depression is difficult to estimate in wild populations because pedigree data are inevitably incomplete and because good data are needed on survival and reproduction. Predicting future population consequences is especially difficult because this also requires projecting future inbreeding levels and their impacts on long-term population dynamics, which are subject to many uncertainties. We illustrate how such projections can be derived through Bayesian state-space modeling methods based on a 26-year data set for North Island Robins (Petroica longipes) reintroduced to Tiritiri Matangi Island in 1992. We used pedigree data to model increases in the average inbreeding level (F) over time based on kinship of possible breeding pairs and to estimate empirically Ne /N (effective/census population size). We used multiple imputation to model the unknown components of inbreeding coefficients, which allowed us to estimate effects of inbreeding on survival for all 1458 birds in the data set while modeling density dependence and environmental stochasticity. This modeling indicated that inbreeding reduced juvenile survival (1.83 lethal equivalents [SE 0.81]) and may have reduced subsequent adult survival (0.44 lethal equivalents [0.81]) but had no apparent effect on numbers of fledglings produced. Average inbreeding level increased to 0.10 (SE 0.001) as the population grew from 33 (0.3) to 160 (6) individuals over the 25 years, giving a Ne/N ratio of 0.56 (0.01). Based on a model that also incorporated habitat regeneration, the population was projected to reach a maximum of 331-1144 birds (median 726) in 2130, then to begin a slow decline. Without inbreeding, the population would be expected stabilize at 887-1465 birds (median 1131). Such analysis, therefore, makes it possible to empirically derive the information needed for rational decisions about inbreeding management while accounting for multiple sources of uncertainty.

Uso de Datos a Largo Plazo de una Población Reintroducida para Estimar Empíricamente las Consecuencias Futuras de la Endogamia Resumen La depresión endogámica es una amenaza importante a largo plazo para las poblaciones reintroducidas. Sin embargo, es complicado estimar la fuerza de la depresión endogámica en las poblaciones silvestres porque los datos sobre el linaje sin duda estarán incompletos y porque se necesitan datos sólidos sobre la supervivencia y la reproducción. Es especialmente difícil predecir las consecuencias poblacionales a futuro pues esto requiere proyectar a futuro los niveles de endogamia y sus impactos sobre las dinámicas poblacionales a largo plazo, las cuales están sujetas a muchas incertidumbres. Ilustramos cómo dichas proyecciones pueden derivarse mediante métodos de modelado bayesiano de estado-espacio basados en un conjunto de datos obtenidos durante 26 años para los tordos de la Isla del Norte (Petroica longipes) reintroducidos a la isla Tiritiri Matangi en 1992. Usamos datos de linaje para modelar los incrementos en el nivel promedio de endogamia ( F̲ ) a lo largo del tiempo con base en el parentesco de las posibles parejas reproductoras y para estimar empíricamente Ne/N (tamaño poblacional efectivo/por censo). Usamos una imputación múltiple para modelar los componentes desconocidos de los coeficientes de endogamia, lo que nos permitió estimar los efectos de la endogamia sobre la supervivencia para todas las aves (1458) incluidas en el conjunto de datos a la vez que modelamos la dependencia de la densidad y la estocasticidad ambiental. Este modelado indicó que la endogamia redujo la supervivencia juvenil (1.83 equivalentes letales [SE 0.81]) y podría haber reducido la subsecuente supervivencia adulta (0.44 equivalentes letales [0.81]) pero no tuvo un efecto aparente sobre los números de polluelos producidos. El nivel promedio de endogamia incrementó a 0.10 (SE 0.001) conforme la población creció de 33 (0.3) a 160 (6) individuos a lo largo de los 25 años, lo que resultó en una proporción Ne/N de 0.56 (0.01). Con base en un modelo que también incorporó la regeneración del hábitat, se proyectó que la población alcanzaría un máximo de 331-1144 aves (mediana: 726) para 2130 y después comenzaría una lenta disminución. Sin la endogamia, se esperaría que la población se estabilizaría con 887-1465 (mediana: 1131) aves. Por lo tanto, dicho análisis hace posible la derivación empírica de la información necesaria para las decisiones racionales sobre el manejo de la endogamia a la vez que considera a varias fuentes de incertidumbre.

