Heterogeneous genomic differentiation between walking-stick ecotypes: "isolation by adaptation" and multiple roles for divergent selection

Genetic differentiation can be highly variable across the genome. For example, loci under divergent selection and those tightly linked to them may exhibit elevated differentiation compared to neutral regions. These represent "outlier loci" whose differentiation exceeds neutral expectations. Adaptive divergence can also increase genome-wide differentiation by promoting general barriers to neutral gene flow, thereby facilitating genomic divergence via genetic drift. This latter process can yield a positive correlation between adaptive phenotypic divergence and neutral genetic differentiation (described here as "isolation-by-adaptation"). Here, we examine both these processes by combining an AFLP genome scan of two host plant ecotypes of Timema cristinae walking-sticks with existing data on adaptive phenotypic divergence and ecological speciation in these insects. We found that about 8% of loci are outliers in multiple population comparisons. Replicated comparisons between population-pairs using the same versus different host species revealed that 1-2% of loci are subject to host-related selection specifically. Locus-specific analyses revealed that up to 10% of putatively neutral (nonoutlier) AFLP loci exhibit significant isolation-by-adaptation. Our results suggest that selection may affect differentiation directly, via linkage, or by facilitating genetic drift. They thus illustrate the varied and sometimes nonintuitive contributions of selection to heterogeneous genomic differentiation.     

Selection and genomic differentiation during ecological speciation: isolating the contributions of host association via a comparative genome scan of Neochlamisus bebbianae leaf beetles

This study uses a comparative genome scan to evaluate the contributions of host plant related divergent selection to genetic differentiation and ecological speciation in maple- and willow-associated populations of Neochlamisus bebbianae leaf beetles. For each of 15 pairwise population comparisons, we identified "outlier loci" whose strong differentiation putatively reflects divergent selection. Of 447 AFLP loci, 15% were outliers across multiple population comparisons, and low linkage disequilibrium indicated that these outliers derived from multiple regions of the genome. Outliers were further classified as "host-specific" if repeatedly observed in "different-host" population comparisons but never in "same-host" comparisons. Outliers exhibiting the opposite pattern were analogously classified as "host-independent." Host-specific outliers represented 5% of all loci and were more frequent than host-independent outliers, thus revealing a large role for host-adaptation in population genomic differentiation. Evidence that host-related selection can promote divergence despite gene flow was provided by population trees. These were structured by host-association when datasets included host-specific outliers, but not when based on neutral loci, which united sympatric populations. Lastly, three host-specific outliers were highly differentiated in all nine different-host comparisons. Because host-adaptation promotes reproductive isolation in these beetles, these loci provide promising candidate gene regions for future molecular studies of ecological speciation.     

Natural Selection and Neutral Evolution Jointly Drive Population Divergence between Alpine and Lowland Ecotypes of the Allopolyploid Plant Anemone multifida (Ranunculaceae)

Population differentiation can be driven in large part by natural selection, but selectively neutral evolution can play a prominent role in shaping patters of population divergence. The decomposition of the evolutionary history of populations into the relative effects of natural selection and selectively neutral evolution enables an understanding of the causes of population divergence and adaptation. In this study, we examined heterogeneous genomic divergence between alpine and lowland ecotypes of the allopolyploid plant, Anemone multifida. Using peak height and dominant AFLP data, we quantified population differentiation at non-outlier (neutral) and outlier loci to determine the potential contribution of natural selection and selectively neutral evolution to population divergence. We found 13 candidate loci, corresponding to 2.7% of loci, with signatures of divergent natural selection between alpine and lowland populations and between alpine populations (Fst  = 0.074–0.445 at outlier loci), but neutral population differentiation was also evident between alpine populations (F_ST  = 0.041–0.095 at neutral loci). By examining population structure at both neutral and outlier loci, we determined that the combined effects of selection and neutral evolution are associated with the divergence of alpine populations, which may be linked to extreme abiotic conditions and isolation between alpine sites. The presence of outlier levels of genetic variation in structured populations underscores the importance of separately analyzing neutral and outlier loci to infer the relative role of divergent natural selection and neutral evolution in population divergence.     

