Conservation genetics as a management tool: The five best-supported paradigms to assist the management of threatened species.

About 50 y ago, Crow and Kimura [An Introduction to Population Genetics Theory (1970)] and Ohta and Kimura [Genet. Res. 22, 201-204 (1973)] laid the foundations of conservation genetics by predicting the relationship between population size and genetic marker diversity. This work sparked an enormous research effort investigating the importance of population dynamics, in particular small population size, for population mean performance, population viability, and evolutionary potential. In light of a recent perspective [J. C. Teixeira, C. D. Huber, Proc. Natl. Acad. Sci. U.S.A. 118, 10 (2021)] that challenges some fundamental assumptions in conservation genetics, it is timely to summarize what the field has achieved, what robust patterns have emerged, and worthwhile future research directions. We consider theory and methodological breakthroughs that have helped management, and we outline some fundamental and applied challenges for conservation genetics.

Evolutionary potential under heat and drought stress at the southern range edge of North American Arabidopsis lyrata.

The warm edges of species' distributions are vulnerable to global warming. Evidence is the recent range retraction from there found in many species. It is unclear why populations cannot easily adapt to warmer, drier, or combined hot and dry conditions and locally persist. Here, we assessed the ability to adapt to these stressors in the temperate species Arabidopsis lyrata. We grew plants from replicate seed families of a central population with high genetic diversity under a temperature and precipitation regime typical of the low-latitude margin or under hotter and/or drier conditions within naturally occurring amplitudes. We then estimated genetic variance-covariance (G-) matrices of traits depicting growth and allocation as well as selection vectors to compare the predicted adaptation potential under the different climate-stress regimes. We found that the sum of genetic variances and genetic correlations were not significantly different under stress as compared to benign conditions. However, under drought and heat drought, the predicted ability to adapt was severely constrained due to strong selection and selection pointing in a direction with less multivariate genetic variation. The much-reduced ability to adapt to dry and hot-dry conditions is likely to reduce the persistence of populations at the low-latitude margin of the species' distribution and contribute to the local extinction of the species under further warming.

Negative interaction effect of heat and drought stress at the warm end of species distribution.

Geographic range limits of species are often a reflection of their ecological niche limits. In many organisms, important niche limits that coincide with distribution limits are warm and warm-dry conditions. We investigated the effects of heat and drought, as they can occur at the warm end of distribution. In a greenhouse experiment, we raised North American Arabidopsis lyrata from the centre of its distribution as well as from low- and high-latitude limits under average and extreme conditions. We assessed plant growth and development, as well as leaf and root functional traits, and tested for a decline in performance and selection acting on growth, leaf, and root traits. Drought and heat, when applied alone, lowered plant performance, while combined stress caused synergistically negative effects. Plants from high latitudes did not survive under combined stress, whereas plants originating from central and low latitudes had low to moderate survival, indicating divergent adaptation. Traits positively associated with survival under drought, with or without heat, were delayed and slowed growth, though plastic responses in these traits were generally antagonistic to the direction of selection. In line, higher tolerance of stress in southern populations did not involve aspects of growth but rather a higher root-to-shoot ratio and thinner leaves. In conclusion, combined heat and drought, as can occur at southern range edges and presumably more so under global change, seriously impede the long-term persistence of A. lyrata, even though they impose selection and populations may adapt, though under likely interference by considerable maladaptive plasticity.

Drivers of linkage disequilibrium across a species' geographic range.

While linkage disequilibrium (LD) is an important parameter in genetics and evolutionary biology, the drivers of LD remain elusive. Using whole-genome sequences from across a species' range, we assessed the impact of demographic history and mating system on LD. Both range expansion and a shift from outcrossing to selfing in North American Arabidopsis lyrata were associated with increased average genome-wide LD. Our results indicate that range expansion increases short-distance LD at the farthest range edges by about the same amount as a shift to selfing. However, the extent over which LD in genic regions unfolds was shorter for range expansion compared to selfing. Linkage among putatively neutral variants and between neutral and deleterious variants increased to a similar degree with range expansion, providing support that genome-wide LD was positively associated with mutational load. As a consequence, LD combined with mutational load may decelerate range expansions and set range limits. Finally, a small number of genes were identified as LD outliers, suggesting that they experience selection by either of the two demographic processes. These included genes involved in flowering and photoperiod for range expansion, and the self-incompatibility locus for mating system.

