Homoploid hybrid speciation is challenging to document because hybridization can lead to outcomes other than speciation. Thus, some authors have argued that establishment of homoploid hybrid speciation should include evidence that reproductive barriers isolating the hybrid neo-species from its parental species were derived from hybridization. While this criterion is difficult to satisfy, several recent papers have successfully employed a common pipeline to identify candidate genes underlying such barriers and (in one case) to validate their function. We describe this pipeline, its application to several plant and animal species, and what we have learned about homoploid hybrid speciation as a consequence. We argue that—-given the ubiquity of admixture and the polygenic basis of reproductive isolation—-homoploid hybrid speciation could be much more common and more protracted than suggested by earlier conceptual arguments and theoretical studies.

Organisms inhabiting highly seasonal environments must cope with a wide range of environmentally induced challenges. Many seasonal challenges require extensive physiological modification to survive. In winter, to survive extreme cold and limited resources, insects commonly enter diapause, which is an endogenously derived dormant state associated with minimized cellular processes and low energetic expenditure. Due to the high degree of complexity involved in diapause, substantial cellular regulation is required, of which our understanding primarily derives from the transcriptome via messenger RNA expression dynamics. Here we aim to advance our understanding of diapause by investigating microRNA (miRNA) expression in diapausing and direct developing pupae of the butterfly Pieris napi. We identified coordinated patterns of miRNA expression throughout diapause in both head and abdomen tissues of pupae, and via miRNA target identification, found several expression patterns to be enriched for relevant diapause-related physiological processes. We also identified two candidate miRNAs, miR-14-5p and miR-2a-3p, that are likely involved in diapause progression through the ecdysone synthesis pathway, a critical regulator of diapause termination. miR-14-5p targets phantom, a gene in the ecdysone synthesis pathway, and miR-2a-3p, which has been found to be expressed in response to ecdysone. Together, the expression patterns of these two miRNAs match our current understanding of the timing of hormonal regulation of diapause in P. napi and provide interesting candidates to further explore the mechanistic role of microRNAs in diapause regulation.

The evolution of animals and their gut symbionts is a complex phenomenon, obscured by lability and diversity. In social organisms, transmission of symbionts among relatives may yield systems with more stable associations. Here we study the history of a social insect symbiosis involving cephalotine ants and their extracellular gut bacteria, which come predominantly from host-specialized lineages. We perform multi-locus phylogenetics for symbionts from nine bacterial orders, and map prior amplicon sequence data to lineage-assigned symbiont genomes, studying distributions of rigorously defined symbionts across 20 host species. Based on monophyly and additional hypothesis testing, we estimate that these specialized gut bacteria belong to 18 distinct lineages, of which 15 have been successfully isolated and cultured. Several symbiont lineages showed evidence for domestication events that occurred later in cephalotine evolutionary history, and only one lineage was ubiquitously detected in all 20 host species and 48 colonies sampled with amplicon 16S rRNA sequencing. We found evidence for phylogenetically constrained distributions in four symbionts, suggesting historical or genetic impacts on community composition. Two lineages showed evidence for frequent intra-lineage co-infections, highlighting the potential for niche divergence after initial domestication. Nearly all symbionts showed evidence for occasional host switching, but four may, more often, co-diversify with their hosts. Through our further assessment of symbiont localization and genomic functional profiles, we demonstrate distinct niches for symbionts with shared evolutionary histories, prompting further questions on the forces underlying the evolution of hosts and their gut microbiomes.

Generalist butterflies are characterized by a broad host repertoire that can comprise several families or even different orders of plants. The genetic and physiological mechanisms underlying the use of such a wide host range are still not fully understood. Earlier studies indicate that the consumption of different host plants is associated with host-specific gene expression profiles. It remained, however, unclear if and how larvae can alter these profiles in the case of a changing host environment. Using the polyphagous comma butterfly (Polygonia c-album) we show that larvae can adjust their transcriptional profiles in response to a new host plant. The switch to some of the host plants, however, resulted in a larger transcriptional response and, thus, seems to be more challenging. At a physiological level, no correspondence for these patterns could be found in larval performance. This suggests that a high transcriptional but also phenotypic flexibility are essential for the use of a broad and diverse host range. We furthermore propose that host switch tests in the laboratory followed by transcriptomic investigations can be a valuable tool to examine not only plasticity in host use but also subtle and/or transient trade-offs in the evolution of host plant repertoires. key words: insect-plant association, host plant adaptation, gene expression, phenotypic plasticity