Selection on outlier loci and their association with adaptive phenotypes in Littorina saxatilis contact zones

A fundamental issue in speciation research is to evaluate phenotypic variation and the genomics driving the evolution of reproductive isolation between sister taxa. Above all, hybrid zones are excellent study systems for researchers to examine the association of genetic differentiation, phenotypic variation and the strength of selection. We investigated two contact zones in the marine gastropod Littorina saxatilis and utilized landmark‐based geometric morphometric analysis together with amplified fragment length polymorphism (AFLP) markers to assess phenotypic and genomic divergence between ecotypes under divergent selection. From genetic markers, we calculated the cline width, linkage disequilibrium and the average effective selection on a locus. Additionally, we conducted an association analysis linking the outlier loci and phenotypic variation between ecotypes and show that a proportion of outlier loci are associated with key adaptive phenotypic traits.     

Evidence for ecological speciation via a host shift in the holly leaf miner,Phytomyza glabricola (Diptera: Agromyzidae)

Evolutionary radiations have been well documented in plants and insects, and natural selection may often underly these radiations. If radiations are adaptive, the diversity of species could be due to ecological speciation in these lineages. Agromyzid flies exhibit patterns of repeated host‐associated radiations. We investigated whether host‐associated population divergence and evidence of divergent selection exist in the leaf miner Phytomyza glabricola on its sympatric host plants, the holly species, Ilex coriacea and I. glabra. Using AFLPs and nuclear sequence data, we found substantial genetic divergence between host‐associated populations of these flies throughout their geographic range. Genome scans using the AFLP data identified 13 loci under divergent selection, consistent with processes of ecological speciation. EF‐1α data suggest that I. glabra is the original host of P. glabricola and that I. coriacea is the novel host, but the AFLP data are ambiguous with regard to directionality of the host shift.     

AFLP genome scans suggest divergent selection on colour patterning in allopatric colour morphs of a cichlid fish

Genome scan-based tests for selection are directly applicable to natural populations to study the genetic and evolutionary mechanisms behind phenotypic differentiation. We conducted AFLP genome scans in three distinct geographic colour morphs of the cichlid fish Tropheus moorii to assess whether the extant, allopatric colour pattern differentiation can be explained by drift and to identify markers mapping to genomic regions possibly involved in colour patterning. The tested morphs occupy adjacent shore sections in southern Lake Tanganyika and are separated from each other by major habitat barriers. The genome scans revealed significant genetic structure between morphs, but a very low proportion of loci fixed for alternative AFLP alleles in different morphs. This high level of polymorphism within morphs suggested that colour pattern differentiation did not result exclusively from neutral processes. Outlier detection methods identified six loci with excess differentiation in the comparison between a bluish and a yellow-blotch morph and five different outlier loci in comparisons of each of these morphs with a red morph. As population expansions and the genetic structure of Tropheus make the outlier approach prone to false-positive signals of selection, we examined the correlation between outlier locus alleles and colour phenotypes in a genetic and phenotypic cline between two morphs. Distributions of allele frequencies at one outlier locus were indeed consistent with linkage to a colour locus. Despite the challenges posed by population structure and demography, our results encourage the cautious application of genome scans to studies of divergent selection in subdivided and recently expanded populations.     

Population‐genomic approach reveals adaptive floral divergence in discrete populations of a hawk moth‐pollinated violet

Local adaptation to contrasting biotic or abiotic environments is an important evolutionary step that presumably precedes floral diversification at the species level, yet few studies have demonstrated the adaptive nature of intraspecific floral divergence in wild plant populations. We combine a population‐genomic approach with phenotypic information on floral traits to examine whether the differentiation in metric floral traits exhibited by 14 populations of the southern Spanish hawk moth‐pollinated violet Viola cazorlensis reflects adaptive divergence. Screening of many amplified fragment length polymorphism (AFLP) loci using a multiple‐marker‐based neutrality test identified nine outlier loci (2.6% of the total) that departed from neutral expectations and were potentially under selection. Generalized analysis of molecular variance revealed significant relationships between genetic distance and population divergence in three floral traits when genetic distance was based on outlier loci, but not when it was based on neutral ones. Population means of floral traits were closely correlated with population scores on the first principal coordinate axis of the genetic distance matrix using outlier loci, and with the allelic frequencies of four of the outlier loci. Results strongly support the adaptive nature of intraspecific floral divergence exhibited by V. cazorlensis and illustrate the potential of genome scans to identify instances of adaptive divergence when used in combination with phenotypic information.     

