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.

Insects and recent climate change.

Insects have diversified through more than 450 million y of Earth's changeable climate, yet rapidly shifting patterns of temperature and precipitation now pose novel challenges as they combine with decades of other anthropogenic stressors including the conversion and degradation of land. Here, we consider how insects are responding to recent climate change while summarizing the literature on long-term monitoring of insect populations in the context of climatic fluctuations. Results to date suggest that climate change impacts on insects have the potential to be considerable, even when compared with changes in land use. The importance of climate is illustrated with a case study from the butterflies of Northern California, where we find that population declines have been severe in high-elevation areas removed from the most immediate effects of habitat loss. These results shed light on the complexity of montane-adapted insects responding to changing abiotic conditions. We also consider methodological issues that would improve syntheses of results across long-term insect datasets and highlight directions for future empirical work.

Population genomic evidence of selection on structural variants in a natural hybrid zone.

Structural variants (SVs) can promote speciation by directly causing reproductive isolation or by suppressing recombination across large genomic regions. Whereas examples of each mechanism have been documented, systematic tests of the role of SVs in speciation are lacking. Here, we take advantage of long-read (Oxford nanopore) whole-genome sequencing and a hybrid zone between two Lycaeides butterfly taxa (L. melissa and Jackson Hole Lycaeides) to comprehensively evaluate genome-wide patterns of introgression for SVs and relate these patterns to hypotheses about speciation. We found >100,000 SVs segregating within or between the two hybridizing species. SVs and SNPs exhibited similar levels of genetic differentiation between species, with the exception of inversions, which were more differentiated. We detected credible variation in patterns of introgression among SV loci in the hybrid zone, with 562 of 1419 ancestry-informative SVs exhibiting genomic clines that deviated from null expectations based on genome-average ancestry. Overall, hybrids exhibited a directional shift towards Jackson Hole Lycaeides ancestry at SV loci, consistent with the hypothesis that these loci experienced more selection on average than SNP loci. Surprisingly, we found that deletions, rather than inversions, showed the highest skew towards excess ancestry from Jackson Hole Lycaeides. Excess Jackson Hole Lycaeides ancestry in hybrids was also especially pronounced for Z-linked SVs and inversions containing many genes. In conclusion, our results show that SVs are ubiquitous and suggest that SVs in general, but especially deletions, might disproportionately affect hybrid fitness and thus contribute to reproductive isolation.

Repeated Genetic and Adaptive Phenotypic Divergence across Tidal Elevation in a Foundation Plant Species.

AbstractMicrogeographic genetic divergence can create fine-scale trait variation. When such divergence occurs within foundation species, then it might impact community structure and ecosystem function and cause other cascading ecological effects. We tested for parallel microgeographic trait and genetic divergence in Spartina alterniflora, a foundation species that dominates salt marshes of the US Atlantic and Gulf coasts. Spartina is characterized by tall-form (1-2 m) plants at lower tidal elevations and short-form (<0.5 m) plants at higher tidal elevations, yet whether this trait variation reflects plastic and/or genetically differentiated responses to these environmental conditions remains unclear. In the greenhouse, seedlings raised from tall-form plants grew taller than those from short-form plants, indicating a heritable difference in height. When we reciprocally transplanted seedlings back into the field for a growing season, composite fitness (survivorship and seed production) and key plant traits (plant height and biomass allocation) differed interactively across origin and transplant zones in a manner indicative of local adaptation. Further, a survey of single nucleotide polymorphisms revealed repeated, independent genetic differentiation between tall- and short-form Spartina at five of six tested marshes across the native range. The observed parallel, microgeographic genetic differentiation in Spartina likely underpins marsh health and functioning and provides an underappreciated mechanism that might increase capacity of marshes to adapt to rising sea levels.

Founder effects shape linkage disequilibrium and genomic diversity of a partially clonal invader.

The genomic variation of an invasive species may be affected by complex demographic histories and evolutionary changes during the invasion. Here, we describe the relative influence of bottlenecks, clonality, and population expansion in determining genomic variability of the widespread red macroalga Agarophyton vermiculophyllum. Its introduction from mainland Japan to the estuaries of North America and Europe coincided with shifts from predominantly sexual to partially clonal reproduction and rapid adaptive evolution. A survey of 62,285 SNPs for 351 individuals from 35 populations, aligned to 24 chromosome-length scaffolds indicate that linkage disequilibrium (LD), observed heterozygosity (Ho ), Tajima's D, and nucleotide diversity (Pi) were greater among non-native than native populations. Evolutionary simulations indicate LD and Tajima's D were consistent with a severe population bottleneck. Also, the increased rate of clonal reproduction in the non-native range could not have produced the observed patterns by itself but may have magnified the bottleneck effect on LD. Elevated marker diversity in the genetic source populations could have contributed to the increased Ho and Pi observed in the non-native range. We refined the previous invasion source region to a ~50 km section of northeastern Honshu Island. Outlier detection methods failed to reveal any consistently differentiated loci shared among invaded regions, probably because of the complex A. vermiculophyllum demographic history. Our results reinforce the importance of demographic history, specifically founder effects, in driving genomic variation of invasive populations, even when localized adaptive evolution and reproductive system shifts are observed.

Genetic structure in Louisiana Iris species reveals patterns of recent and historical admixture.

PREMISE: When divergent lineages come into secondary contact, reproductive isolation may be incomplete, thus providing an opportunity to investigate how speciation is manifested in the genome. The Louisiana Irises (Iris, series Hexagonae) comprise a group of three or more ecologically and reproductively divergent lineages that can produce hybrids where they come into contact. We estimated standing genetic variation to understand the current distribution of population structure in the Louisiana Irises. METHODS: We used genotyping-by-sequencing techniques to sample the genomes of Louisiana Iris species across their ranges. We sampled 20 populations (n = 632 individuals) across 11,249 loci and used Entropy and PCA models to assess population genetic data. RESULTS: We discovered evidence for interspecific gene flow in parts of the range. Our analysis revealed patterns of population structure at odds with widely accepted nominal taxonomy. We discovered undescribed hybrid populations, designated as belonging to the I. brevicaulis lineage. Iris nelsonii shared significant ancestry with only one of the purported parent species, I. fulva, evidence inconsistent with a hybrid origin. CONCLUSIONS: This study provides several key findings important to the investigation of standing genetic variation in the Louisiana Iris species complex. Compared to the other nominal species, I. brevicaulis contains a large amount of genetic diversity. In addition, we discovered a previously unknown hybrid zone between I. brevicaulis and I. hexagona along the Texas coast. Finally, our results do not support the long-standing hypothesis that I. nelsonii has mixed ancestry from three parental taxa.

Using RAD-seq to develop sex-linked markers in a haplodiplontic alga.

For many taxa, including isomorphic haplodiplontic macroalgae, determining sex and ploidy is challenging, thereby limiting the scope of some population demographic and genetic studies. Here, we used double-digest restriction site-associated DNA sequencing (ddRAD-seq) to identify sex-linked molecular markers in the widespread red alga Agarophyton vermiculophyllum. In the ddRAD-seq library, we included 10 female gametophytes, 10 male gametophytes, and 16 tetrasporophytes from one native and one non-native site (N = 40 gametophytes and N = 32 tetrasporophytes total). We identified seven putatively female-linked and 19 putatively male-linked sequences. Four female- and eight male-linked markers amplified in all three life cycle stages. Using one female- and one male-linked marker that were sex-specific, we developed a duplex PCR and tested the efficacy of this assay on a subset of thalli sampled at two sites in the non-native range. We confirmed ploidy based on the visual observation of reproductive structures and previous microsatellite genotyping at 10 polymorphic loci. For 32 vegetative thalli, we were able to assign sex and confirm ploidy in these previously genotyped thalli. These markers will be integral to ongoing studies of A. vermiculophyllum invasion. We discuss the utility of RAD-seq over other approaches previously used, such as RAPDs (random amplified polymorphic DNA), for future work designing sex-linked markers in other haplodiplontic macroalgae for which genomes are lacking.

Host plant associations and geography interact to shape diversification in a specialist insect herbivore.

Disentangling the processes underlying geographic and environmental patterns of biodiversity challenges biologists as such patterns emerge from eco-evolutionary processes confounded by spatial autocorrelation among sample units. The herbivorous insect, Belonocnema treatae (Hymenoptera: Cynipidae), exhibits regional specialization on three plant species whose geographic distributions range from sympatry through allopatry across the southern United States. Using range-wide sampling spanning the geographic ranges of the three host plants and genotyping-by-sequencing of 1,217 individuals, we tested whether this insect herbivore exhibited host plant-associated genomic differentiation while controlling for spatial autocorrelation among the 58 sample sites. Population genomic structure based on 40,699 SNPs was evaluated using the hierarchical Bayesian model entropy to assign individuals to genetic clusters and estimate admixture proportions. To control for spatial autocorrelation, distance-based Moran's eigenvector mapping was used to construct regression variables summarizing spatial structure inherent among sample sites. Distance-based redundancy analysis (dbRDA) incorporating the spatial variables was then applied to partition host plant-associated differentiation (HAD) from spatial autocorrelation. By combining entropy and dbRDA to analyse SNP data, we unveiled a complex mosaic of highly structured differentiation within and among gall-former populations finding evidence that geography, HAD and spatial autocorrelation all play significant roles in explaining patterns of genomic differentiation in B. treatae. While dbRDA confirmed host association as a significant predictor of patterns of genomic variation, spatial autocorrelation among sites explained the largest proportion of variation. Our results demonstrate the value of combining dbRDA with hierarchical structural analyses to partition spatial/environmental patterns of genomic variation.

Extreme heterogeneity of population response to climatic variation and the limits of prediction.

Certain general facets of biotic response to climate change, such as shifts in phenology and geographic distribution, are well characterized; however, it is not clear whether the observed similarity of responses across taxa will extend to variation in other population-level processes. We examined population response to climatic variation using long-term incidence data (collected over 42 years) encompassing 149 butterfly species and considerable habitat diversity (10 sites along an elevational gradient from sea level to over 2,700 m in California). Population responses were characterized by extreme heterogeneity that was not attributable to differences in species composition among sites. These results indicate that habitat heterogeneity might be a buffer against climate change and highlight important questions about mechanisms maintaining interpopulation differences in responses to weather. Despite overall heterogeneity of response, population dynamics were accurately predicted by our model for many species at each site. However, the overall correlation between observed and predicted incidence in a cross validation analysis was moderate (Pearson's r = 0.23, SE 0.01), and 97% of observed data fell within the predicted 95% credible intervals. Prediction was most successful for more abundant species as well as for sites with lower annual turnover. Population-level heterogeneity in response to climate variation and the limits of our predictive power highlight the challenges for a future of increasing climatic variability.

Vertical differentiation in tropical forest butterflies: a novel mechanism generating insect diversity?

Many tropical fruit-feeding nymphalid butterflies are associated with either the forest canopy or the understorey; however, the exceptions offer insights into the origins of tropical diversity. As it occurs in both habitats of tropical forests in Ecuador and Peru, Archaeoprepona demophon is one such exception. We compared patterns of occurrence of A. demophon in the canopy and understorey and population genomic variation for evidence of ecological and genetic differentiation between habitats. We found that butterfly occurrences in the canopy were largely uncorrelated with occurrences in the understorey at both localities, indicating independent demographic patterns in the two habitats. We also documented modest, significant genome-level differentiation at both localities. Genetic differentiation between habitat types (separated by approx. 20 m in elevation) was comparable to levels of differentiation between sampling locations (approx. 1500 km). We conclude that canopy and understorey populations of A. demophon represent incipient independent evolutionary units. These findings support the hypothesis that divergence between canopy and understorey-associated populations might be a mechanism generating insect diversity in the tropics.

Population Genomic Evidence Reveals Subtle Patterns of Differentiation in the Trophically Polymorphic Cuatro Ciénegas Cichlid, Herichthys minckleyi.

In recent decades, an increased understanding of molecular ecology has led to a reinterpretation of the role of gene flow during the evolution of reproductive isolation and biological novelty. For example, even in the face of ongoing gene flow strong selection may maintain divergent polymorphisms, or gene flow may introduce novel biological diversity via hybridization and introgression from a divergent species. Herein, we elucidate the evolutionary history and genomic basis of a trophically polymorphic trait in a species of cichlid fish, Herichthys minckleyi. We explored genetic variation at 3 hierarchical levels; between H. minckleyi (n = 69) and a closely related species Herichthys cyanoguttatus (n = 10), between H. minckleyi individuals from 2 geographic locations, and finally between individuals with alternate morphotypes at both a genome-wide and locus-specific scale. We found limited support for the hypothesis that the H. minckleyi polymorphism is the result of ongoing hybridization between the 2 species. Within H. minckleyi we found evidence of geographic genetic structure, and using traditional population genetic analyses found that individuals of alternate morphotypes within a pool appear to be panmictic. However, when we used a locus-specific approach to examine the relationship between multi-locus genotype, tooth size, and geographic sampling, we found the first evidence for molecular genetic differences between the H. minckleyi morphotypes.

Integrating Bayesian genomic cline analyses and association mapping of morphological and ecological traits to dissect reproductive isolation and introgression in a Louisiana Iris hybrid zone.

Hybrid zones provide unique opportunities to examine reproductive isolation and introgression in nature. We utilized 45,384 single nucleotide polymorphism (SNP) loci to perform association mapping of 14 floral, vegetative and ecological traits that differ between Iris hexagona and Iris fulva, and to investigate, using a Bayesian genomic cline (BGC) framework, patterns of genomic introgression in a large and phenotypically diverse hybrid zone in southern Louisiana. Many loci of small effect size were consistently found to be associated with phenotypic variation across all traits, and several individual loci were revealed to influence phenotypic variation across multiple traits. Patterns of genomic introgression were quite heterogeneous throughout the Louisiana Iris genome, with I. hexagona alleles tending to be favoured over those of I. fulva. Loci that were found to have exceptional patterns of introgression were also found to be significantly associated with phenotypic variation in a small number of morphological traits. However, this was the exception rather than the rule, as most loci that were associated with morphological trait variation were not significantly associated with excess ancestry. These findings provide insights into the complexity of the genomic architecture of phenotypic differences and are a first step towards identifying loci that are associated with both trait variation and reproductive isolation in nature.

The predictability of genomic changes underlying a recent host shift in Melissa blue butterflies.

Despite accumulating evidence that evolution can be predictable, studies quantifying the predictability of evolution remain rare. Here, we measured the predictability of genome-wide evolutionary changes associated with a recent host shift in the Melissa blue butterfly (Lycaeides melissa). We asked whether and to what extent genome-wide patterns of evolutionary change in nature could be predicted (i) by comparisons among instances of repeated evolution and (ii) from SNP × performance associations in a laboratory experiment. We delineated the genetic loci (SNPs) most strongly associated with host use in two L. melissa lineages that colonized alfalfa. Whereas most SNPs were strongly associated with host use in none or one of these lineages, we detected a an approximately twofold excess of SNPs associated with host use in both lineages. Similarly, we found that host-associated SNPs in nature could also be partially predicted from SNP × performance (survival and weight) associations in a laboratory rearing experiment. But the extent of overlap, and thus degree of predictability, was somewhat reduced. Although we were able to predict (to a modest extent) the SNPs most strongly associated with host use in nature (in terms of parallelism and from the experiment), we had little to no ability to predict the direction of evolutionary change during the colonization of alfalfa. Our results show that different aspects of evolution associated with recent adaptation can be more or less predictable and highlight how stochastic and deterministic processes interact to drive patterns of genome-wide evolutionary change.

Intraspecific phytochemical variation shapes community and population structure for specialist caterpillars.

Chemically mediated plant-herbivore interactions contribute to the diversity of terrestrial communities and the diversification of plants and insects. While our understanding of the processes affecting community structure and evolutionary diversification has grown, few studies have investigated how trait variation shapes genetic and species diversity simultaneously in a tropical ecosystem. We investigated secondary metabolite variation among subpopulations of a single plant species, Piper kelleyi (Piperaceae), using high-performance liquid chromatography (HPLC), to understand associations between plant phytochemistry and host-specialized caterpillars in the genus Eois (Geometridae: Larentiinae) and associated parasitoid wasps and flies. In addition, we used a genotyping-by-sequencing approach to examine the genetic structure of one abundant caterpillar species, Eois encina, in relation to host phytochemical variation. We found substantive concentration differences among three major secondary metabolites, and these differences in chemistry predicted caterpillar and parasitoid community structure among host plant populations. Furthermore, E. encina populations located at high elevations were genetically different from other populations. They fed on plants containing high concentrations of prenylated benzoic acid. Thus, phytochemistry potentially shapes caterpillar and wasp community composition and geographic variation in species interactions, both of which can contribute to diversification of plants and insects.

Increasing neonicotinoid use and the declining butterfly fauna of lowland California.

The butterfly fauna of lowland Northern California has exhibited a marked decline in recent years that previous studies have attributed in part to altered climatic conditions and changes in land use. Here, we ask if a shift in insecticide use towards neonicotinoids is associated with butterfly declines at four sites in the region that have been monitored for four decades. A negative association between butterfly populations and increasing neonicotinoid application is detectable while controlling for land use and other factors, and appears to be more severe for smaller-bodied species. These results suggest that neonicotinoids could influence non-target insect populations occurring in proximity to application locations, and highlights the need for mechanistic work to complement long-term observational data.

Understanding a migratory species in a changing world: climatic effects and demographic declines in the western monarch revealed by four decades of intensive monitoring.

Migratory animals pose unique challenges for conservation biologists, and we have much to learn about how migratory species respond to drivers of global change. Research has cast doubt on the stability of the eastern monarch butterfly (Danaus plexippus) population in North America, but the western monarchs have not been as intensively examined. Using a Bayesian hierarchical model, sightings of western monarchs over approximately 40 years were investigated using summer flight records from ten sites along an elevational transect in Northern California. Multiple weather variables were examined, including local and regional temperature and precipitation. Population trends from the ten focal sites and a subset of western overwintering sites were compared to summer and overwintering data from the eastern migration. Records showed western overwintering grounds and western breeding grounds had negative trends over time, with declines concentrated early in the breeding season, which were potentially more severe than in the eastern population. Temporal variation in the western monarch also appears to be largely independent of (uncorrelated with) the dynamics in the east. For our focal sites, warmer temperatures had positive effects during winter and spring, and precipitation had a positive effect during spring. These climatic associations add to our understanding of biotic-abiotic interactions in a migratory butterfly, but shifting climatic conditions do not explain the overall, long-term, negative population trajectory observed in our data.

Population genomics reveals a possible history of backcrossing and recombination in the gynogenetic fish Poecilia formosa.

Unisexual sperm-dependent vertebrates are of hybrid origins, rare, and predicted to be short-lived as a result of several challenges arising from their mode of reproduction. In particular, because of a lack of recombination, clonal species are predicted to have a low potential to respond to natural selection. However, many unisexual sperm-dependent species persist, and assessing the genetic diversity present in these species is fundamental to understanding how they avoid extinction. We used population genomic methods to assess genotypic variation within the unisexual fish Poecilia formosa. Measures of admixture and population differentiation, as well as clustering analyses, indicate that the genomes of individuals of P. formosa are admixed and intermediate between Poecilia latipinna and Poecilia mexicana, consistent with the hypothesis of their hybrid origins. Bayesian genomic cline analyses indicate that about 12% of sampled loci exhibit patterns consistent with inheritance from only one parent. The estimation of observed heterozygosity clearly suggests that P. formosa is not comprised of direct descendants of a single nonrecombining asexual F1 hybrid individual. Additionally, the estimation of observed heterozygosity provides support for the hypothesis that the history of this unisexual species has included backcrossing with the parent species before the onset of gynogenesis. We also document high levels of variation among asexual individuals, which is attributable to recombination (historical or ongoing) and the accumulation of mutations. The high genetic variation suggests that this unisexual vertebrate has more potential to respond to natural selection than if they were frozen F1 hybrids.

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.

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.

A hierarchical perspective on the diversity of butterfly species' responses to weather in the Sierra Nevada Mountains.

An important and largely unaddressed issue in studies of biotic-abiotic relationships is the extent to which closely related species, or species living in similar habitats, have similar responses to weather. We addressed this by applying a hierarchical, Bayesian analytical framework to a long-term data set for butterflies which allowed us to simultaneously investigate responses of the entire fauna and individual species. A small number of variables had community-level effects. In particular, higher total annual snow depth had a positive effect on butterfly occurrences, while spring minimum temperature and El Niño-Southern Oscillation (ENSO) sea-surface variables for April-May had negative standardized coefficients. Our most important finding was that variables with large impacts at the community-level did not necessarily have a consistent response across all species. Species-level responses were much more similar to each other for snow depth compared to the other variables with strong community effects. This variation in species-level responses to weather variables raises important complications for the prediction of biotic responses to shifting climatic conditions. In addition, we found that clear associations with weather can be detected when considering ecologically delimited subsets of the community. For example, resident species and non-ruderal species had a much more unified response to weather variables compared to non-resident species and ruderal species, which suggests local adaptation to climate. These results highlight the complexity of biotic-abiotic interactions and confront that complexity with methodological advances that allow ecologists to understand communities and shifting climates while simultaneously revealing species-specific variation in response to climate.