Prediction of genomic breeding values of milk traits in Brazilian Saanen goats.

The study's objective was to compare the genomic prediction ability methods for the traits milk yield, milk composition and somatic cell count of Saanen Brazilian goats. Nine hundred forty goats, genotyped with an Axiom_OviCap (Caprine) panel, Affimetrix customized array with 62,557 single nucleotide polymorphisms (SNPs), were used for the genomic selection analyses. The genomic methods studied to estimate the effects of SNPs and direct genomic values (DGV) were as follows: (a) genomic BLUP (GBLUP), (b) Bayes Cπ and (c) Bayesian Lasso (BLASSO). Estimated breeding values (EBV) and deregressed estimated breeding values (dEBV) were used as response variables for the genomic predictions. The prediction ability was assessed by Pearson's correlation between DGV and response variables (EBV and dEBV). Regression coefficients of the response variables on the DGV were obtained to verify if the genomic predictions were biased. In addition, the mean square error of prediction (MSE) was used as a measure of verification of model fit to the data. The means of prediction accuracy, when EBV was used as a response variable, were 0.68, 0.68 and 0.67 for GBLUP, Bayes Cπ and BLASSO, respectively. With dEBV, the mean prediction accuracy was 0.50 for all models. The averages of the EBV regression coefficients on DGV were 1.08 for all models (GBLUP, Bayes Cπ and BLASSO), higher than those obtained for the regression coefficient of dEBV on DGV, which presented values of 1.05, 1.05 and 1.08 for GBLUP, Bayes Cπ and BLASSO, respectively. None of the methods stood out in terms of prediction ability; however, the GBLUP method was the most appropriate for estimating the DGV, in a slightly more reliable and less biased way, besides presenting the lowest computational cost. In the context of the present study, EBV was the preferred response variables considering the genomic prediction accuracy despite dEBV also presented lower bias.

Moderate Amounts of Epistasis are Not Evolutionarily Stable in Small Populations.

High mutation rates select for the evolution of mutational robustness where populations inhabit flat fitness peaks with little epistasis, protecting them from lethal mutagenesis. Recent evidence suggests that a different effect protects small populations from extinction via the accumulation of deleterious mutations. In drift robustness, populations tend to occupy peaks with steep flanks and positive epistasis between mutations. However, it is not known what happens when mutation rates are high and population sizes are small at the same time. Using a simple fitness model with variable epistasis, we show that the equilibrium fitness has a minimum as a function of the parameter that tunes epistasis, implying that this critical point is an unstable fixed point for evolutionary trajectories. In agent-based simulations of evolution at finite mutation rate, we demonstrate that when mutations can change epistasis, trajectories with a subcritical value of epistasis evolve to decrease epistasis, while those with supercritical initial points evolve towards higher epistasis. These two fixed points can be identified with mutational and drift robustness, respectively.

Adaptive and maladaptive genetic diversity in small populations: Insights from the Brook Charr (Salvelinus fontinalis) case study.

Investigating the relative importance of neutral versus selective processes governing the accumulation of genetic variants is a key goal in both evolutionary and conservation biology. This is particularly true in the context of small populations, where genetic drift can counteract the effect of selection. Using Brook Charr (Salvelinus fontinalis) from Québec, Canada, as a case study, we investigated the importance of demographic versus selective processes governing the accumulation of both adaptive and maladaptive mutations in closed versus open and connected populations to assess gene flow effect. This was achieved by using 14,779 high-quality filtered SNPs genotyped among 1,416 fish representing 50 populations from three life history types: lacustrine (closed populations), riverine and anadromous (connected populations). Using the PROVEAN algorithm, we observed a considerable accumulation of putative deleterious mutations across populations. The absence of correlation between the occurrence of putatively beneficial or deleterious mutations and local recombination rate supports the hypothesis that genetic drift might be the main driver of the accumulation of such variants. However, despite a lower genetic diversity observed in lacustrine than in riverine or anadromous populations, lacustrine populations do not exhibit more deleterious mutations than the two other history types, suggesting that the negative effect of genetic drift in lacustrine populations may be mitigated by that of relaxed purifying selection. Moreover, we also identified genomic regions associated with anadromy, as well as an overrepresentation of transposable elements associated with variation in environmental variables, thus supporting the importance of transposable elements in adaptation.