Analysis of F ST outliers at allozyme loci in Pacific salmon: implications for natural selection

Natural selection has been invoked to explain the observed geographic distribution of allozyme allele frequencies for a number of teleost species. The effects of selection on allozyme loci in three species of Pacific salmon were tested. A simulation-based approach to estimate the null distribution of population differentiation (F _ST) and test for F _ST outliers was used. This approach showed that a majority of allozyme loci conform to neutral expectations predicted by the simulation model, with relatively few F _ST outliers found. No consistent F _ST outlier loci were found across species. Analysis of population sub-groups based on geography and genetic identity reduced the number of outlier loci for some species, indicating that large geographic groups may include genetically divergent populations and/or that there is geographic heterogeneity in selection pressure upon allozyme loci. Two outlier allozyme loci found in this analysis, lactate dehydrogenase-B and malic enzyme, have been shown to be influenced by selection in other teleost species. This approach is also useful in identifying allozyme loci (or other genetic markers) that meet assumptions for population genetic study.     

Isolation by adaptation in Neochlamisus leaf beetles: host-related selection promotes neutral genomic divergence

This study tests how divergent natural selection promotes genomic differentiation during ecological speciation. Specifically, we use adaptive ecological divergence (here, population divergence in host plant use and preference) as a proxy for selection strength and evaluate the correlation between levels of adaptive and genetic differentiation across pairwise population comparisons. Positive correlations would reveal the pattern predicted by our hypothesis, that of 'isolation by adaptation' (IBA). Notably, IBA is predicted not only for selected loci but also for neutral loci. This may reflect the effects of divergent selection on neutral loci that are 'loosely linked' to divergently selected loci or on geneflow restriction that facilitates genetic drift at all loci, including neutral loci that are completely unlinked to those evolving under divergent selection. Here, we evaluate IBA in maple- and willow-associated populations of Neochlamisus bebbianae leaf beetles. To do so, we collected host preference data to construct adaptive divergence indices and used AFLPs (amplified fragment length polymorphisms) and mitochondrial sequences to quantify genetic differentiation. Partial Mantel tests showed significant IBA in 'pooled' analyses of putatively neutral and of putatively selected ('outlier') AFLP loci. This pattern was also recovered in 12% of 'locus-specific' analyses that separately evaluated genetic differentiation at individual neutral loci. These results provided evidence for widespread effects of selection on neutral genomic divergence. Our collective findings indicate that host-related selection may play important roles in the population genomic differentiation of both neutral and selected gene regions in herbivorous insects.     

Identifying insecticide resistance genes in mosquito by combining AFLP genome scans and 454 pyrosequencing

AFLP‐based genome scans are widely used to study the genetics of adaptation and to identify genomic regions potentially under selection. However, this approach usually fails to detect the actual genes or mutations targeted by selection owing to the difficulties of obtaining DNA sequences from AFLP fragments. Here, we combine classical AFLP outlier detection with 454 sequencing of AFLP fragments to obtain sequences from outlier loci. We applied this approach to the study of resistance to Bacillus thuringiensis israelensis (Bti) toxins in the dengue vector Aedes aegypti. A genome scan of Bti‐resistant and Bti‐susceptible A. aegypti laboratory strains was performed based on 432 AFLP markers. Fourteen outliers were detected using two different population genetic algorithms. Out of these, 11 were successfully sequenced. Three contained transposable elements (TEs) sequences, and the 10 outliers that could be mapped at a unique location in the reference genome were located on different supercontigs. One outlier was in the vicinity of a gene coding for an aminopeptidase potentially involved in Bti toxin‐binding. Patterns of sequence variability of this gene showed significant deviation from neutrality in the resistant strain but not in the susceptible strain, even after taking into account the known demographic history of the selected strain. This gene is a promising candidate for future functional analysis.     

Genome‐wide association analyses reveal polygenic genomic architecture underlying divergent shell morphology in Spanish Littorina saxatilis ecotypes

Gene flow between diverging populations experiencing dissimilar ecological conditions can theoretically constrain adaptive evolution. To minimize the effect of gene flow, alleles underlying traits essential for local adaptation are predicted to be located in linked genome regions with reduced recombination. Local reduction in gene flow caused by selection is expected to produce elevated divergence in these regions. The highly divergent crab‐adapted and wave‐adapted ecotypes of the marine snail Littorina saxatilis present a model system to test these predictions. We used genome‐wide association (GWA) analysis of geometric morphometric shell traits associated with microgeographic divergence between the two L. saxatilis ecotypes within three separate sampling sites. A total of 477 snails that had individual geometric morphometric data and individual genotypes at 4,066 single nucleotide polymorphisms (SNPs) were analyzed using GWA methods that corrected for population structure among the three sites. This approach allowed dissection of the genomic architecture of shell shape divergence between ecotypes across a wide geographic range, spanning two glacial lineages. GWA revealed 216 quantitative trait loci (QTL) with shell size or shape differences between ecotypes, with most loci explaining a small proportion of phenotypic variation. We found that QTL were evenly distributed across 17 linkage groups, and exhibited elevated interchromosomal linkage, suggesting a genome‐wide response to divergent selection on shell shape between the two ecotypes. Shell shape trait‐associated loci showed partial overlap with previously identified outlier loci under divergent selection between the two ecotypes, supporting the hypothesis of diversifying selection on these genomic regions. These results suggest that divergence in shell shape between the crab‐adapted and wave‐adapted ecotypes is produced predominantly by a polygenic genomic architecture with positive linkage disequilibrium among loci of small effect.     

Host‐race formation: promoted by phenology,constrained by heritability

Host‐race formation is promoted by genetic trade‐offs in the ability of herbivores to use alternate hosts, including trade‐offs due to differential timing of host‐plant availability. We examined the role of phenology in limiting host‐plant use in the goldenrod gall fly (Eurosta solidaginis) by determining: (1) whether phenology limits alternate host use, leading to a trade‐off that could cause divergent selection on Eurosta emergence time and (2) whether Eurosta has the genetic capacity to respond to such selection in the face of existing environmental variation. Experiments demonstrated that oviposition and gall induction on the alternate host, Solidago canadensis, were the highest on young plants, whereas the highest levels of gall induction on the normal host, Solidago gigantea, occurred on intermediate‐age plants. These findings indicate a phenological trade‐off for host‐plant use that sets up the possibility of divergent selection on emergence time. Heritability, estimated by parent–offspring regression, indicated that host‐race formation is impeded by the amount of genetic variation, relative to environmental, for emergence time.     

The role of local ecology during hybridization at the initial stages of ecological speciation in a marine snail

Hybrid zones of ecologically divergent populations are ideal systems to study the interaction between natural selection and gene flow during the initial stages of speciation. Here, we perform an amplified fragment length polymorphism (AFLP) genome scan in parallel hybrid zones between divergent ecotypes of the marine snail Littorina saxatilis, which is considered a model case for the study of ecological speciation. Ridged‐Banded (RB) and Smooth‐Unbanded (SU) ecotypes are adapted to different shore levels and microhabitats, although they present a sympatric distribution at the mid‐shore where they meet and mate (partially assortatively). We used shell morphology, outlier and nonoutlier AFLP loci from RB, SU and hybrid specimens captured in sympatry to determine the level of phenotypic and genetic introgression. We found different levels of introgression at parallel hybrid zones and nonoutlier loci showed more gene flow with greater phenotypic introgression. These results were independent from the phylogeography of the studied populations, but not from the local ecological conditions. Genetic variation at outlier loci was highly correlated with phenotypic variation. In addition, we used the relationship between genetic and phenotypic variation to estimate the heritability of morphological traits and to identify potential Quantitative Trait Loci to be confirmed in future crosses. These results suggest that ecology (exogenous selection) plays an important role in this hybrid zone. Thus, ecologically based divergent natural selection is responsible, simultaneously, for both ecotype divergence and hybridization. On the other hand, genetic introgression occurs only at neutral loci (nonoutliers). In the future, genome‐wide studies and controlled crosses would give more valuable information about this process of speciation in the face of gene flow.     

Scanning the European corn borer (Ostrinia spp.) genome for adaptive divergence between host-affiliated sibling species

It has recently been shown that the European corn borer, a major pest of maize crops, is actually composed of two genetically differentiated and reproductively isolated taxa, which are found in sympatry over a wide geographical range in Eurasia. Each taxon is adapted to specific host plants: Ostrinia nubilalis feeds mainly on maize, while O. scapulalis feeds mainly on hop or mugwort. Here, we present a genome scan approach as a first step towards an integrated molecular analysis of the adaptive genomic divergence between O. nubilalis and O. scapulalis. We analysed 609 AFLP marker loci in replicate samples of sympatric populations of Ostrinia spp. collected on maize, hop and mugwort, in France. Using two genome scan methods based on the analysis of population differentiation, we found a set of 28 outlier loci that departed from the neutral expectation in one or the other method (of which a subset of 14 loci were common to both methods), which showed a significantly increased differentiation between O. nubilalis and O. scapulalis, when compared to the rest of the genome. A subset of 12 outlier loci were sequenced, of which 7 were successfully re‐amplified as target candidate loci. Three of these showed homology with annotated lepidopteran sequences from public nucleotide databases.     

Climatic adaptation and ecological divergence between two closely related pine species in Southeast China

Climate is one of the most important drivers for adaptive evolution in forest trees. Climatic selection contributes greatly to local adaptation and intraspecific differentiation, but this kind of selection could also have promoted interspecific divergence through ecological speciation. To test this hypothesis, we examined intra‐ and interspecific genetic variation at 25 climate‐related candidate genes and 12 reference loci in two closely related pine species, Pinus massoniana Lamb. and Pinus hwangshanensis Hisa, using population genetic and landscape genetic approaches. These two species occur in Southeast China but have contrasting ecological preferences in terms of several environmental variables, notably altitude, although hybrids form where their distributions overlap. One or more robust tests detected signals of recent and/or ancient selection at two‐thirds (17) of the 25 candidate genes, at varying evolutionary timescales, but only three of the 12 reference loci. The signals of recent selection were species specific, but signals of ancient selection were mostly shared by the two species likely because of the shared evolutionary history. F_ST outlier analysis identified six SNPs in five climate‐related candidate genes under divergent selection between the two species. In addition, a total of 24 candidate SNPs representing nine candidate genes showed significant correlation with altitudinal divergence in the two species based on the covariance matrix of population history derived from reference SNPs. Genetic differentiation between these two species was higher at the candidate genes than at the reference loci. Moreover, analysis using the isolation‐with‐migration model indicated that gene flow between the species has been more restricted for climate‐related candidate genes than the reference loci, in both directions. Taken together, our results suggest that species‐specific and divergent climatic selection at the candidate genes might have counteracted interspecific gene flow and played a key role in the ecological divergence of these two closely related pine species.     

Outlier loci highlight the direction of introgression in oaks

Loci considered to be under selection are generally avoided in attempts to infer past demographic processes as they do not fit neutral model assumptions. However, opportunities to better reconstruct some aspects of past demography might thus be missed. Here we examined genetic differentiation between two sympatric European oak species with contrasting ecological dynamics (Quercus robur and Quercus petraea) with both outlier (i.e. loci possibly affected by divergent selection between species or by hitchhiking effects with genomic regions under selection) and nonoutlier loci. We sampled 855 individuals in six mixed forests in France and genotyped them with a set of 262 SNPs enriched with markers showing high interspecific differentiation, resulting in accurate species delimitation. We identified between 13 and 74 interspecific outlier loci, depending on the coalescent simulation models and parameters used. Greater genetic diversity was predicted in Q. petraea (a late‐successional species) than in Q. robur (an early successional species) as introgression should theoretically occur predominantly from the resident species to the invading species. Remarkably, this prediction was verified with outlier loci but not with nonoutlier loci. We suggest that the lower effective interspecific gene flow at loci showing high interspecific divergence has better preserved the signal of past asymmetric introgression towards Q. petraea caused by the species' contrasting dynamics. Using markers under selection to reconstruct past demographic processes could therefore have broader potential than generally recognized.     

Differential introgression from a sister species explains high FST outlier loci within a mussel species

Scanning genomes for loci with high levels of population differentiation has become a standard of population genetics. F_ST outlier loci are most often interpreted as signatures of local selection, but outliers might arise for many other reasons too often left unexplored. Here, we tried to identify further the history and genetic basis underlying strong differentiation at F_ST outlier loci in a marine mussel. A genome scan of genetic differentiation has been conducted between Atlantic and Mediterranean populations of Mytilus galloprovincialis. The differentiation was low overall (F_ST = 0.03), but seven loci (2%) were strong F_ST outliers. We then analysed DNA sequence polymorphism at two outlier loci. The genetic structure proved to be the consequence of differential introgression of alleles from the sister‐hybridizing species Mytilus edulis. Surprisingly, the Mediterranean population was the most introgressed at these two loci, although the contact zone between the two species is nowadays localized along the Atlantic coasts of France and the British Isles. A historical contact between M. edulis and Mediterranean M. galloprovincialis should have happened during glacial periods. It proved difficult to disentangle two hypotheses: (i) introgression was adaptive, implying edulis alleles have been favoured in Mediterranean populations, or (ii) the genetic architecture of the barrier to edulis gene flow is different between the two M. galloprovincialis backgrounds. Five of the seven outliers between M. galloprovincialis populations were also outliers between M. edulis and Atlantic M. galloprovincialis, which would support the latter hypothesis. Differential introgression across semi‐permeable barriers to gene flow is a neglected scenario to interpret outlying loci that may prove more widespread than anticipated.     