Numerous studies have tested for geographically congruent spatial genetic structures and population units in codistributed species. Yet, few have elucidated the relative importance of biogeographic influences versus ecological interactions in determining the congruence of genetic structure in coevolving species. Here, we present the first study testing for genetic codifferentiation in a widely distributed and highly-coevolved mutualism, in which symbiont gene dispersal is expected to be positively correlated. In the fig, Ficus petiolaris, and its host-specific Pegoscapus pollinating wasp, we evaluated the extent to which geographical patterns of differentiation in each species are similar and explained by shared sources of vicariance, co-dispersal, or species-dependent factors. In both species, the Trans-Mexican Volcanic Belt was a major source of vicariance differentiating southern and northern phylogroups. Within these phylogroups, however, fig and pollinator showed surprisingly different population genetic structure. In F. petiolaris, the Gulf of California was a strong phylogeographic break in the northern phylogroup. In contrast, within its northern phylogroup, Pegoscapus sp. showed no genetic structure and only weak isolation by distance over a 1500 km range. In the southern phylogroup, exceptional genetic differentiation was observed among populations separated by as little as 300 km. Despite mutual selective pressure between figs and fig wasps, and the role of fig wasps in fig gene flow, we conclude that range-wide patterns of genetic differentiation are primarily influenced by biological features unique to each species rather than by shared sources of vicariance or correlated gene dispersal.

The balaenid whale, comprising three species of right whales and the bowhead whale, represents an ancient and highly endangered lineage of marine mammals. To unravel the evolutionary history of balaenid whales with respect to gene flow, a comprehensive analysis based on whole-genome data was conducted for all species within this group. Employing population genomic methodologies, we revealed the polytomic nature of extant right whales, identified passage of historical trans-equatorial migration, and provided estimates to the age of the group. Furthermore, we investigated the impact of glacial cycles on the connectivity of bowhead whale populations. By employing multiple complementary approaches to detect gene flow, we identified and characterized gene flow events from bowhead whales to North Atlantic right whales, offering detailed insights into the process. Lastly, we assessed the phenotypic consequences of interspecies gene flow. The outcomes of our study shed light on the intricate evolutionary history of modern balaenid whales, which have been profoundly shaped by ancient climate events.

Invasive species are a growing global economic and ecological problem. However, it is not well understood how environmental factors mediate invasive range expansion. In this study, we investigated the recent and rapid range expansion of common tansy across environmental gradients in Minnesota, U.S.A. We densely sampled individuals across the expanding range and performed reduced representation sequencing to generate a dataset of 3071 polymorphic loci for 176 individuals. We used non-spatial and spatially-explicit analyses to determine the relative influences of geographic distance and environmental variation on patterns of genomic variation. We found no evidence for isolation-by-distance (IBD) but strong evidence for isolation-by-environment (IBE), indicating that environmental factors may have modulated patterns of range expansion. Land use classification and soils were particularly important variables related to population structure although they operated on different spatial scales; land-use classification was related to broad-scale patterns and soils were related to fine-scale patterns. All analyses indicated a distinctive genetic cluster in the most recently invaded portion of the range. Individuals from the far northwestern range margin were separated from the remainder of the range by reduced migration, which was associated with environmental resistance. This portion of the range was invaded primarily in the last 15 years. Ecological niche models also indicated that this cluster was associated with expansion of the niche. While invasion is often assumed to be primarily influenced by dispersal limitation, our results suggest that ongoing invasion and range shifts with climate change may be strongly affected by environmental heterogeneity.

Priority effects, where the order and timing of species arrival influence the assembly of ecological communities, have been observed in a variety of taxa and habitats. However, the genetic and molecular basis of priority effects remains unclear in most cases, hindering the mechanistic understanding of priority effects. We sought to gain such understanding for the common nectar yeast Metschnikowia reukaufii, which engages in strong priority effects with other species of nectar yeast, including M. rancensis, another species commonly found in the nectar of our study plant, the hummingbird-pollinated Diplacus (Mimulus) aurantiacus. After inoculation into two contrasting types of synthetic nectar simulating early arrival of M. rancensis, we conducted whole-transcriptome sequencing of 108 genetically diverse strains of M. reukaufii. We found that several genes were differentially expressed in M. reukaufii strains when the nectar had been conditioned by growth of M. rancensis. Many of these genes were associated with amino acid metabolism, consistent with our previous finding that early-arriving species limit late-arriving species’ growth by reducing amino acid availability. Furthermore, investigation of expression quantitative trait loci (eQTLs) revealed that genes involved in amino acid transport and resistance to antifungal compounds were enriched in genetic variants, with differing effects on gene expression based on priority effects of M. rancensis. These results demonstrate that intraspecific variation in the ability of nectar yeasts to respond to nutrient limitation and direct fungal competition may underpin the molecular mechanisms of priority effects.