Latitudinal divergence in a widespread amphibian: Contrasting patterns of neutral and adaptive genomic variation.

Stochastic effects from demographic processes and selection are expected to shape the distribution of genetic variation in spatially heterogeneous environments. As the amount of genetic variation is central for long-term persistence of populations, understanding how these processes affect variation over large-scale geographical gradients is pivotal. We investigated the distribution of neutral and putatively adaptive genetic variation, and reconstructed demographic history in the moor frog (Rana arvalis) using 136 individuals from 15 populations along a 1,700-km latitudinal gradient from northern Germany to northern Sweden. Using double digest restriction-site associated DNA sequencing we obtained 27,590 single nucleotide polymorphisms (SNPs), and identified differentiation outliers and SNPs associated with growing season length. The populations grouped into a southern and a northern cluster, representing two phylogeographical lineages from different post-glacial colonization routes. Hybrid index estimation and demographic model selection showed strong support for a southern and northern lineage and evidence of gene flow between regions located on each side of a contact zone. However, patterns of past gene flow over the contact zone differed between neutral and putatively adaptive SNPs. While neutral nucleotide diversity was higher along the southern than the northern part of the gradient, nucleotide diversity in differentiation outliers showed the opposite pattern, suggesting differences in the relative strength of selection and drift along the gradient. Variation associated with growing season length decreased with latitude along the southern part of the gradient, but not along the northern part where variation was lower, suggesting stronger climate-mediated selection in the north. Outlier SNPs included loci involved in immunity and developmental processes.

Conservation of old individual trees and small populations is integral to maintain species' genetic diversity of a historically fragmented woody perennial.

Historically fragmented and specialized habitats such as granite outcrops are understudied globally unique hot spots of plant evolution. In contrast to predictions based on mainstream population genetic theory, some granite outcrop plants appear to have persisted as very small populations despite prolonged geographic and genetic isolation. Eucalyptus caesia Benth. is a long-lived lignotuberous tree endemic with a naturally fragmented distribution on granite outcrops in south-western Australia. To quantify population to landscape-level genetic structure, we employed microsatellite genotyping at 14 loci of all plants in 18 stands of E. caesia. Sampled stands were characterized by low levels of genetic diversity, small absolute population sizes, localized clonality and strong fine-scale genetic subdivision. There was no significant relationship between population size and levels of heterozygosity. At the landscape scale, high levels of population genetic differentiation were most pronounced among representatives of the two subspecies in E. caesia as originally circumscribed. Past genetic interconnection was evident between some geographic neighbours separated by up to 20 km. Paradoxically, other pairs of neighbouring stands as little as 7 km apart were genetically distinct. There was no consistent pattern of isolation by distance across the 280 km range of E. caesia. Low levels of gene flow, together with strong drift within stands, provide some explanation of the patterns of genetic differentiation we observed. Individual genet longevity via the ability to repeatedly resprout and expand from a lignotuber may enhance the persistence of some woody perennial endemic plants despite small population size, minimal genetic interconnection and low heterozygosity.

Noninvasive Sampling Reveals Short-Term Genetic Rescue in an Insular Red Fox Population.

