The Arsenault Lab

Most ant species have two distinct female castes—queens and workers—yet the developmental and genetic mechanisms that produce these alternative phenotypes remain poorly understood. Working with a clonal ant, we discovered a variant strain that expresses queen-like traits in individuals that would normally become workers. The variants show changes in morphology, behavior, and fitness that cause them to rely on workers in wild-type (WT) colonies for survival. Overall, they resemble the queens of many obligately parasitic ants that have evolutionarily lost the worker caste and live inside colonies of closely related hosts. The prevailing theory for the evolution of these workerless social parasites is that they evolve from reproductively isolated populations of facultative intermediates that acquire parasitic phenotypes in a stepwise fashion. However, empirical evidence for such facultative ancestors remains weak, and it is unclear how reproductive isolation could gradually arise in sympatry. In contrast, we isolated these variants just a few generations after they arose within their WT parent colony, implying that the complex phenotype reported here was induced in a single genetic step. This suggests that a single genetic module can decouple the coordinated mechanisms of caste development, allowing an obligately parasitic variant to arise directly from a free-living ancestor. Consistent with this hypothesis, the variants have lost one of the two alleles of a putative supergene that is heterozygous in WTs. These findings provide a plausible explanation for the evolution of ant social parasites and implicate new candidate molecular mechanisms for ant caste differentiation.

Indirect genetic effects describe phenotypic variation that results from differences in the genotypic composition of social partners. Such effects represent heritable sources of environmental variation in eusocial organisms because individuals are typically reared by their siblings. In the fire ant Solenopsis invicta, a social supergene exhibits striking indirect genetic effects on worker regulation of colony queen number, such that the genotypic composition of workers at the supergene determines whether colonies contain a single or multiple queens. We assessed the direct and indirect genetic effects of this supergene on gene expression in brains and abdominal tissues from lab-reared workers and compared these with previously published data from field-collected pre-reproductive queens. We found that direct genetic effects caused larger gene expression changes and were more consistent across tissue types and castes than indirect genetic effects. Indirect genetic effects influenced the expression of many loci but were generally restricted to the abdominal tissues. Further, indirect genetic effects were only detected when the genotypic composition of social partners differed throughout the development and adult life of focal workers, and were often only significant with relatively lenient statistical cutoffs. Our study provides insight into direct and indirect genetic effects of a social supergene on gene regulatory dynamics across tissues and castes in a complex society.

The fire ant Solenopsis invicta exists in two alternate social forms: monogyne nests contain a single reproductive queen and polygyne nests contain multiple reproduc- tive queens. This colony-level social polymorphism corresponds with individual dif- ferences in queen physiology, queen dispersal patterns and worker discrimination behaviours, all evidently regulated by an inversion-based supergene that spans more than 13 Mb of a “social chromosome,” contains over 400 protein-coding genes and rarely undergoes recombination. The specific mechanisms by which this supergene influences expression of the many distinctive features that characterize the alternate forms remain almost wholly unknown. To advance our understanding of these mech- anisms, we explore the effects of social chromosome genotype and natal colony so- cial form on gene expression in queens sampled as they embarked on nuptial flights, using RNA-sequencing of brains and ovaries. We observe a large effect of natal social form, that is, of the social/developmental environment, on gene expression profiles, with similarly substantial effects of genotype, including: (a) supergene-associated gene upregulation, (b) allele-specific expression and (c) pronounced extra-supergene trans-regulatory effects. These findings, along with observed spatial variation in dif- ferential and allele-specific expression within the supergene region, highlight the complex gene regulatory landscape that emerged following divergence of the inver- sion-mediated Sb haplotype from its homologue, which presumably largely retained the ancestral gene order. The distinctive supergene-associated gene expression tra- jectories we document at the onset of a queen’s reproductive life expand the known record of relevant molecular correlates of a complex social polymorphism and point to putative genetic factors underpinning the alternate social syndromes.