Intraspecific variation in reproductive barriers between two closely related Arabidopsis sister species.

Reproductive isolation (RI) is a critical component of speciation and varies strongly in timing and strength among different sister taxa, depending on, for example the geography of speciation and divergence time. However, these factors may also produce variation in timing and strength among populations within species. Here we tested for variation in the expression of RI among replicate population pairs between the sister taxa Arabidopsis lyrata subsp. lyrata and A. arenicola. While the former is predominantly outcrossing, the latter is predominantly selfing. We focused on intrinsic prezygotic and postzygotic RI as both species occur largely in allopatry. We assessed RI by performing within-population crosses and interspecific between-population crosses, and by raising offspring. RI was generally high between all interspecific population pairs, but it varied in timing and strength depending on population history. Prezygotic isolation was strongest between the closest-related population pair, while early postzygotic isolation was high for all other population pairs. Furthermore, the timing and strength of RI depended strongly on cross direction. Our study provides empirical support that reproductive barriers between species are highly variable among population pairs and asymmetric within population pairs, and this variation seems to follow patterns typically described across species pairs.

Environment dependence of the expression of mutational load and species' range limits.

Theoretical and empirical research on the causes of species' range limits suggest the contribution of several intrinsic and extrinsic factors, with potentially complex interactions among them. An intrinsic factor proposed by recent theory is mutational load increasing towards range edges because of genetic drift. Furthermore, environmental quality may decline towards range edges and enhance the expression of load. Here, we tested whether the expression of mutational load associated with range limits in the North American plant Arabidopsis lyrata was enhanced under stressful environmental conditions by comparing the performance of within- versus between-population crosses at common garden sites across the species' distribution and beyond. Heterosis, reflecting the expression of load, increased with heightened estimates of genomic load and with environmental stress caused by warming, but the interaction was not significant. We conclude that range-edge populations suffer from a twofold genetic Allee effect caused by increased mutational load and stress-dependent load linked to general heterozygote deficiency, but there is no synergistic effect between them.

Reduced pollinator service in small populations of Arabidopsis lyrata at its southern range limit.

Even though a high fraction of angiosperm plants depends on animal pollinators for sexual reproduction, little is known how pollinator service changes across the ranges of plant species and whether it may contribute to range limits. Here, we tested for variation in pollinator service in the North American Arabidopsis lyrata from its southern to northern range edge and evaluated the driving mechanisms. We monitored insect pollinators using time-lapse cameras in 13 populations over two years and spotted 67 pollinating insect taxa, indicating the generalist nature of this plant-pollinator system. Pollinator service was highest at intermediate local flower densities and higher in large compared to small plant populations. Southern populations had generally smaller population sizes, and visitation rate and pollination ratio decreased with latitude. We also found that pollinator visitation was positively correlated with the richness of other flowering plants. This study indicates that plant populations at southern range edges receive only marginal pollinator service if they are small, and the effect of lower pollination is also detectable within populations across the range when the local flower density is low. Results, therefore, suggest the potential for an Allee effect in pollination that manifests itself across spatial scales.

Lineage-specific adaptation to climate involves flowering time in North American Arabidopsis lyrata.

Adaptation to local climatic conditions is commonly found within species, but whether it involves the same intraspecific genomic variants is unknown. We studied this question in North American Arabidopsis lyrata, whose current distribution is shaped by post-glacial range expansion from two refugia, resulting in two distinct genetic clusters covering comparable climatic gradients. Using pooled whole-genome sequence data of 41 outcrossing populations, we identified loci associated with three niche-determining climatic variables in the two clusters and compared these outliers. Little evidence was found for parallelism in climate adaptation for single nucleotide polymorphisms (SNPs) and for genes with an accumulation of outlier SNPs. Significantly increased selection coefficients supported them as candidates of climate adaptation. However, the fraction of gene ontology (GO) terms shared between clusters was higher compared to outlier SNPs and outlier genes, suggesting that selection acts on similar pathways but not necessarily the same genes. Enriched GO terms involved responses to abiotic and biotic stress, circadian rhythm and development, with flower development and reproduction being among the most frequently detected. In line with GO enrichment, regulators of flowering time were detected as outlier genes. Our results suggest that while adaptation to environmental gradients on the genomic level are lineage-specific in A. lyrata, similar biological processes seem to be involved. Differential loss of standing genetic variation, probably driven by genetic drift, can in part account for the lack of parallel evolution on the genomic level.

Accumulation of Mutational Load at the Edges of a Species Range.

Why species have geographically restricted distributions is an unresolved question in ecology and evolutionary biology. Here, we test a new explanation that mutation accumulation due to small population size or a history of range expansion can contribute to restricting distributions by reducing population growth rate at the edge. We examined genomic diversity and mutational load across the entire geographic range of the North American plant Arabidopsis lyrata, including old, isolated populations predominantly at the southern edge and regions of postglacial range expansion at the northern and southern edges. Genomic diversity in intergenic regions declined toward distribution edges and signatures of mutational load in exon regions increased. Genomic signatures of mutational load were highly linked to phenotypically expressed load, measured as reduced performance of individual plants and lower estimated rate of population growth. The geographic pattern of load and the connection between load and population growth demonstrate that mutation accumulation reduces fitness at the edge and helps restrict species' distributions.

A Practical Guide to the Study of Distribution Limits.

Factors that limit the geographic distribution of species are broadly important in ecology and evolutionary biology, and understanding distribution limits is imperative for predicting how species will respond to environmental change. Good data indicate that factors such as dispersal limitation, small effective population size, and isolation are sometimes important. But empirical research highlights no single factor that explains the ubiquity of distribution limits. In this article, we outline a guide to tackling distribution limits that integrates established causes, such as dispersal limitation and spatial environmental heterogeneity, with understudied causes, such as mutational load and genetic or developmental integration of traits limiting niche expansion. We highlight how modeling and quantitative genetic and genomic analyses can provide insight into sources of distribution limits. Our practical guide provides a framework for considering the many factors likely to determine species distributions and how the different approaches can be integrated to predict distribution limits using eco-evolutionary modeling. The framework should also help predict distribution limits of invasive species and of species under climate change.

Postglacial ecotype formation under outcrossing and self-fertilization in Arabidopsis lyrata.

The formation of ecotypes has been invoked as an important driver of postglacial biodiversity, because many species colonized heterogeneous habitats and experienced divergent selection. Ecotype formation has been predominantly studied in outcrossing taxa, while far less attention has been paid to the implications of mating system shifts. Here, we addressed whether substrate-related ecotypes exist in selfing and outcrossing populations of Arabidopsis lyrata subsp. lyrata and whether the genomic footprint differs between mating systems. The North American subspecies colonized both rocky and sandy habitats during postglacial range expansion and shifted the mating system from predominantly outcrossing to predominantly selfing in a number of regions. We performed an association study on pooled whole-genome sequence data of 20 selfing or outcrossing populations, which suggested genes involved in adaptation to substrate. Motivated by enriched gene ontology terms, we compared root growth between plants from the two substrates in a common environment and found that plants originating from sand grew roots faster and produced more side roots, independent of mating system. Furthermore, single nucleotide polymorphisms associated with substrate-related ecotypes were more clustered among selfing populations. Our study provides evidence for substrate-related ecotypes in A. lyrata and divergence in the genomic footprint between mating systems. The latter is the likely result of selfing populations having experienced divergent selection on larger genomic regions due to higher genome-wide linkage disequilibrium.

Accumulation of transposable elements in selfing populations of Arabidopsis lyrata supports the ectopic recombination model of transposon evolution.

Transposable elements (TE) can constitute a large fraction of plant genomes, yet our understanding of their evolution and fitness effect is still limited. Here we tested several models of evolution that make specific predictions about differences in TE abundance between selfing and outcrossing taxa, and between small and large populations. We estimated TE abundance in multiple populations of North American Arabidopsis lyrata differing in mating system and long-term size, using transposon insertion display on several TE families. Selfing populations had higher TE copy numbers per individual and higher TE allele frequencies, supporting models which assume that selection against TEs acts predominantly against heterozygotes via the process of ectopic recombination. In outcrossing populations differing in long-term size, the data supported neither a model of density-regulated transposition nor a model of direct deleterious effect. Instead, the population structure of TEs revealed that outcrossing populations tended to split into western and eastern groups - as previously detected using microsatellite markers - whereas selfing populations from west and east were less differentiated. This, too, agrees with the model of ectopic recombination. Overall, our results suggest that TE elements are nearly neutral except for their deleterious potential to disturb meiosis in the heterozygous state.

Genetic differentiation in life history traits and thermal stress performance across a heterogeneous dune landscape in Arabidopsis lyrata.

Background and Aims: Over very short spatial scales, the habitat of a species can differ in multiple abiotic and biotic factors. These factors may impose natural selection on several traits and can cause genetic differentiation within a population. We studied multivariate genetic differentiation in a plant species of a sand dune landscape by linking environmental variation with differences in genotypic trait values and gene expression levels to find traits and candidate genes of microgeographical adaptation. Methods: Maternal seed families of Arabidopsis lyrata were collected in Saugatuck Dunes State Park, Michigan, USA, and environmental parameters were recorded at each collection site. Offspring plants were raised in climate chambers and exposed to one of three temperature treatments: regular occurrence of frost, heat, or constant control conditions. Several traits were assessed: plant growth, time to flowering, and frost and heat resistance. Key Results: The strongest trait-environment association was between a fast switch to sexual reproduction and weaker growth under frost, and growing in the open, away from trees. The second strongest association was between the trait combination of small plant size and early flowering under control conditions combined with large size under frost, and the combination of environmental conditions of growing close to trees, at low vegetation cover, on dune bottoms. Gene expression analysis by RNA-seq revealed candidate genes involved in multivariate trait differentiation. Conclusions: The results support the hypothesis that in natural populations, many environmental factors impose selection, and that they affect multiple traits, with the relative direction of trait change being complex. The results highlight that heterogeneity in the selection environment over small spatial scales is a main driver of the maintenance of adaptive genetic variation within populations.

Quantitative Genetic Architecture at Latitudinal Range Boundaries: Reduced Variation but Higher Trait Independence.

Species distribution limits are hypothesized to be caused by small population size and limited genetic variation in ecologically relevant traits, but earlier studies have not evaluated genetic variation in multivariate phenotypes. We asked whether populations at the latitudinal edges of the distribution have altered quantitative genetic architecture of ecologically relevant traits compared with midlatitude populations. We calculated measures of evolutionary potential in nine Arabidopsis lyrata populations spanning the latitudinal range of the species in eastern and midwestern North America. Environments at the latitudinal extremes have reduced water availability, and therefore plants were assessed under wet and dry treatments. We estimated genetic variance-covariance (G-) matrices for 10 traits related to size, development, and water balance. Populations at southern and northern distribution edges had reduced levels of genetic variation across traits, but their G-matrices were more spherical; G-matrix orientation was unrelated to latitude. As a consequence, the predicted short-term response to selection was at least as strong in edge populations as in central populations. These results are consistent with genetic drift eroding variation and reducing the effectiveness of correlational selection at distribution margins. We conclude that genetic variation of isolated traits poorly predicts the capacity to evolve in response to multivariate selection and that the response to selection may frequently be greater than expected at species distribution margins because of genetic drift.

Detecting genetic responses to environmental change.

Changes in environmental conditions can rapidly shift allele frequencies in populations of species with relatively short generation times. Frequency shifts might be detectable in neutral genetic markers when stressful conditions cause a population decline. However, frequency shifts that are diagnostic of specific conditions depend on isolating sets of genes that are involved in adaptive responses. Shifts at candidate loci underlying adaptive responses and DNA regions that control their expression have now been linked to evolutionary responses to pollution, global warming and other changes. Conversely, adaptive constraints, particularly in physiological traits, are recognized through DNA decay in candidate genes. These approaches help researchers and conservation managers understand the power and constraints of evolution.

Latitudinal trait variation and responses to drought in Arabidopsis lyrata.

Species may respond in three ways to environmental change: adapt, migrate, or go extinct. Studies of latitudinal clines can provide information on whether species have adapted to abiotic stress such as temperature and drought in the past and what the traits underlying adaptation are. We investigated latitudinal trait variation and response to drought in North American populations of Arabidopsis lyrata. Plants from nine populations collected over 13° latitude were grown under well-watered and dry conditions. A total of 1,620 seedlings were raised and 12 phenological, physiological, morphological, and life history traits were measured. Two traits, asymptotic rosette size and the propensity to flower, were significantly associated with latitude: plants from northern locations grew to a larger size and were more likely to flower in the first season. Most traits displayed a plastic response to drought, but plasticity was never related linearly with latitude nor was it enhanced in populations from extreme latitudes with reduced water availability. Populations responded to drought by adopting mixed strategies of resistance, tolerance, and escape. The study shows that latitudinal adaptation in A. lyrata involves the classic life history traits, size at and timing of reproduction. Contrary to recent theoretical predictions, adaptation to margins is based on fixed trait differences and not on phenotypic plasticity, at least with respect to drought.

The battle of the sexes over seed size: support for both kinship genomic imprinting and interlocus contest evolution.

Outcrossing creates a venue for parental conflict. When one sex provides parental care to offspring fertilized by several partners, the nonproviding sex is under selection to maximally exploit the caring sex. The caring sex may counteradapt, and a coevolutionary arms race ensues. Genetic models of this conflict include the kinship theory of genomic imprinting (parent-of-origin-specific expression of maternal-care effectors) and interlocus conflict evolution (interaction between male selfish signals and female abatement). Predictions were tested by measuring the sizes of seeds produced by within-population crosses (diallel design) and between-population crosses in outcrossing and selfing populations of Arabidopsis lyrata. Within-population diallel crosses revealed substantial maternal variance in seed size in most populations. The comparison of between- and within-population crosses showed that seeds were larger when pollen came from another outcrossing population than when pollen came from a selfing or the same population, supporting interlocus contest evolution between male selfish genes and female recognition genes. Evidence for kinship genomic imprinting came from complementary trait means of seed size in reciprocal between-population crosses independent of whether populations were predominantly selfing or outcrossing. Hence, both kinship genomic imprinting and interlocus contest are supported in outcrossing Arabidopsis, whereas only kinship genomic imprinting is important in selfing populations.

Asynchronous life cycles contribute to reproductive isolation between two Alpine butterflies.

Geographic isolation often leads to the emergence of distinct genetic lineages that are at least partially reproductively isolated. Zones of secondary contact between such lineages are natural experiments that allow investigation of how reproductive isolation evolves and co-existence is maintained. While temporal isolation through allochrony has been suggested to promote reproductive isolation in sympatry, its potential for isolation upon secondary contact is far less understood. Sampling two contact zones of a pair of mainly allopatric Alpine butterflies over several years and taking advantage of museum samples, we show that the contact zones have remained geographically stable over several decades. Furthermore, they seem to be maintained by the asynchronous life cycles of the two butterflies, with one reaching adulthood primarily in even and the other primarily in odd years. Genomic inferences document that allochrony is leaky and that gene flow from allopatric sites scales with the degree of geographic isolation. Overall, we show that allochrony has the potential to contribute to the maintenance of secondary contact zones of lineages that diverged in allopatry.

Trait divergence and trade-offs among Brassicaceae species differing in elevational distribution.

Species have restricted geographic distributions and the causes are still largely unknown. Temperature has long been associated with distribution limits, suggesting that there are ubiquitous constraints to the evolution of the climate niche. Here, we investigated the traits involved in such constraints by macroevolutionary comparisons involving 100 Brassicaceae species differing in elevational distribution. Plants were grown under three temperature treatments (regular frost, mild, regular heat) and phenotyped for phenological, morphological, and thermal resistance traits. Trait values were analyzed by assessing the effect of temperature and elevational distribution, by comparing models of evolutionary trajectories, and by correlative approaches to identify trade-offs. Analyses pointed to size, leaf morphology, and growth under heat as among the most discriminating traits between low- and high-elevation species, with high-elevation species growing faster under the occurrence of regular heat bouts, at the cost of reduced size. Mixed models and evolutionary models supported adaptive divergence for these traits, and correlation analysis indicated their involvement in moderate trade-offs. Finally, we found asymmetry in trait evolution, with evolvability across traits being 50% less constrained under regular frost. Overall, results suggest that trade-offs between traits under adaptive divergence contribute to the disparate distribution of species along the elevational gradient.