Signals of natural selection can be quickly eroded in high gene-flow systems, severely challenging efforts to understand how and when genetic adaptation occurs in the ocean. This long-standing, unresolved topic in ecology has renewed importance because rapidly changing environmental conditions are driving range expansions that, in many cases, necessitate rapid evolutionary responses. To test for genetic adaptation in a coastal marine species with high dispersal potential, we performed a series of crosses on Kellet’s whelk (Kelletia kelletii) collected from its historical and recently colonized range, and conducted RNA-Seq on offspring that we reared in a common garden environment. We identified 2,770 differentially expressed genes between 54 samples with historical-range and expanded-range ancestry. Using SNPs called directly from the differentially expressed genes, we revealed parental population structure that enabled us to assign “unknown” samples back to their range of origin with unprecedented accuracy for a marine species (92.6 to 94.5%). The SNP with the highest predictive importance occurred on triosephosphate isomerase (TPI), an essential enzyme for glycolysis and glucogenesis, which also plays a role in cold stress response. TPI is both highly upregulated and contains a non-synonymous mutation in the expanded range, where ocean temperatures are colder than in the historical range. Our findings pave the way for accurately identifying patterns of dispersal, gene flow, and population connectivity in the ocean by demonstrating that rapid genetic adaptation can occur even in high gene flow species and that experimental transcriptomics can reveal mechanisms for how marine organisms respond to changing environmental conditions.

Insect immune response plays a crucial role in how external threats influence overall fitness through life history traits. An understudied question is how the use of different host plants might affect the ability of herbivorous insects to resist viral pathogens. The Melissa blue butterfly (Lycaeides melissa) has colonized the exotic legume Medicago sativa as a larval host within the past 200 years. Here we investigate how novel host plant use affects the immune response of L. melissa when infected with the lepidopteran virus, Junonia coenia densovirus (JcDV). We measured immune strength in response to JcDV in two ways: 1) direct measurement of phenoloxidase activity and melanization, and 2) transcriptional sequencing of individuals exposed to different viral and host plant treatments. Viral infection caused total phenoloxidase (total PO) to increase. We detected an interaction between viral infection and host plant for total PO: for control larvae, host plant use had no effect on total PO, whereas for infected larvae, total PO was significantly higher for larvae consuming the native host. Within the exotic host plant treatment, few genes were differentially regulated due to viral infection. Approximately two times more genes were differentially regulated in response to infection for larvae eating the native or exotic host, with differential expression of few putative immune genes. These results demonstrate that consumption of a novel host plant can alter both physiological and transcriptional responses to infection, emphasizing the importance of understanding diet when studying the molecular basis of immune function.

Understanding gene flow can help biodiversity to mitigate habitat changes by contributing to inform and design protected areas. The brown trout, Salmo trutta, displays a multitude of life-history strategies and represents an ideal model for applications in conservation genetics. Using a panel of 185-single nucleotide polymorphism markers, the present study aimed to explore the population structure of the brown trout and in the English Channel. The genotypes of 2,729 individual trout from 88 rivers were obtained across England and France. Population structure revealed the presence of genetic clusters following an east/west gradient. The maximum threshold distance between genetic distance and geographic distance was 344 km. The measure appeared relative to the studied spatial environment and reflected Salmo trutta capacity to achieve long migration distances. A machine-learning framework derived from a gradient forest analysis was used to generate a resistance surface using changes in allelic frequencies and environmental predicators. The resulting surface identified areas limiting gene flow. On the British coast, a genetic break was observed along the Jurassic coast, whereas the Cotentin peninsula acted as a physical barrier among French coastal populations. Salmo trutta populations appeared to be differently affected by environmental factors reflecting demes preference to specific breeding ground. Using our resistance map, the distance of maximum correlation using cost distance were computed allowing the pruning of our genetic graph. The resulting least cost path connections were mapped to reveal the main dispersal routes. Finally, a prioritization analysis using connectivity surface was implemented to design potential protected areas.

Coral populations across the Great Barrier Reef (GBR) could rapidly adapt to the warming climate if they have standing genetic variation for thermal adaptation. Here, we describe a locus likely involved in acclimatization and adaptation of Acropora millepora to cooler temperatures at higher latitudes. This locus shows a strong signal of selection in the A. millepora genome, with a steep latitudinal gradient of derived allele frequency, and harbors a cluster of eight tandemly repeated Δ9-desaturase genes adjacent to a region where a hard sweep likely occurred. In colonies reciprocally transplanted across 4.5 degrees of latitude, the expression of Δ9-desaturase was upregulated at the cooler high-latitude reef. Furthermore, corals from the warmer low-latitude reef with one or two copies of the “cold-adapted” Δ9-desaturase allele expressed the gene more and grew faster than their peers when transplanted to the cooler reef. In other organisms ranging from bacteria to fish, Δ9-desaturase is upregulated under cold conditions to adjust membrane fluidity by introducing double bonds into fatty acid chains of membrane lipids. While all these lines of evidence are suggestive rather than conclusive, they collectively make Δ9-desaturase a strong candidate marker gene for coral thermal acclimatization and adaptation.

Feathers comprise a series of evolutionary innovations but also harbor colour, a key biological trait known to co-vary with life history or complex traits. Those relationships are particularly true in melanin-based pigmentation species due to known pleiotropic effects of the melanocortin pathway – originating from melanin-associated phenotypes. Here we explore the molecular basis of melanin coloration and expected co-variation at the molecular level in the melanin-based, colour polymorphic system of the tawny owl (Strix aluco). An extensive body of literature has revealed that grey and brown tawny owl colour morphs differ in a series of life history and behavioral traits. Thus, it is plausible to expect co-variation also at molecular level between colour morphs. To investigate this possibility, we assembled the first draft genome of the species against which we mapped ddRADseq reads from 220 grey and 150 brown morphs - representing 10 years of pedigree data from a population in Southern Finland - and explored genome-wide associations with colour phenotype. Our results revealed putative molecular signatures of cold adaptation strongly associated with the grey phenotype, namely a non-synonymous substitution in MCHR1, plus 2 substitutions in non-coding regions of FTCD and FAM135A whose genotype combinations obtained a predictive power of up to 100% (predicting grey colour). These suggest molecular basis of cold environment adaptations predicted to be grey-morph specific. Our results potentially reveal part of the molecular machinery of melanin-associated phenotypes and provide novel insights towards understanding the functional genomics of colour polymorphism in melanin-based pigmented species.

Biological invasions have caused the loss of freshwater biodiversity worldwide. The interplay between adaptive responses and demographic characteristics is expected to be important for the resilience of populations to biological invasions, but the interaction between these factors is poorly understood. The native freshwater gastropod Amnicola limosus is distributed along spatial variation in impact from an invasive molluscivorous fish (Neogobius melanostomus), as well as in calcium concentration, which limits the distribution of this invader and thus provides refuges for the gastropods. We investigated if refuge populations could provide migrants to declining invaded gastropod populations through gene flow (i.e., demographic rescue), which could also help maintain genetic diversity (i.e., genetic rescue). We also tested for genetic adaptation of A. limosus to the invasive predator and the low calcium habitats. We conducted pooled whole-genome sequencing of twelve gastropod populations from the Upper St. Lawrence River, complemented with a laboratory reciprocal transplant of wild F0 A. limosus to measure survival and fecundity in treatments of water calcium concentration (low/high) and round goby cue (present/absent). We found that gene flow is restricted from the low-calcium uninvaded refugia towards high-calcium invaded populations, implying that the potential for demographic and genetic rescue is limited. We also detected signatures of divergent selection between habitat types and evidence of low fitness of individuals from refugia populations in both habitat types, which could be either a cause or consequence of the population structure between habitat types and highlights the potential conflict between demographic/genetic rescue and adaptation.

Mitonuclear interactions have been proposed as evolutionary drivers of sexual reproduction, sexual selection, adaptation, and speciation. We investigated the role of pre-mating isolation in maintaining functional mitonuclear interactions in a wild population with divergent sets of proposed co-adapted mitonuclear genotypes. Two lineages have been identified in the eastern yellow robin Eopsaltria australis - putatively climate-adapted to ‘inland’ and ‘coastal’ climates. The lineages differ by ~7% of mitochondrial DNA positions, whereas nuclear genome differences are concentrated into a sex-linked region enriched with mitochondrial genes. This pattern can be explained by female-linked selection accompanied by male-mediated gene flow across the narrow hybrid zone in which the two lineages coexist. It remains unknown whether lineage divergence is driven by intrinsic incompatibilities (particularly in females, under Haldane’s rule), extrinsic incompatibilities, or both. We tested whether non-random mating with respect to partners’ mitolineages or Z-linked variation could facilitate lineage divergence. We used field data, Z-linked and mitolineage genetic markers from two locations where the lineages hybridize, to test whether females choose to mate with (1) males of their own mitolineage and/or bearing similar Z-linked variation, as might be expected if hybrids experience intrinsic incompatibilities, or (2) putatively locally-adapted males, as expected under environmental selection. Comparisons of field observations and simulations present no evidence of non-random mating: the observed reduced female gene flow likely operates via post-mating isolation. Future studies testing for female-biased mortality at different life stages and female habitat selection should clarify the mechanisms of selection.

Natural plant populations often exhibit marked differences in gene expression patterns that can reflect heterogeneity in selective pressures. Analyzing gene expression as a quantitative trait provides a unique opportunity to evaluate the underlying genomic basis of a plethora of traits and their interactions in driving adaptive evolution. We investigated patterns and processes driving expression differentiation under conditions mimicking future climates by combining common garden experiments with transcriptome-wide datasets obtained from hybrid populations of Pinus strobiformis and P. flexilis. We found strong signals of genotype-environment interactions (GEI) at the individual transcript and the co-expression module levels suggesting a marked influence of drought related variables on adaptive evolution. Overall, survival was positively associated with P. flexilis ancestry, but it exhibited an environment-specific pattern. Co-expression modules exhibiting strong associations with survival and genomic ancestry were representative of similar functional categories across both gardens. Using network topology measures, putatively adaptive garden-specific expression traits were pleiotropic and belonged to modules exhibiting high population differentiation yet low preservation across gardens. Overall, our study suggests the presence of substantial genetic variation underlying univariate and multivariate traits in novel climates that may enable populations of long-lived forest trees to respond to rapid shifts in climatic conditions in early seedling stages when mortality tends to be the highest. Our finding of pleiotropic trait architectures underlying adaptive traits, however, implies rapid adaptive responses to changing selection pressures depend on whether trait covariances align with the direction of change in selection pressures.

The olfactory sense is crucial for organisms, facilitating environmental recognition and inter-individual communication. Ithomiini butterflies exemplify this importance not only because they rely strongly on olfactory cues in both inter- and intra-sexual behaviours, but also because they show re-emergence of macro-glomerular complexes (MGCs). These specialized structures within the antennal lobe, lost in butterflies, but present in moths where they enable the integration of information from various types of pheromones, refining responses to specific cues. In this study we present high-quality genome assemblies for four Ithomiini species, investigating chemosensory evolution and associating expression profiles with neuroanatomical differences. We found that antennal transcriptomes across species exhibit profound divergence, indicating distinct species adaptations in environmental sensing. Noteworthy, sexual dimorphism is also characterised both in chemosensory genes and non-chemosensory genes, suggesting their relevance in behaviour. Lipid-related genes exhibit sexual dimorphism, potentially linked to pheromone production or host selection. The study broadens the understanding of antennal chemosensory adaptations in butterflies, highlighting the intricate interplay between genetic diversity, ecological specialization, and sensory perception with neuro-anatomical differences. Insights into chemosensory gene evolution, expression patterns, and potential functional implications enhance our knowledge of sensory adaptations in butterflies, laying the foundation for future investigations into the genetic drivers of behaviour, adaptation, and speciation in insects.

Most dioecious plants are trees. However, because of the difficulty in determining sex from vegetative morphology, previous investigations of the sex ratios of dioecious trees were limited to flowering individuals, leading to inadequate and potentially unreliable data on patterns of sex ratios and the underlying mechanisms driving their variation. Here, we applied sex-specific molecular markers to investigate the sex ratio of a fully mapped population of the dioecious tree Diospyros morrisiana (Ebenaceae) in a subtropical forest. We also investigated the sexual dimorphism of life-history traits and spatial association between male and female trees to determine potential processes shaping the sex ratio at different life stages. Molecular sexing revealed a female-biased population sex ratio for this D. morrisiana population, contrasting with the male-biased operational (i.e., flowering) sex ratio. The sex ratio of D. morrisiana shifted from female-biased to male-biased over older life stages. We found that reproduction had a larger impact on the growth of female trees, which may account for the ontogenetic shift in sex ratio. There was no evidence of spatial segregation of the sexes beyond a scale of 2 m. Through molecular sexing of all individuals across all life stages, our work revealed for the first time a shift from a female- to a male-biased sex ratio in a huge population of a dioecious tree species. To better understand variation in sex ratios and the underlying mechanisms in dioecious trees, the sex of non-flowering and juvenile individuals should be included in future studies.