Genetic factors in the decline of small populations are extremely difficult to study in nature. We leveraged a natural experiment to investigate evidence of inbreeding depression and genetic rescue in a remnant population of subalpine-specialized Sierra Nevada red foxes (Vulpes vulpes necator) using noninvasive genetic monitoring during 2010-2017. Only 7 individuals were detected in the first 2 years. These individuals assigned genetically to the historical population and exhibited genetic hallmarks of inbreeding and no evidence of reproduction. Two years into the study, we detected 2 first-generation immigrant males from a recently expanding population of red foxes in the Great Basin Desert. Through annual resampling of individuals (634 red fox DNA samples, 41 individuals) and molecular reconstruction of pedigrees, we documented 1-3 litters/year for 5 years, all descended directly or indirectly from matings involving immigrant foxes. The observed heterozygosity and allelic richness of the population nearly doubled in 2 years. Abundance increased, indicative of a rapidly expanding population. Throughout the study, adult survival was high. Restoration of gene flow apparently improved the demographic trajectory of this population in the short term. Whether these benefits continue in the longer term could depend on numerous factors, such as maintenance of any locally adapted alleles. This study highlights the value of noninvasive genetic monitoring to assess rapidly shifting conditions in small populations. Uncertainties about the longer-term trajectory of this population underscore the need to continue monitoring and to research potential for both negative and positive aspects of continued genetic infusion.

Methods for the analysis of multiple endpoints in small populations: A review.

While current guidelines generally recommend single endpoints for primary analyses of confirmatory clinical trials, it is recognized that certain settings require inference on multiple endpoints for comprehensive conclusions on treatment effects. Furthermore, combining treatment effect estimates from several outcome measures can increase the statistical power of tests. Such an efficient use of resources is of special relevance for trials in small populations. This paper reviews approaches based on a combination of test statistics or measurements across endpoints as well as multiple testing procedures that allow for confirmatory conclusions on individual endpoints. We especially focus on feasibility in trials with small sample sizes and do not solely rely on asymptotic considerations. A systematic literature search in the Scopus database, supplemented by a manual search, was performed to identify research papers on analysis methods for multiple endpoints with relevance to small populations. The identified methods were grouped into approaches that combine endpoints into a single measure to increase the power of statistical tests and methods to investigate differential treatment effects in several individual endpoints by multiple testing.

Fear creates an Allee effect: experimental evidence from seasonal populations.

Allee effects driven by predation can play a strong role in the decline of small populations but are conventionally thought to occur when generalist predators target specific prey (i.e. type II functional response). However, aside from direct consumption, fear of predators could also increase vigilance and reduce time spent foraging as population size decreases, as has been observed in wild mammals living in social groups. To investigate the role of fear on fitness in relation to population density in a species with limited sociality, we exposed varying densities of Drosophila melanogaster to mantid predators either during an experimental breeding season or non-breeding season. The presence of mantids in either season decreased the reproductive performance of individuals but only at low breeding densities, providing evidence for an Allee effect. We then used our experimental results to parametrize a mathematical model to examine the population consequences of fear at low densities. Fear tended to destabilize population dynamics and increase the risk of extinction up to sevenfold. Our study provides unique experimental evidence that the indirect effects of the presence of predators can cause an Allee effect and has important consequences for our understanding of the dynamics of small populations.

Unexpected high genetic diversity in small populations suggests maintenance by associative overdominance.

The effective population size (Ne ) is a central factor in determining maintenance of genetic variation. The neutral theory predicts that loss of variation depends on Ne , with less genetic drift in larger populations. We monitored genetic drift in 42 Drosophila melanogaster populations of different adult census population sizes (10, 50 or 500) using pooled RAD sequencing. In small populations, variation was lost at a substantially lower rate than expected. This observation was consistent across two ecological relevant thermal regimes, one stable and one with a stressful increase in temperature across generations. Estimated ratios between Ne and adult census size were consistently higher in small than in larger populations. The finding provides evidence for a slower than expected loss of genetic diversity and consequently a higher than expected long-term evolutionary potential in small fragmented populations. More genetic diversity was retained in areas of low recombination, suggesting that associative overdominance, driven by disfavoured homozygosity of recessive deleterious alleles, is responsible for the maintenance of genetic diversity in smaller populations. Consistent with this hypothesis, the X-chromosome, which is largely free of recessive deleterious alleles due to hemizygosity in males, fits neutral expectations even in small populations. Our experiments provide experimental answers to a range of unexpected patterns in natural populations, ranging from variable diversity on X-chromosomes and autosomes to surprisingly high levels of nucleotide diversity in small populations.

