Selection leads to remarkable variability in the outcomes of hybridisation across replicate hybrid zones.

Hybrid zones have been viewed as an opportunity to see speciation in action. When hybrid zones are replicated, it is assumed that if the same genetic incompatibilities are maintaining reproductive isolation across all instances of secondary contact, those incompatibilities should be identifiable by consistent patterns in the genome. In contrast, changes in allele frequencies due to genetic drift should be idiosyncratic for each hybrid zone. To test this assumption, we simulated 20 replicates of each of 12 hybrid zone scenarios with varied genetic incompatibilities, rates of migration, selection and different starting population size ratios of parental species. We found remarkable variability in the outcomes of hybridisation in replicate hybrid zones, particularly with Bateson-Dobzhansky-Muller incompatibilities and strong selection. We found substantial differences among replicates in the overall genomic composition of individuals, including admixture proportions, inter-specific ancestry complement and number of ancestry junctions. Additionally, we found substantial variation in genomic clines among replicates at focal loci, regardless of locus-specific selection. We conclude that processes other than selection are responsible for some consistent outcomes of hybridisation, whereas selection on incompatibilities can lead to genomically widespread and highly variable outcomes. We highlight the challenge of mapping between pattern and process in hybrid zones and call attention to how selection against incompatibilities will commonly lead to variable outcomes. We hope that this study informs future research on replicate hybrid zones and encourages further development of statistical techniques, theoretical models and exploration of additional axes of variation to understand reproductive isolation.

Anthropogenic Change and the Process of Speciation.

Anthropogenic impacts on the environment alter speciation processes by affecting both geographical contexts and selection patterns on a worldwide scale. Here we review evidence of these effects. We find that human activities often generate spatial isolation between populations and thereby promote genetic divergence but also frequently cause sudden secondary contact and hybridization between diverging lineages. Human-caused environmental changes produce new ecological niches, altering selection in diverse ways that can drive diversification; but changes also often remove niches and cause extirpations. Human impacts that alter selection regimes are widespread and strong in magnitude, ranging from local changes in biotic and abiotic conditions to direct harvesting to global climate change. Altered selection, and evolutionary responses to it, impacts early-stage divergence of lineages, but does not necessarily lead toward speciation and persistence of separate species. Altogether, humans both promote and hinder speciation, although new species would form very slowly relative to anthropogenic hybridization, which can be nearly instantaneous. Speculating about the future of speciation, we highlight two key conclusions: (1) Humans will have a large influence on extinction and "despeciation" dynamics in the short term and on early-stage lineage divergence, and thus potentially speciation in the longer term, and (2) long-term monitoring combined with easily dated anthropogenic changes will improve our understanding of the processes of speciation. We can use this knowledge to preserve and restore ecosystems in ways that promote (re-)diversification, increasing future opportunities of speciation and enhancing biodiversity.

Diverse data sources and new statistical models offer prospects for improving the predictability of anthropogenic hybridization.

Human disturbance can theoretically influence the rates of hybridization, but few studies have convincingly identified a causal link. Grabenstein et al. (2022) used a genomic and phenotypic study of chickadees to associate hybridization with human disturbance. Additionally, this is consistent with citizen science reports of chickadee hybrids across the range. We highlight the exciting aspects of this work and make suggestions about a role for broad geographic and genomic sampling, and new statistical methods to better connect hybridization outcomes to anthropogenic disturbance in diverse study systems.

Admixture mapping reveals loci for carcass mass in red deer x sika hybrids in Kintyre, Scotland.

We deployed admixture mapping on a sample of 386 deer from a hybrid swarm between native red deer (Cervus elaphus) and introduced Japanese sika (Cervus nippon) sampled in Kintyre, Scotland to search for quantitative trait loci (QTLs) underpinning phenotypic differences between the species. These two species are highly diverged genetically [Fst between pure species, based on 50K single nucleotide polymorphism (SNPs) = 0.532] and phenotypically: pure red have on average twice the carcass mass of pure sika in our sample (38.7 kg vs 19.1 kg). After controlling for sex, age, and population genetic structure, we found 10 autosomal genomic locations with QTL for carcass mass. Effect sizes ranged from 0.191 to 1.839 kg and as expected, in all cases the allele derived from sika conferred lower carcass mass. The sika population was fixed for all small carcass mass alleles, whereas the red deer population was typically polymorphic. GO term analysis of genes lying in the QTL regions are associated with oxygen transport. Although body mass is a likely target of selection, none of the SNPs marking QTL are introgressing faster or slower than expected in either direction.

Low Heritability but Significant Early Environmental Effects on Resting Metabolic Rate in a Wild Passerine.

AbstractPredicting the impact of climate change on biodiversity requires understanding the adaptation potential of wild organisms. Evolutionary responses depend on the additive genetic variation associated with the phenotypic traits targeted by selection. We combine 5 years of cross-fostering experiments, measurements of resting metabolic rate (RMR) on nearly 200 wild collared flycatcher (Ficedula albicollis) nestlings, and animal models using a 17-year pedigree to evaluate the potential for an evolutionary response to changing environmental conditions. Contrary to other avian studies, we find no significant heritability of whole-organism, mass-independent, or mass-specific RMR, but we report a strong effect of nest environment instead. We therefore conclude that variation in nestling RMR is explained by variation in the early-life environment provided by the parents. We discuss possible underlying specific parental effects and the importance of taking different mechanisms into account to understand how animals phenotypically adapt (or fail to adapt) to climate change.

Locus-specific introgression in young hybrid swarms: Drift may dominate selection.

Closely related species that have previously inhabited geographically separated ranges are hybridizing at an increasing rate due to human disruptions. These human-mediated hybrid zones can be used to study reproductive isolation between species at secondary contact, including examining locus-specific rates of introgression. Introgression is expected to be heterogenous across the genome, reflecting variation in selection. Those loci that introgress especially slowly are good candidates for being involved in reproductive isolation, while those loci that introgress quickly may be involved in adaptive introgression. In the context of conservation, policy makers are especially concerned about introduced alleles moving quickly into the background of a native or endemic species, as these alleles could replace the native alleles in the population, leading to extinction via hybridization. We applied genomic cline analyses to 44,997 SNPs to identify loci introgressing more or less when compared to the genome wide expectation in a human-mediated hybridizing population of red deer and sika in Kintyre Scotland. We found 11.4% of SNPs had cline centres that were significantly different from the genome wide expectation, and 17.6% of all SNPs had excess rates of introgression. Based on simulations, we believe that many of these markers have diverged from the genome-wide average due to drift, rather than because of selection, and we suggest that these simulations can be useful as a null distribution for future studies of genomic clines. Future work on red deer and sika could determine the policy implications of allelic-replacement due to drift rather than selection, and could use replicate, geographically distinct hybrid zones to narrow down those loci that are responding to selection.

Fluctuating optimum and temporally variable selection on breeding date in birds and mammals.

Temporal variation in natural selection is predicted to strongly impact the evolution and demography of natural populations, with consequences for the rate of adaptation, evolution of plasticity, and extinction risk. Most of the theory underlying these predictions assumes a moving optimum phenotype, with predictions expressed in terms of the temporal variance and autocorrelation of this optimum. However, empirical studies seldom estimate patterns of fluctuations of an optimum phenotype, precluding further progress in connecting theory with observations. To bridge this gap, we assess the evidence for temporal variation in selection on breeding date by modeling a fitness function with a fluctuating optimum, across 39 populations of 21 wild animals, one of the largest compilations of long-term datasets with individual measurements of trait and fitness components. We find compelling evidence for fluctuations in the fitness function, causing temporal variation in the magnitude, but not the direction of selection. However, fluctuations of the optimum phenotype need not directly translate into variation in selection gradients, because their impact can be buffered by partial tracking of the optimum by the mean phenotype. Analyzing individuals that reproduce in consecutive years, we find that plastic changes track movements of the optimum phenotype across years, especially in bird species, reducing temporal variation in directional selection. This suggests that phenological plasticity has evolved to cope with fluctuations in the optimum, despite their currently modest contribution to variation in selection.

Tissue-specific patterns of regulatory changes underlying gene expression differences among Ficedula flycatchers and their naturally occurring F1 hybrids.

Changes in interacting cis- and trans-regulatory elements are important candidates for Dobzhansky-Muller hybrid incompatibilities and may contribute to hybrid dysfunction by giving rise to misexpression in hybrids. To gain insight into the molecular mechanisms and determinants of gene expression evolution in natural populations, we analyzed the transcriptome from multiple tissues of two recently diverged Ficedula flycatcher species and their naturally occurring F1 hybrids. Differential gene expression analysis revealed that the extent of differentiation between species and the set of differentially expressed genes varied across tissues. Common to all tissues, a higher proportion of Z-linked genes than autosomal genes showed differential expression, providing evidence for a fast-Z effect. We further found clear signatures of hybrid misexpression in brain, heart, kidney, and liver. However, while testis showed the highest divergence of gene expression among tissues, it showed no clear signature of misexpression in F1 hybrids, even though these hybrids were found to be sterile. It is therefore unlikely that incompatibilities between cis-trans regulatory changes explain the observed sterility. Instead, we found evidence that cis-regulatory changes play a significant role in the evolution of gene expression in testis, which illustrates the tissue-specific nature of cis-regulatory evolution bypassing constraints associated with pleiotropic effects of genes.

Differences in incubation behaviour and niche separation of two competing flycatcher species.

ABSTRACT: Food availability sets the stage for incubation behaviour of a female bird and thereby indirectly determines the nest temperature, which in turn affects development and metabolism of avian embryos. Changes in development and metabolism in turn are known to influence offspring's ability to adjust to environmental changes later in life. However, few studies have investigated the role of interspecific differences in incubation behaviour in relation to niche separation between competing sibling species. We studied the effects of habitat quality (in terms of caterpillar availability) on incubation behaviour of two ecologically similar and closely related species, collared and pied flycatchers (Ficedula albicollis and F. hypoleuca), in their hybrid zone on the island of Öland, Sweden. Even though both species prefer caterpillar-rich deciduous forests as nesting sites, collared flycatchers, whose nestlings have higher energetic demands, are able to nest only in deciduous forests, whereas pied flycatchers have more flexible habitat requirements. Overall, higher food availability was associated with increased nest attendance, higher incubation temperature and a lower number of foraging trips across species. In addition, collared flycatchers had more frequent and shorter foraging trips across habitat types, allocated more heat to eggs and therefore maintained higher nest temperatures compared to pied flycatchers. We argue that the higher heat allocation or the need to maintain a higher nest temperature for embryo development may constrain collared flycatchers to focus on relatively more profitable prey. Our results highlight the importance of considering incubation behaviour in the context of understanding species differences in niche use. SIGNIFICANCE STATEMENT: Niche separation plays an important role in mitigating effects of competition between closely related species. Whether species differences in incubation behaviour relate to differences in niche use remains unknown. We compared incubation behaviour of two sympatric flycatcher species that differ in sensitivity to food availability. The competitively more dominant and larger species, the collared flycatcher, whose nestlings are more sensitive to food shortages, made more frequent foraging trips but allocated more heat to eggs, leading to higher nest temperature despite lower nest attendance, compared to pied flycatchers. These interspecific differences may be a result of differences in embryo sensitivity or female physiology and contribute to the niche separation between the species, which in turn can facilitate coexistence.

Increased genetic marker density reveals high levels of admixture between red deer and introduced Japanese sika in Kintyre, Scotland.

Hybridization is a natural process at species range boundaries, but increasing numbers of species are hybridizing due to direct or indirect human activities. In such cases of anthropogenic hybridization, subsequent introgression can threaten the survival of native species. To date, many such systems have been studied with too few genetic markers to assess the level of threat resulting from advanced backcrossing. Here, we use 44,999 single nucleotide polymorphisms (SNPs) and the ADMIXTURE program to study two areas of Scotland where a panel of 22 diagnostic microsatellites previously identified introgression between native red deer (Cervus elaphus) and introduced Japanese sika (Cervus nippon). In Kintyre, we reclassify 26% of deer from the pure species categories to the hybrid category whereas in the NW Highlands we only reclassify 2%. As expected, the reclassified individuals are mostly advanced backcrosses. We also investigate the ability of marker panels selected on different posterior allele frequency criteria to find hybrids assigned by the full marker set and show that in our data, ancestry informative markers (i.e. those that are highly differentiated between the species, but not fixed) are better than diagnostic markers (those markers that are fixed between the species) because they are more evenly distributed in the genome. Diagnostic loci are concentrated on the X chromosome to the detriment of autosomal coverage.

Detecting the True Extent of Introgression during Anthropogenic Hybridization.

Hybridization among naturally separate taxa is increasing owing to human impact, and can result in taxon loss. Previous classification of anthropogenic hybridization has largely ignored the case of bimodal hybrid zones, in which hybrids commonly mate with parental species, resulting in many backcrossed individuals with a small proportion of introgressed genome. Genetic markers can be used to detect such hybrids, but until recently too few markers have been used to detect the true extent of introgression. Recent studies of wolves and trout have employed thousands of markers to reveal previously undetectable backcrosses. This improved resolution will lead to increased detection of late-generation backcrosses, shed light on the consequences of anthropogenic hybridization, and pose new management issues for conservation scientists.

Difference in plasticity of resting metabolic rate - the proximate explanation to different niche breadth in sympatric Ficedula flycatchers.

Variation in relative fitness of competing recently formed species across heterogeneous environments promotes coexistence. However, the physiological traits mediating such variation in relative fitness have rarely been identified. Resting metabolic rate (RMR) is tightly associated with life history strategies, thermoregulation, diet use, and inhabited latitude and could therefore moderate differences in fitness responses to fluctuations in local environments, particularly when species have adapted to different climates in allopatry. We work in a long-term study of collared (Ficedula albicollis) and pied flycatchers (Ficedula hypoleuca) in a recent hybrid zone located on the Swedish island of Öland in the Baltic Sea. Here, we explore whether differences in RMR match changes in relative performance of growing flycatcher nestlings across environmental conditions using an experimental approach. The fitness of pied flycatchers has previously been shown to be less sensitive to the mismatch between the peak in food abundance and nestling growth among late breeders. Here, we find that pied flycatcher nestlings have lower RMR in response to higher ambient temperatures (associated with low food availability). We also find that experimentally relaxed nestling competition is associated with an increased RMR in this species. In contrast, collared flycatcher nestlings did not vary their RMR in response to these environmental factors. Our results suggest that a more flexible nestling RMR in pied flycatchers is responsible for the better adaptation of pied flycatchers to the typical seasonal changes in food availability experienced in this hybrid zone. Generally, subtle physiological differences that have evolved when species were in allopatry may play an important role to patterns of competition, coexistence, or displacements between closely related species in secondary contact.

Phenological shifts in North American red squirrels: disentangling the roles of phenotypic plasticity and microevolution.

Phenological shifts are the most widely reported ecological responses to climate change, but the requirements to distinguish their causes (i.e. phenotypic plasticity vs. microevolution) are rarely met. To do so, we analysed almost two decades of parturition data from a wild population of North American red squirrels (Tamiasciurus hudsonicus). Although an observed advance in parturition date during the first decade provided putative support for climate change-driven microevolution, a closer look revealed a more complex pattern. Parturition date was heritable [h2  = 0.14 (0.07-0.21 (HPD interval)] and under phenotypic selection [ß = -0.14 ± 0.06 (SE)] across the full study duration. However, the early advance reversed in the second decade. Further, selection did not act on the genetic contribution to variation in parturition date, and observed changes in predicted breeding values did not exceed those expected due to genetic drift. Instead, individuals responded plastically to environmental variation, and high food [white spruce (Picea glauca) seed] production in the first decade appears to have produced a plastic advance. In addition, there was little evidence of climate change affecting the advance, as there was neither a significant influence of spring temperature on parturition date or evidence of a change in spring temperatures across the study duration. Heritable traits not responding to selection in accordance with quantitative genetic predictions have long presented a puzzle to evolutionary ecologists. Our results on red squirrels provide empirical support for one potential solution: phenotypic selection arising from an environmental, as opposed to genetic, covariance between the phenotypic trait and annual fitness.

Climate-driven build-up of temporal isolation within a recently formed avian hybrid zone.

Divergence in the onset of reproduction can act as an important source of reproductive isolation (i.e., allochronic isolation) between co-occurring young species, but evidence for the evolutionary processes leading to such divergence is often indirect. While advancing spring seasons strongly affect the onset of reproduction in many taxa, it remains largely unexplored whether contemporary spring advancement directly affects allochronic isolation between young species. We examined how increasing spring temperatures affected onset of reproduction and thereby hybridization between pied and collared flycatchers (Ficedula spp.) across habitat types in a young secondary contact zone. We found that both species have advanced their timing of breeding in 14 years. However, selection on pied flycatchers to breed earlier was weaker, resulting in a slower response to advancing springs compared to collared flycatchers and thereby build-up of allochronic isolation between the species. We argue that a preadaptation to a broader niche use (diet) of pied flycatchers explains the slower response to raising spring temperature, but that reduced risk to hybridize may contribute to further divergence in the onset of breeding in the future. Our results show that minor differences in the response to environmental change of co-occurring closely related species can quickly cause allochronic isolation.

Correction: Hybrid Dysfunction Expressed as Elevated Metabolic Rate in Male Ficedula Flycatchers.

[This corrects the article DOI: 10.1371/journal.pone.0161547.].

Hybrid Dysfunction Expressed as Elevated Metabolic Rate in Male Ficedula Flycatchers.

Studies of ecological speciation are often biased towards extrinsic sources of selection against hybrids, resulting from intermediate hybrid morphology, but the knowledge of how genetic incompatibilities accumulate over time under natural conditions is limited. Here we focus on a physiological trait, metabolic rate, which is central to life history strategies and thermoregulation but is also likely to be sensitive to mismatched mitonuclear interactions. We measured the resting metabolic rate of male collared, and pied flycatchers as well as of naturally occurring F1 hybrid males, in a recent hybrid zone. We found that hybrid males had a higher rather than intermediate metabolic rate, which is indicative of hybrid physiological dysfunction. Fitness costs associated with elevated metabolic rate are typically environmentally dependent and exaggerated under harsh conditions. By focusing on male hybrid dysfunction in an eco-physiological trait, our results contribute to the general understanding of how combined extrinsic and intrinsic sources of hybrid dysfunction build up under natural conditions.

Increasing the power of genome wide association studies in natural populations using repeated measures - evaluation and implementation.

Genomewide association studies (GWAS) enable detailed dissections of the genetic basis for organisms' ability to adapt to a changing environment. In long-term studies of natural populations, individuals are often marked at one point in their life and then repeatedly recaptured. It is therefore essential that a method for GWAS includes the process of repeated sampling. In a GWAS, the effects of thousands of single-nucleotide polymorphisms (SNPs) need to be fitted and any model development is constrained by the computational requirements. A method is therefore required that can fit a highly hierarchical model and at the same time is computationally fast enough to be useful.Our method fits fixed SNP effects in a linear mixed model that can include both random polygenic effects and permanent environmental effects. In this way, the model can correct for population structure and model repeated measures. The covariance structure of the linear mixed model is first estimated and subsequently used in a generalized least squares setting to fit the SNP effects. The method was evaluated in a simulation study based on observed genotypes from a long-term study of collared flycatchers in Sweden.The method we present here was successful in estimating permanent environmental effects from simulated repeated measures data. Additionally, we found that especially for variable phenotypes having large variation between years, the repeated measurements model has a substantial increase in power compared to a model using average phenotypes as a response.The method is available in the r package RepeatABEL. It increases the power in GWAS having repeated measures, especially for long-term studies of natural populations, and the R implementation is expected to facilitate modelling of longitudinal data for studies of both animal and human populations.

Competition-driven build-up of habitat isolation and selection favoring modified dispersal patterns in a young avian hybrid zone.

Competition-driven evolution of habitat isolation is an important mechanism of ecological speciation but empirical support for this process is often indirect. We examined how an on-going displacement of pied flycatchers from their preferred breeding habitat by collared flycatchers in a young secondary contact zone is associated with (a) access to an important food resource (caterpillar larvae), (b) immigration of pied flycatchers in relation to habitat quality, and (c) the risk of hybridization in relation to habitat quality. Over the past 12 years, the estimated access to caterpillar larvae biomass in the habitat surrounding the nests of pied flycatchers has decreased by a fifth due to shifted establishment possibilities, especially for immigrants. However, breeding in the high quality habitat has become associated with such a high risk of hybridization for pied flycatchers that overall selection currently favors pied flycatchers that were forced to immigrate into the poorer habitats (despite lower access to preferred food items). Our results show that competition-driven habitat segregation can lead to fast habitat isolation, which per se caused an opportunity for selection to act in favor of future "voluntarily" altered immigration patterns and possibly strengthened habitat isolation through reinforcement.

Song discrimination by nestling collared flycatchers during early development.

Pre-zygotic isolation is often maintained by species-specific signals and preferences. However, in species where signals are learnt, as in songbirds, learning errors can lead to costly hybridization. Song discrimination expressed during early developmental stages may ensure selective learning later in life but can be difficult to demonstrate before behavioural responses are obvious. Here, we use a novel method, measuring changes in metabolic rate, to detect song perception and discrimination in collared flycatcher embryos and nestlings. We found that nestlings as early as 7 days old respond to song with increased metabolic rate, and, by 9 days old, have increased metabolic rate when listening to conspecific when compared with heterospecific song. This early discrimination between songs probably leads to fewer heterospecific matings, and thus higher fitness of collared flycatchers living in sympatry with closely related species.

Females discriminate against heterospecific sperm in a natural hybrid zone.

When hybridization is maladaptive, species-specific mate preferences are selectively favored, but low mate availability may constrain species-assortative pairing. Females paired to heterospecifics may then benefit by copulating with multiple males and subsequently favoring sperm of conspecifics. Whether such mechanisms for biasing paternity toward conspecifics act as important reproductive barriers in socially monogamous vertebrate species remains to be determined. We use a combination of long-term breeding records from a natural hybrid zone between collared and pied flycatchers (Ficedula albicollis and F. hypoleuca), and an in vitro experiment comparing conspecific and heterospecific sperm performance in female reproductive tract fluid, to evaluate the potential significance of female cryptic choice. We show that the females most at risk of hybridizing (pied flycatchers) frequently copulate with multiple males and are able to inhibit heterospecific sperm performance. The negative effect on heterospecific sperm performance was strongest in pied flycatcher females that were most likely to have been previously exposed to collared flycatcher sperm. We thus demonstrate that a reproductive barrier acts after copulation but before fertilization in a socially monogamous vertebrate. While the evolutionary history of this barrier is unknown, our results imply that there is opportunity for it to be accentuated via a reinforcement-like process.