Additive genetic effects in interacting species jointly determine the outcome of caterpillar herbivory.

Plant-insect interactions are common and important in basic and applied biology. Trait and genetic variation can affect the outcome and evolution of these interactions, but the relative contributions of plant and insect genetic variation and how these interact remain unclear and are rarely subject to assessment in the same experimental context. Here, we address this knowledge gap using a recent host-range expansion onto alfalfa by the Melissa blue butterfly. Common garden rearing experiments and genomic data show that caterpillar performance depends on plant and insect genetic variation, with insect genetics contributing to performance earlier in development and plant genetics later. Our models of performance based on caterpillar genetics retained predictive power when applied to a second common garden. Much of the plant genetic effect could be explained by heritable variation in plant phytochemicals, especially saponins, peptides, and phosphatidyl cholines, providing a possible mechanistic understanding of variation in the species interaction. We find evidence of polygenic, mostly additive effects within and between species, with consistent effects of plant genotype on growth and development across multiple butterfly species. Our results inform theories of plant-insect coevolution and the evolution of diet breadth in herbivorous insects and other host-specific parasites.

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.

A polygenic architecture with habitat-dependent effects underlies ecological differentiation in Silene.

Ecological differentiation can drive speciation but it is unclear how the genetic architecture of habitat-dependent fitness contributes to lineage divergence. We investigated the genetic architecture of cumulative flowering, a fitness component, in second-generation hybrids between Silene dioica and Silene latifolia transplanted into the natural habitat of each species. We used reduced-representation sequencing and Bayesian sparse linear mixed models (BSLMMs) to analyze the genetic control of cumulative flowering in each habitat. Our results point to a polygenic architecture of cumulative flowering. Allelic effects were mostly beneficial or deleterious in one habitat and neutral in the other. Positive-effect alleles often were derived from the native species, whereas negative-effect alleles, at other loci, tended to originate from the non-native species. We conclude that ecological differentiation is governed and maintained by many loci with small, habitat-dependent effects consistent with conditional neutrality. This pattern may result from differences in selection targets in the two habitats and from environmentally dependent deleterious load. Our results further suggest that selection for native alleles and against non-native alleles acts as a barrier to gene flow between species.

Admixture mapping in interspecific Populus hybrids identifies classes of genomic architectures for phytochemical, morphological and growth traits.

The genomic architecture of functionally important traits is key to understanding the maintenance of reproductive barriers and trait differences when divergent populations or species hybridize. We conducted a genome-wide association study (GWAS) to study trait architecture in natural hybrids of two ecologically divergent Populus species. We genotyped 472 seedlings from a natural hybrid zone of Populus alba and Populus tremula for genome-wide markers from reduced representation sequencing, phenotyped the plants in common gardens for 46 phytochemical (phenylpropanoid), morphological and growth traits, and used a Bayesian polygenic model for mapping. We detected three classes of genomic architectures: traits with finite, detectable associations of genetic loci with phenotypic variation in addition to highly polygenic heritability; traits with indications for polygenic heritability only; and traits with no detectable heritability. For the first class, we identified genome regions with plausible candidate genes for phenylpropanoid biosynthesis or its regulation, including MYB transcription factors and glycosyl transferases. GWAS in natural, recombinant hybrids represent a promising step towards resolving the genomic architecture of phenotypic traits in long-lived species. This facilitates the fine-mapping and subsequent functional characterization of genes and networks causing differences in hybrid performance and fitness.

Genetic evidence for species cohesion, substructure and hybrids in spruce.

The origin and history of species are shaped by various evolutionary dynamics, including their persistence in the face of potential gene flow from related taxa. In this study, we use broad geographical and taxonomic sampling (2,219 individuals) to establish the distribution of species, hybrids and cryptic genetic variation within the conifer genus Picea (spruce) across western North America. We demonstrate that the six species of spruce in this region are distinguishable based on their genetic composition, and that the more closely related Engelmann spruce (P. engelmannii) and white spruce (P. glauca) have generated numerous and widespread hybrids. These hybrids occur in the central Rocky Mountains, well to the south of the well-established region of admixture in Canada. Additionally, we provide evidence for subdivision within Engelmann spruce, manifested as a southern Rocky Mountains form, and a northern Rocky Mountain and Cascade mountains (western) form. In the intervening central Rocky Mountains region (forests in Wyoming and adjacent states) we found primarily individuals with admixed ancestry. Following their origin, these species of spruce have interacted repeatedly and in different geographical contexts. Multiple pairs of species have been shown to hybridize, yet the species persist and retain distinguishable compositions. At the same time, large geographical areas exist where hybrids are pervasive. Consequently, spruce provide a case study for the maintenance of species boundaries, particularly for how widespread hybridization need not lead to the collapse and loss of species.

Genome divergence and diversification within a geographic mosaic of coevolution.

Despite substantial interest in coevolution's role in diversification, examples of coevolution contributing to speciation have been elusive. Here, we build upon past studies that have shown both coevolution between South Hills crossbills and lodgepole pine (Pinus contorta), and high levels of reproductive isolation between South Hills crossbills and other ecotypes in the North American red crossbill (Loxia curvirostra) complex. We used genotyping by sequencing to generate population genomic data and applied phylogenetic and population genetic analyses to characterize the genetic structure within and among nine of the ecotypes. Although genome-wide divergence was slight between ecotypes (FST  = 0.011-0.035), we found evidence of relative genetic differentiation (as measured by FST ) between and genetic cohesiveness within many of them. As expected for nomadic and opportunistic breeders, we detected no evidence of isolation by distance. The one sedentary ecotype, the South Hills crossbill, was genetically most distinct because of elevated divergence at a small number of loci rather than pronounced overall genome-wide divergence. These findings suggest that mechanisms related to recent local coevolution between South Hills crossbills and lodgepole pine (e.g. strong resource-based density dependence limiting gene flow) have been associated with genome divergence in the face of gene flow. Our results further characterize a striking example of coevolution driving speciation within perhaps as little as 6000 years.

Highly variable reproductive isolation among pairs of Catostomus species.

Hybridization between diverged taxa tests the strength of reproductive isolation and can therefore reveal mechanisms of reproductive isolation. However, it remains unclear how consistent reproductive isolation is across species' ranges and to what extent reproductive isolation might remain polymorphic as species diverge. To address these questions, we compared outcomes of hybridization across species pairs of Catostomus fishes in three rivers in the Upper Colorado River basin, where an introduced species, C. commersoni, hybridizes with at least two native species, C. discobolus and C. latipinnis. We observed substantial heterogeneity in outcomes of hybridization, both between species pairs and across geographically separate rivers within each species pair. We also observed hybridization of additional related species with our focal species, suggesting that reproductive isolation in this group involves interactions of multiple evolutionary and ecological factors. These findings suggest that a better understanding of the determinants of variation in reproductive isolation is needed and that studies of reproductive isolation in hybrids should consider how the dynamics and mechanisms of reproductive isolation vary over ecological space and over evolutionary time. Our results also have implications for the conservation and management of native catostomids in the Colorado River basin. Heterogeneity in outcomes of hybridization suggests that the threat posed by hybridization and genetic introgression to the persistence of native species probably varies with extent of reproductive isolation, both across rivers and across species pairs.

The evolution of novel host use is unlikely to be constrained by trade-offs or a lack of genetic variation.

The genetic and ecological factors that shape the evolution of animal diets remain poorly understood. For herbivorous insects, the expectation has been that trade-offs exist, such that adaptation to one host plant reduces performance on other potential hosts. We investigated the genetic architecture of alternative host use by rearing individual Lycaeides melissa butterflies from two wild populations in a crossed design on two hosts (one native and one introduced) and analysing the genetic basis of differences in performance using genomic approaches. Survival during the experiment was highest when butterfly larvae were reared on their natal host plant, consistent with local adaptation. However, cross-host correlations in performance among families (within populations) were not different from zero. We found that L. melissa populations possess genetic variation for larval performance and variation in performance had a polygenic basis. We documented very few genetic variants with trade-offs that would inherently constrain diet breadth by preventing the optimization of performance across hosts. Instead, most genetic variants that affected performance on one host had little to no effect on the other host. In total, these results suggest that genetic trade-offs are not the primary cause of dietary specialization in L. melissa butterflies.

Experimental evidence for ecological selection on genome variation in the wild.

Understanding natural selection's effect on genetic variation is a major goal in biology, but the genome-scale consequences of contemporary selection are not well known. In a release and recapture field experiment we transplanted stick insects to native and novel host plants and directly measured allele frequency changes within a generation at 186,576 genetic loci. We observed substantial, genome-wide allele frequency changes during the experiment, most of which could be attributed to random mortality (genetic drift). However, we also documented that selection affected multiple genetic loci distributed across the genome, particularly in transplants to the novel host. Host-associated selection affecting the genome acted on both a known colour-pattern trait as well as other (unmeasured) phenotypes. We also found evidence that selection associated with elevation affected genome variation, although our experiment was not designed to test this. Our results illustrate how genomic data can identify previously underappreciated ecological sources and phenotypic targets of selection.

Genome-wide association mapping of phenotypic traits subject to a range of intensities of natural selection in Timema cristinae.

The genetic architecture of adaptive traits can reflect the evolutionary history of populations and also shape divergence among populations. Despite this central role in evolution, relatively little is known regarding the genetic architecture of adaptive traits in nature, particularly for traits subject to known selection intensities. Here we quantitatively describe the genetic architecture of traits that are subject to known intensities of differential selection between host plant species in Timema cristinae stick insects. Specifically, we used phenotypic measurements of 10 traits and 211,004 single-nucleotide polymorphisms (SNPs) to conduct multilocus genome-wide association mapping. We identified a modest number of SNPs that were associated with traits and sometimes explained a large proportion of trait variation. These SNPs varied in their strength of association with traits, and both major and minor effect loci were discovered. However, we found no relationship between variation in levels of divergence among traits in nature and variation in parameters describing the genetic architecture of those same traits. Our results provide a first step toward identifying loci underlying adaptation in T. cristinae. Future studies will examine the genomic location, population differentiation, and response to selection of the trait-associated SNPs described here.

Unexpected ancestry of Populus seedlings from a hybrid zone implies a large role for postzygotic selection in the maintenance of species.

In the context of potential interspecific gene flow, the integrity of species will be maintained by reproductive barriers that reduce genetic exchange, including traits associated with prezygotic isolation or poor performance of hybrids. Hybrid zones can be used to study the importance of different reproductive barriers, particularly when both parental species and hybrids occur in close spatial proximity. We investigated the importance of barriers to gene flow that act early vs. late in the life cycle of European Populus by quantifying the prevalence of homospecific and hybrid matings within a mosaic hybrid zone. We obtained genotypic data for 11 976 loci from progeny and their maternal parents and constructed a Bayesian model to estimate individual admixture proportions and hybrid classes for sampled trees and for the unsampled pollen parent. Matings that included one or two hybrid parents were common, resulting in admixture proportions of progeny that spanned the whole range of potential ancestries between the two parental species. This result contrasts strongly with the distribution of admixture proportions in adult trees, where intermediate hybrids and each of the parental species are separated into three discrete ancestry clusters. The existence of the full range of hybrids in seedlings is consistent with weak reproductive isolation early in the life cycle of Populus. Instead, a considerable amount of selection must take place between the seedling stage and maturity to remove many hybrid seedlings. Our results highlight that high hybridization rates and appreciable hybrid fitness do not necessarily conflict with the maintenance of species integrity.

Admixture and the organization of genetic diversity in a butterfly species complex revealed through common and rare genetic variants.

Detailed information about the geographic distribution of genetic and genomic variation is necessary to better understand the organization and structure of biological diversity. In particular, spatial isolation within species and hybridization between them can blur species boundaries and create evolutionary relationships that are inconsistent with a strictly bifurcating tree model. Here, we analyse genome-wide DNA sequence and genetic ancestry variation in Lycaeides butterflies to quantify the effects of admixture and spatial isolation on how biological diversity is organized in this group. We document geographically widespread and pervasive historical admixture, with more restricted recent hybridization. This includes evidence supporting previously known and unknown instances of admixture. The genome composition of admixed individuals varies much more among than within populations, and tree- and genetic ancestry-based analyses indicate that multiple distinct admixed lineages or populations exist. We find that most genetic variants in Lycaeides are rare (minor allele frequency <0.5%). Because the spatial and taxonomic distributions of alleles reflect demographic and selective processes since mutation, rare alleles, which are presumably younger than common alleles, were spatially and taxonomically restricted compared with common variants. Thus, we show patterns of genetic variation in this group are multifaceted, and we argue that this complexity challenges simplistic notions concerning the organization of biological diversity into discrete, easily delineated and hierarchically structured entities.

Analyses of genetic ancestry enable key insights for molecular ecology.

Gene flow and recombination in admixed populations produce genomes that are mosaic combinations of chromosome segments inherited from different source populations, that is, chromosome segments with different genetic ancestries. The statistical problem of estimating genetic ancestry from DNA sequence data has been widely studied, and analyses of genetic ancestry have facilitated research in molecular ecology and ecological genetics. In this review, we describe and compare different model-based statistical methods used to infer genetic ancestry. We describe the conceptual and mathematical structure of these models and highlight some of their key differences and shared features. We then discuss recent empirical studies that use estimates of genetic ancestry to analyse population histories, the nature and genetic basis of species boundaries, and the genetic architecture of traits. These diverse studies demonstrate the breadth of applications that rely on genetic ancestry estimates and typify the genomics-enabled research that is becoming increasingly common in molecular ecology. We conclude by identifying key research areas where future studies might further advance this field.

A road map for molecular ecology.

The discipline of molecular ecology has undergone enormous changes since the journal bearing its name was launched approximately two decades ago. The field has seen great strides in analytical methods development, made groundbreaking discoveries and experienced a revolution in genotyping technology. Here, we provide brief perspectives on the main subdisciplines of molecular ecology, describe key questions and goals, discuss common challenges, predict future research directions and suggest research priorities for the next 20 years.

Comparative Analysis of Core Microbiome Assignments: Implications for Ecological Synthesis.

The concept of a core microbiome has been broadly used to refer to the consistent presence of a set of taxa across multiple samples within a given habitat. The assignment of taxa to core microbiomes can be performed by several methods based on the abundance and occupancy (i.e., detection across samples) of individual taxa. These approaches have led to methodological inconsistencies, with direct implications for ecological interpretation. Here, we reviewed a set of methods most commonly used to infer core microbiomes in divergent systems. We applied these methods using large data sets and analyzed simulations to determine their accuracy in core microbiome assignments. Our results show that core taxa assignments vary significantly across methods and data set types, with occupancy-based methods most accurately defining true core membership. We also found the ability of these methods to accurately capture core assignments to be contingent on the distribution of taxon abundance and occupancy in the data set. Finally, we provide specific recommendations for further studies using core taxa assignments and discuss the need for unifying methodical approaches toward data processing to advance ecological synthesis. IMPORTANCE Different methods are commonly used to assign core microbiome membership, leading to methodological inconsistencies across studies. In this study, we review a set of the most commonly used core microbiome assignment methods and compare their core assignments using both simulated and empirical data. We report inconsistent classifications from commonly applied core microbiome assignment methods. Furthermore, we demonstrate the implication that variable core assignments may have on downstream ecological interpretations. Although we still lack a standardized approach to core taxa assignments, our study provides a direction to properly test core assignment methods and offers advances in model parameterization and method choice across distinct data types.

Genomic consequences of multiple speciation processes in a stick insect.

Diverse geographical modes and mechanisms of speciation are known, and individual speciation genes have now been identified. Despite this progress, genome-wide outcomes of different evolutionary processes during speciation are less understood. Here, we integrate ecological and spatial information, mating trials, transplantation data and analysis of 86 130 single nucleotide polymorphisms (SNPs) in eight populations (28 pairwise comparisons) of Timema cristinae stick insects to test the effects of different factors on genomic divergence in a system undergoing ecological speciation. We find patterns consistent with effects of numerous factors, including geographical distance, gene flow, divergence in host plant use and climate, and selection against maladaptive hybridization (i.e. reinforcement). For example, the number of highly differentiated 'outlier loci', allele-frequency clines and the overall distribution of genomic differentiation were recognizably affected by these factors. Although host use has strong effects on phenotypic divergence and reproductive isolation, its effects on genomic divergence were subtler and other factors had pronounced effects. The results demonstrate how genomic data can provide new insights into speciation and how genomic divergence can be complex, yet predictable. Future work could adopt experimental, mapping and functional approaches to directly test which genetic regions are affected by selection and determine their physical location in the genome.

Genome-wide association genetics of an adaptive trait in lodgepole pine.

Pine cones that remain closed and retain seeds until fire causes the cones to open (cone serotiny) represent a key adaptive trait in a variety of pine species. In lodgepole pine, there is substantial geographical variation in serotiny across the Rocky Mountain region. This variation in serotiny has evolved as a result of geographically divergent selection, with consequences that extend to forest communities and ecosystems. An understanding of the genetic architecture of this trait is of interest owing to the wide-reaching ecological consequences of serotiny and also because of the repeated evolution of the trait across the genus. Here, we present and utilize an inexpensive and time-effective method for generating population genomic data. The method uses restriction enzymes and PCR amplification to generate a library of fragments that can be sequenced with a high level of multiplexing. We obtained data for more than 95,000 single nucleotide polymorphisms across 98 serotinous and nonserotinous lodgepole pines from three populations. We used a Bayesian generalized linear model (GLM) to test for an association between genotypic variation at these loci and serotiny. The probability of serotiny varied by genotype at 11 loci, and the association between genotype and serotiny at these loci was consistent in each of the three populations of pines. Genetic variation across these 11 loci explained 50% of the phenotypic variation in serotiny. Our results provide a first genome-wide association map of serotiny in pines and demonstrate an inexpensive and efficient method for generating population genomic data.

Ecological outcomes of hybridization vary extensively in Catostomus fishes.

Hybridization outcomes vary geographically and can depend on the environment. Hybridization can also reshape biotic interactions, leading to ecological shifts. If hybrids function differently ecologically in ways that enhance or reduce fitness, and those ecological roles vary geographically, ecological factors might explain variation in hybridization outcomes. However, relatively few studies have focused on ecological traits of hybrids. We compared the feeding ecology of Catostomus fish species and hybrids by using stable isotopes (δ13 C and δ15 N) as a proxy for diet and habitat use, and compared two native species, an introduced species, and three interspecific hybrid crosses. We included hybrids and parental species from seven rivers where hybridization outcomes vary. Relative isotopic niches of native species varied geographically, but native species did not fully overlap in isotopic space in any river sampled, suggesting little overlap of resource use between historically sympatric species. The introduced species overlapped with one or both native species in every river, suggesting similar resource use and potential competition. Hybrids occupied intermediate, matching, or more transgressive isotopic niches, and varied within and among rivers. Ecological outcomes of hybridization varied across locations, implying that hybridization might have unpredictable, idiosyncratic ecological effects.

Whole-Genome Duplication and Host Genotype Affect Rhizosphere Microbial Communities.

The composition of microbial communities found in association with plants is influenced by host phenotype and genotype. However, the ways in which specific genetic architectures of host plants shape microbiomes are unknown. Genome duplication events are common in the evolutionary history of plants and influence many important plant traits, and thus, they may affect associated microbial communities. Using experimentally induced whole-genome duplication (WGD), we tested the effect of WGD on rhizosphere bacterial communities in Arabidopsis thaliana. We performed 16S rRNA amplicon sequencing to characterize differences between microbiomes associated with specific host genetic backgrounds (Columbia versus Landsberg) and ploidy levels (diploid versus tetraploid). We modeled relative abundances of bacterial taxa using a hierarchical Bayesian approach. We found that host genetic background and ploidy level affected rhizosphere community composition. We then tested to what extent microbiomes derived from a specific genetic background or ploidy level affected plant performance by inoculating sterile seedlings with microbial communities harvested from a prior generation. We found a negative effect of the tetraploid Columbia microbiome on growth of all four plant genetic backgrounds. These findings suggest an interplay between host genetic background and ploidy level and bacterial community assembly with potential ramifications for host fitness. Given the prevalence of ploidy-level variation in both wild and managed plant populations, the effects on microbiomes of this aspect of host genetic architecture could be a widespread driver of differences in plant microbiomes. IMPORTANCE Plants influence the composition of their associated microbial communities, yet the underlying host-associated genetic determinants are typically unknown. Genome duplication events are common in the evolutionary history of plants and affect many plant traits. Using Arabidopsis thaliana, we characterized how whole-genome duplication affected the composition of rhizosphere bacterial communities and how bacterial communities associated with two host plant genetic backgrounds and ploidy levels affected subsequent plant growth. We observed an interaction between ploidy level and genetic background that affected both bacterial community composition and function. This research reveals how genome duplication, a widespread genetic feature of both wild and crop plant species, influences bacterial assemblages and affects plant growth.