Health Disparities Research Among Small Tribal Populations: Describing Appropriate Criteria for Aggregating Tribal Health Data.

In response to community concerns, we used the Tribal Participatory Research framework in collaboration with 5 American-Indian communities in Washington, Idaho, and Montana to identify the appropriate criteria for aggregating health data on small tribes. Across tribal sites, 10 key informant interviews and 10 focus groups (n = 39) were conducted between July 2012 and April 2013. Using thematic analysis of focus group content, we identified 5 guiding criteria for aggregating tribal health data: geographic proximity, community type, environmental exposures, access to resources and services, and economic development. Preliminary findings were presented to focus group participants for validation at each site, and a culminating workshop with representatives from all 5 tribes verified our final results. Using this approach requires critical assessment of research questions and study designs by investigators and tribal leaders to determine when aggregation or stratification is appropriate and how to group data to yield robust results relevant to local concerns. At project inception, tribal leaders should be consulted regarding the validity of proposed groupings. After regular project updates, they should be consulted again to confirm that findings are appropriately contextualized for dissemination.

Effective number of breeders, effective population size and their relationship with census size in an iteroparous species, Salvelinus fontinalis.

The relationship between the effective number of breeders (Nb) and the generational effective size (Ne) has rarely been examined empirically in species with overlapping generations and iteroparity. Based on a suite of 11 microsatellite markers, we examine the relationship between Nb, Ne and census population size (Nc) in 14 brook trout (Salvelinus fontinalis) populations inhabiting 12 small streams in Nova Scotia and sampled at least twice between 2009 and 2015. Unbiased estimates of Nb obtained with individuals of a single cohort, adjusted on the basis of age at first maturation (α) and adult lifespan (AL), were from 1.66 to 0.24 times the average estimates of Ne obtained with random samples of individuals of mixed ages (i.e. [Formula: see text]). In turn, these differences led to adjusted Ne estimates that were from nearly five to 0.7 times the estimates derived from mixed-aged individuals. These differences translate into the same range of variation in the ratio of effective to census population size [Formula: see text] within populations. Adopting [Formula: see text] as the more precise and unbiased estimates, we found that these brook trout populations differ markedly in their effective to census population sizes (range approx. 0.3 to approx. 0.01). Using AgeNe, we then showed that the variance in reproductive success or reproductive skew varied among populations by a factor of 40, from Vk/k ≈ 5 to 200. These results suggest wide differences in population dynamics, probably resulting from differences in productivity affecting the intensity of competition for access to mates or redds, and thus reproductive skew. Understanding the relationship between Ne, Nb and Nc, and how these relate to population dynamics and fluctuations in population size, are important for the design of robust conservation strategies in small populations with overlapping generations and iteroparity.

Fallback tests for co-primary endpoints.

When efficacy of a treatment is measured by co-primary endpoints, efficacy is claimed only if for each endpoint an individual statistical test is significant at level α. While such a strategy controls the family-wise type I error rate (FWER), it is often strictly conservative and allows for no inference if not all null hypotheses can be rejected. In this paper, we investigate fallback tests, which are defined as uniform improvements of the classical test for co-primary endpoints. They reject whenever the classical test rejects but allow for inference also in settings where only a subset of endpoints show a significant effect. Similarly to the fallback tests for hierarchical testing procedures, these fallback tests for co-primary endpoints allow one to continue testing even if the primary objective of the trial was not met. We propose examples of fallback tests for two and three co-primary endpoints that control the FWER in the strong sense under the assumption of multivariate normal test statistics with arbitrary correlation matrix and investigate their power in a simulation study. The fallback procedures for co-primary endpoints are illustrated with a clinical trial in a rare disease and a diagnostic trial. © 2016 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd.