Reference genome for the American rubyspot damselfly, Hetaerina americana.

Damselflies and dragonflies (Order: Odonata) play important roles in both aquatic and terrestrial food webs and can serve as sentinels of ecosystem health and predictors of population trends in other taxa. The habitat requirements and limited dispersal of lotic damselflies make them especially sensitive to habitat loss and fragmentation. As such, landscape genomic studies of these taxa can help focus conservation efforts on watersheds with high levels of genetic diversity, local adaptation, and even cryptic endemism. Here, as part of the California Conservation Genomics Project (CCGP), we report the first reference genome for the American rubyspot damselfly, Hetaerina americana, a species associated with springs, streams and rivers throughout California. Following the CCGP assembly pipeline, we produced two de novo genome assemblies. The primary assembly includes 1,630,044,487 base pairs, with a contig N50 of 5.4 Mb, a scaffold N50 of 86.2 Mb, and a BUSCO completeness score of 97.6%. This is the seventh Odonata genome to be made publicly available and the first for the subfamily Hetaerininae. This reference genome fills an important phylogenetic gap in our understanding of Odonata genome evolution, and provides a genomic resource for a host of interesting ecological, evolutionary, and conservation questions for which the rubyspot damselfly genus Hetaerina is an important model system.

Genomics and ecological modelling clarify species integrity in a confusing group of butterflies.

Recent advances in both genomics and ecological modelling present new, multidisciplinary opportunities for resolving species boundaries and understanding the mechanisms that maintain their integrity in regions of contact. Here, we use a combination of high-throughput DNA sequencing and ecological niche modelling to resolve species boundaries and niche divergence within the Speyeria atlantis-hesperis (Lepidoptera: Nymphalidae) complex, a confusing group of North American butterflies. This complex is notorious for its muddled species delimitations, morphological ambiguity, and extensive mitonuclear discordance. Our admixture and multispecies coalescent-based analyses of single nucleotide polymorphisms identified substantial divergences between S. atlantis and S. hesperis in areas of contact, as well as between distinct northern and southern lineages within S. hesperis. Our results also provide evidence of past introgression relating to another species, S. zerene, which previous work has shown to be more distantly related to the S. atlantis-hesperis complex. We then used ecological models to predict habitat suitability for each of the three recovered genomic lineages in the S. atlantis-hesperis complex and assessed their pairwise niche divergence. These analyses resolved that these three lineages are significantly diverged in their respective niches and are not separated by discontinuities in suitable habitat that might present barriers to gene flow. We therefore infer that ecologically-mediated selection resulting in disparate habitat associations is a principal mechanism reinforcing their genomic integrity. Overall, our results unambiguously support significant evolutionary and ecological divergence between the northern and southern lineages of S. hesperis, sufficient to recognize the southern evolutionary lineage as a distinct species, called S. nausicaa based on taxonomic priority.

Gene flow and climate-associated genetic variation in a vagile habitat specialist.

Previous work in landscape genetics suggests that geographic isolation is of greater importance to genetic divergence than variation in environmental conditions. This is intuitive when configurations of suitable habitat are a dominant factor limiting dispersal and gene flow, but has not been thoroughly examined for habitat specialists with strong dispersal capability. Here, we evaluate the effects of geographic and environmental isolation on genetic divergence for a vagile invertebrate with high habitat specificity and a discrete dispersal life stage: Dod's Old World swallowtail butterfly, Papilio machaon dodi. In Canada, P. m. dodi are generally restricted to eroding habitat along major river valleys where their larval host plant occurs. A series of causal and linear mixed effects models indicate that divergence of genome-wide single nucleotide polymorphisms is best explained by a combination of environmental isolation (variation in summer temperatures) and geographic isolation (Euclidean distance). Interestingly, least-cost path and circuit distances through a resistance surface parameterized as the inverse of habitat suitability were not supported. This suggests that, although habitat associations of many butterflies are specific due to reproductive requirements, habitat suitability and landscape permeability are not equivalent concepts due to considerable adult vagility. We infer that divergent selection related to variation in summer temperatures has produced two genetic clusters within P. m. dodi, differing in voltinism and diapause propensity. Within the next century, temperatures are predicted to rise by amounts greater than the present-day difference between regions of the genetic clusters, potentially affecting the persistence of the northern cluster under continued climate change.

Perceptual Range, Targeting Ability, and Visual Habitat Detection by Greater Fritillary Butterflies Speyeria cybele (Lepidoptera: Nymphalidae) and Speyeria atlantis.

Butterflies are widely invoked as model organisms in studies of metapopulation and dispersal processes. Integral to such investigations are understandings of perceptual range; the maximum distance at which organisms are able to detect patches of suitable habitat. To infer perceptual range, researchers have released butterflies at varying distances from habitat patches and observed their subsequent flight behaviors. It is often assumed that butterflies rely on visual senses for habitat detection; however, this assumption has not been explicitly investigated. Here, we assess the extent and sensory determinants of perceptual range for the great spangled fritillary (Speyeria cybele (Fabricius, 1775)) and Atlantis fritillary (Speyeria atlantis (W.H. Edwards, 1862)). This was achieved by experimentally releasing butterflies over open water at various distances from a lake island, representing an isolated habitat patch in a dichotomous habitat-matrix landscape. To infer whether butterflies rely on vision for habitat detection, we exposed a subset of butterflies to a series of intense light flashes before release to induce flash blindness (bleaching of photoreceptive rhodopsins) without affecting olfaction. Flashed individuals were 30.1 times less likely to successfully navigate to the target island after release, suggesting butterflies rely primarily on visual senses to navigate fragmented landscapes. For unflashed butterflies, the likelihood of successful navigation decreased by a factor of 2.1 for every 10 m increase in release distance. However, no specific distance threshold for perceptual range was observed. We therefore suggest that perceptual range is best viewed as a continuum of probabilities (targeting ability), reflecting the likelihood of habitat detection across a range of distances.

Decoupling habitat fragmentation from habitat loss: butterfly species mobility obscures fragmentation effects in a naturally fragmented landscape of lake islands.

Since the publication of the theory of island biogeography, ecologists have postulated that fragmentation of continuous habitat presents a prominent threat to species diversity. However, negative fragmentation effects may be artifacts; the result of species diversity declining with habitat loss, and habitat loss correlating positively with degree of fragmentation. In this study, we used butterfly assemblages on islands of Lake of the Woods, Ontario, Canada to decouple habitat fragmentation from habitat loss and test two competing hypotheses: (1) the island effect hypothesis, which predicts that decreasing fragment size and increasing fragment isolation reduces species diversity beyond the effects of habitat loss, and (2) the habitat amount hypothesis, which negates fragmentation effects and predicts that only total habitat area determines the diversity of species persisting on fragmented landscapes. Using eight independent size classes of islands (ranging from 0.1 to 8.0 ha) that varied in number of islands while holding total area constant, species diversity comparisons, species accumulation curves, and species-area relationship extrapolations demonstrated that smaller insular habitats contained at least as many butterfly species as continuous habitat. However, when highly mobile species occurring on islands without their larval food plants were excluded from analyses, island effects on potentially reproducing species became apparent. Similarily, generalized linear models suggested that effects of island isolation and vascular plant richness on insular butterfly richness were confounded by species of high mobility. We conclude that inter-fragment movements of highly mobile species may obscure important fragmentation effects on potentially reproducing populations, questioning support for the habitat amount hypothesis.

Moths passing in the night: Phenological and genomic divergences within a forest pest complex.

Temporal separation of reproductive timing can contribute to species diversification both through allochronic speciation and later reinforcement of species boundaries. Such phenological differences are an enigmatic component of evolutionary divergence between two major forest defoliator species of the spruce budworm complex: Choristoneura fumiferana and C. occidentalis. While these species interbreed freely in laboratory settings, natural hybridization rates have not been reliably quantified due to their indistinguishable morphology. To assess whether temporal isolation is contributing to reproductive isolation, we collected adult individuals throughout their expected zone of sympatry in western Canada at 10-day intervals over two successive years, assigning taxonomic identities using thousands of single nucleotide polymorphisms. We found unexpectedly broad sympatry between C. fumiferana and C. occidentalis biennis and substantial overlap of regional flight periods. However, flight period divergence was much more apparent on a location-by-location basis, highlighting the importance of considering spatial scale in these analyses. Phenological comparisons were further complicated by the biennial life cycle of C. o. biennis, the main subspecies of C. occidentalis in the region, and the occasional occurrence of the annually breeding subspecies C. o. occidentalis. Nonetheless, we demonstrate that biennialism is not a likely contributor to reproductive isolation within the species complex. Overall, interspecific F1 hybrids comprised 2.9% of sequenced individuals, confirming the genomic distinctiveness of C. fumiferana and C. occidentalis, while also showing incomplete reproductive isolation of lineages. Finally, we used F ST-based outlier and genotype-environment association analyses to identify several genomic regions under putative divergent selection. These regions were disproportionately located on the Z linkage region of C. fumiferana, and contained genes, particularly antifreeze proteins, that are likely to be associated with overwintering success and diapause. In addition to temporal isolation, we conclude that other mechanisms, including ecologically mediated selection, are contributing to evolutionary divergence within the spruce budworm species complex.

Host association, environment, and geography underlie genomic differentiation in a major forest pest.

Diverse geographic, environmental, and ecological factors affect gene flow and adaptive genomic variation within species. With recent advances in landscape ecological modelling and high-throughput DNA sequencing, it is now possible to effectively quantify and partition their relative contributions. Here, we use landscape genomics to identify determinants of genomic differentiation in the forest tent caterpillar, Malacosoma disstria, a widespread and irruptive pest of numerous deciduous tree species in North America. We collected larvae from multiple populations across Eastern Canada, where the species experiences a diversity of environmental gradients and feeds on a number of different host tree species, including trembling aspen (Populus tremuloides), sugar maple (Acer saccharum), red oak (Quercus rubra), and white birch (Betula papyrifera). Using a combination of reciprocal causal modelling (RCM) and distance-based redundancy analyses (dbRDA), we show that differentiation of thousands of genome-wide single nucleotide polymorphisms (SNPs) among individuals is best explained by a combination of isolation by distance, isolation by environment (spatial variation in summer temperatures and length of the growing season), and differences in host association. Configuration of suitable habitat inferred from ecological niche models was not significantly related to genomic differentiation, suggesting that M. disstria dispersal is agnostic with respect to habitat quality. Although population structure was not discretely related to host association, our modelling framework provides the first molecular evidence of host-associated differentiation in M. disstria, congruent with previous documentation of reduced growth and survival of larvae moved between natal host species. We conclude that ecologically mediated selection is contributing to variation within M. disstria, and that divergent adaptation related to both environmental conditions and host association should be considered in ongoing research and management of this important forest pest.