Supergenes are clusters of linked genetic loci that jointly affect the expression of complex phenotypes, such as social organization. Little is known about the origin and evolution of these intriguing genomic elements. Here we analyse whole-genome sequences of males from native populations of six fire ant species and show that variation in social organization is under the control of a novel supergene haplotype (termed Sb), which evolved by sequential incorporation of three inversions spanning half of a ‘social chromosome’. Two of the inversions interrupt protein-coding genes, resulting in the increased expression of one gene and modest truncation in the primary protein structure of another. All six socially polymorphic species studied harbour the same three inversions, with the single origin of the supergene in their common ancestor inferred by phylogenomic analyses to have occurred half a million years ago. The persistence of Sb along with the ancestral SB haplotype through multiple speciation events provides a striking example of a functionally important trans-species social polymorphism presumably maintained by balancing selection. We found that while recombination between the Sb and SB haplotypes is severely restricted in all species, a low level of gene flux between the haplotypes has occurred following the appearance of the inversions, potentially mitigating the evolutionary degeneration expected at genomic regions that cannot freely recombine. These results provide a detailed picture of the structural genomic innovations involved in the formation of a supergene controlling a complex social phenotype.

The study of the major transition to eusociality presents several challenges to researchers, largely resulting from the importance of complex behavioral phenotypes and the shift from individual to group level selection. These challenges are being met with corresponding technological improvements. Advances in resource development for non-model taxa, behavioral tracking, nucleic acid sequencing, and reverse genetics are facilitating studies of hypotheses that were previously intractable. These innovations are resulting in the development of new model systems tailored to the exploration of specific behavioral phenotypes and the querying of underlying molecular mechanisms that drive eusocial behaviors. Here, we present a brief overview of how methodological innovations are advancing our understanding of the evolution of eusociality.

Developmental plasticity describes the influence of environmental factors on phenotypic variation. An important mediator of developmental plasticity in many animals is parental care. Here, we examine the consequences of maternal care on offspring after the initial mass provisioning of brood in the small carpenter bee, Ceratina calcarata. Removal of the mother during larval development leads to increased aggression and avoidance in adulthood. This corresponds with changes in expression of over one thousand genes, alternative splicing of hundreds of genes, and significant changes to DNA methylation. We identify genes related to metabolic and neuronal functions that may influence developmental plasticity and aggression. We observe no genome-wide association between differential DNA methylation and differential gene expression or splicing, though indirect relationships may exist between these factors. Our results provide insight into the gene regulatory context of DNA methylation in insects and the molecular avenues through which variation in maternal care influences developmental plasticity.

Changes in gene regulation that underlie phenotypic evolution can be encoded directly in the DNA sequence or mediated by chromatin modifications such as DNA methylation. It has been hypothesized that the evolution of eusocial division of labor is associated with enhanced gene regulatory potential, which may include expansions in DNA methylation in the genomes of Hymenoptera (bees, ants, wasps, and sawflies). Recently, this hypothesis garnered support from analyses of a commonly used metric to estimate DNA methylation in silico, CpG content. Here, we test this hypothesis using direct, nucleotide-level measures of DNAmethylationacross ninespeciesofHymenoptera. In doing so,wegeneratednewDNAmethylomes for three speciesof interest, including one solitary and one facultatively eusocial halictid bee and a sawfly. We demonstrate that the strength of correlation between CpG content and DNA methylation varies widely among hymenopteran taxa, highlighting shortcomings in the utility of CpG content as a proxy for DNA methylation in comparative studies of taxa with sparse DNAmethylomes. We observed strikingly high levels of DNAmethylation in the sawfly relative to other investigated hymenopterans. Analyses of molecular evolution suggest the relatively distinct sawfly DNAmethylome may be associatedwith positive selection on functional DNMT3domains. Sawflies are an outgroup to all ants, bees, and wasps, and no sawfly species are eusocial. We find no evidence that either global expansions or variation within individual ortholog groups in DNA methylation are consistently associated with the evolution of social behavior.