Genetic evidence for ecological divergence in kokanee salmon

The evolution of locally adapted phenotypes among populations that experience divergent selective pressures is a central mechanism for generating and maintaining biodiversity. Recently, the advent of high‐throughput DNA sequencing technology has provided tools for investigating the genetic basis of this process in natural populations of nonmodel organisms. Kokanee, the freshwater form of sockeye salmon (Oncorhynchus nerka), occurs as two reproductive ecotypes, which differ in spawning habitat (tributaries vs. shorelines); however, outside of the spawning season the two ecotypes co‐occur in many lakes and lack diagnostic morphological characteristics. We used restriction site‐associated DNA (RAD) sequencing to identify 6145 SNPs and genotype kokanee from multiple spawning sites in Okanagan Lake (British Columbia, Canada). Outlier tests revealed 18 loci putatively under divergent selection between ecotypes, all of which exhibited temporally stable allele frequencies within ecotypes. Six outliers were annotated to sequences in the NCBI database, two of which matched genes associated with early development. There was no evidence for neutral genetic differentiation; however, outlier loci demonstrated significant structure with respect to ecotype and had high assignment accuracy in mixed composition simulations. The absence of neutral structure combined with a small number of highly divergent outlier loci is consistent with theoretical predictions for the early stages of ecological divergence. These outlier loci were then applied to a realistic fisheries scenario in which additional RAD sequencing was used to genotype kokanee collected by trawl in Okanagan Lake, providing preliminary evidence that this approach may be an effective tool for conservation and management.     

Genomic scanning using AFLP to detect loci under selection in the moss Funaria hygrometrica along a climate gradient in the Sierra Nevada Mountains,Spain

The common cord moss Funaria hygrometrica has a worldwide distribution and thrives in a wide variety of environments. Here, we studied the genetic diversity in F. hygrometrica along an abiotic gradient in the Mediterranean high mountain of Sierra Nevada (Spain) using a genome scan method. Eighty‐four samples from 17 locations from 24 to 2700 m were fingerprinted based on their amplified fragment length polymorphism (AFLP) banding pattern. Using PCA and Bayesian inference we found that the genetic diversity was structured in three or four clusters, respectively. Using a genome scan method we identified 13 outlier loci, which showed a signature of positive selection. Partial Mantel tests were performed between the Euclidean distance matrices of geographic and climatic variables, versus the pair‐wise genetic distance of the AFLP dataset and AFLP‐positive outliers dataset. AFLP‐positive outlier data were significantly correlated with the gradient of the climatic variables, suggesting adaptive variation among populations of F. hygrometrica along the Sierra Nevada Mountains. We highlight the additional analyses necessary to identify the nature of these loci, and their biological role in the adaptation process.     

Adaptive differentiation in floral traits in the presence of high gene flow in scarlet gilia (Ipomopsis aggregata)

Plant–pollinator interactions are thought to be major drivers of floral trait diversity. However, the relative importance of divergent pollinator‐mediated selection vs. neutral processes in floral character evolution has rarely been explored. We tested for adaptive floral trait evolution by comparing differentiation at neutral genetic loci to differentiation at quantitative floral traits in a putative Ipomopsis aggregata hybrid zone. Typical I. aggregata subsp. candida displays slender white tubular flowers that are typical of flowers pollinated by hawkmoths, and subsp. collina displays robust red tubular flowers typical of flowers pollinated by hummingbirds; yet, hybrid flower morphs are abundant across the East Slope of the Colorado Rockies. We estimated genetic differentiation (F_ST) for nuclear and chloroplast microsatellite loci and used a half‐sib design to calculate quantitative trait divergence (Q_ST) from collection sites across the morphological hybrid zone. We found little evidence for population structure and estimated mean F_ST to be 0.032. Q_ST values for several floral traits including corolla tube length and width, colour, and nectar volume were large and significantly greater than mean F_ST. We performed multivariate comparisons of neutral loci to genetic correlations within and between populations and found a strong signal for divergent selection, suggesting that specific combinations of floral display and reward traits may be the targets of selection. Our results show little support for historical subspecies categories, yet floral traits are more diverged than expected due to drift alone. Non‐neutral divergence for multivariate quantitative traits suggests that selection by pollinators is maintaining a correlation between display and reward traits.