Iterative evolution of supergene-based social polymorphism in ants.

Species commonly exhibit alternative morphs, with individual fate being determined during development by either genetic factors, environmental cues or a combination thereof. Ants offer an interesting case study because many species are polymorphic in their social structure. Some colonies contain one queen while others contain many queens. This variation in queen number is generally associated with a suite of phenotypic and life-history traits, including mode of colony founding, queen lifespan, queen-worker dimorphism and colony size. The basis of this social polymorphism has been studied in five ant lineages, and remarkably social morph seems to be determined by a supergene in all cases. These 'social supergenes' tend to be large, having formed through serial inversions, and to comprise hundreds of linked genes. They have persisted over long evolutionary timescales, in multiple lineages following speciation events, and have spread between closely related species via introgression. Their evolutionary dynamics are unusually complex, combining recessive lethality, spatially variable selection, selfish genetic elements and non-random mating. Here, we synthesize the five cases of supergene-based social polymorphism in ants, highlighting interesting commonalities, idiosyncrasies and implications for the evolution of polymorphisms in general. This article is part of the theme issue 'Genomic architecture of supergenes: causes and evolutionary consequences'.

Characterization of Queen Supergene Pheromone in the Red Imported Fire Ant Using Worker Discrimination Assays.

Ants use chemical signals to communicate for various purposes related to colony function. Social organization in the red imported fire ant, Solenopsis invicta, is determined by the Sb supergene, with colonies of the monogyne (single-queen) form lacking the element and colonies of the polygyne (multiple-queen) form possessing it. Polygyne workers accept new reproductive queens in their nest, but only those carrying Sb; young winged queens lacking this genetic element are executed as they mature sexually in their natal nest or as they attempt to enter a foreign nest to initiate reproduction after mating and shedding their wings. It has been suggested that queen supergene genotype status is signaled to workers by unsaturated cuticular hydrocarbons, while queen reproductive status is signaled by piperidines (venom alkaloids). We used high-throughput behavioral assays to study worker acceptance of paper dummies dosed with fractions of extracts of polygyne queens, or blends of synthetic counterparts of queen cuticular compounds. We show that the queen supergene pheromone comprises a blend of monoene and diene unsaturated hydrocarbons. Our assays also reveal that unsaturated hydrocarbons elicit discrimination by polygyne workers only when associated with additional compounds that signal queen fertility. This synergistic effect was obtained with a polar fraction of queen extracts, but not by the piperidine alkaloids, suggesting that the chemical(s) indicating queen reproductive status are compounds more polar than cuticular hydrocarbons but are not the piperidine alkaloids. Our results advance understanding of the role of chemical signaling that is central to the regulation of social organization in an important invasive pest and model ant species.

Multi-level social organization and nest-drifting behaviour in a eusocial insect.

Stable social groups usually consist of families. However, recent studies have revealed higher level social structure, with interactions between family groups across different levels of social organization in multiple species. The explanations for why this apparently paradoxical behaviour arises appear to be varied and remain untested. Here, we use automated radio-tagging data from over 1000 wasps from 93 nests and social network analyses of over 30 000 nest visitation records to describe and explain interactions across levels of social organization in the eusocial paper wasp Polistes canadensis. We detected three levels of social organization (nest, aggregation and community) which exchange 'drifter' individuals within and between levels. The highest level (community) may be influenced by the patchiness of high-quality nesting habitats in which these insects exist. Networks of drifter movements were explained by the distance between nests, the group size of donor nests and the worker-to-brood ratios on donor and recipient nests. These findings provide some explanation for the multi-level social interactions, which may otherwise seem paradoxical. Fitness benefits across multiple levels of social organization should be considered when trying to understand animal societies.

Evolution of a supergene that regulates a trans-species social polymorphism.

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.

Doublesex Evolution Is Correlated with Social Complexity in Ants.

The Dmrt (doublesex and mab-3-related transcription factor) genes are transcription factors crucial for sex determination and sexual differentiation. In some social insects, doublesex (dsx) exhibits widespread caste-specific expression across different tissues and developmental stages and has been suggested as a candidate gene for regulating division of labor in social insects. We therefore conducted a molecular evolution analysis of the Dmrt gene family in 20 ants. We found that the insect-specific oligomerization domain of DSX, oligomerization domain 2, was absent in all ants, except for the two phylogenetically basal ant species (Ponerinae), whose social structure and organization resemble the presumed ancestral condition in ants. Phylogenetic reconstruction and selection analysis revealed that dsx evolved faster than the other three members of the Dmrt family. We found evidence for positive selection for dsx in the ant subfamilies with more advanced social organization (Myrmicinae and Formicinae), but not in the Ponerinae. Furthermore, we detected expression of two Dmrt genes, dsx and DMRT11E, in adult ants, and found a clear male-biased expression pattern of dsx in most species for which data are available. Interestingly, we did not detect male-biased expression of dsx in the two ant species that possess a genetic caste determination system. These results possibly suggest an association between the evolution of dsx and social organization as well as reproductive division of labor in ants.

Genetics and Evolution of Social Behavior in Insects.

The study of insect social behavior has offered tremendous insight into the molecular mechanisms mediating behavioral and phenotypic plasticity. Genomic applications to the study of eusocial insect species, in particular, have led to several hypotheses for the processes underlying the molecular evolution of behavior. Advances in understanding the genetic control of social organization have also been made, suggesting an important role for supergenes in the evolution of divergent behavioral phenotypes. Intensive study of social phenotypes across species has revealed that behavior and caste are controlled by an interaction between genetic and environmentally mediated effects and, further, that gene expression and regulation mediate plastic responses to environmental signals. However, several key methodological flaws that are hindering progress in the study of insect social behavior remain. After reviewing the current state of knowledge, we outline ongoing challenges in experimental design that remain to be overcome in order to advance the field.

Social chromosome variants differentially affect queen determination and the survival of workers in the fire ant Solenopsis invicta.

Intraspecific variation in social organization is common, yet the underlying causes are rarely known. An exception is the fire ant Solenopsis invicta in which the existence of two distinct forms of social colony organization is under the control of the two variants of a pair of social chromosomes, SB and Sb. Colonies containing exclusively SB/SB workers accept only one single queen and she must be SB/SB. By contrast, when colonies contain more than 10% of SB/Sb workers, they accept several queens but only SB/Sb queens. The variants of the social chromosome are associated with several additional important phenotypic differences, including the size, fecundity and dispersal strategies of queens, aggressiveness of workers, and sperm count in males. However, little is known about whether social chromosome variants affect fitness in other life stages. Here, we perform experiments to determine whether differential selection occurs during development and in adult workers. We find evidence that the Sb variant of the social chromosome increases the likelihood of female brood to develop into queens and that adult SB/Sb workers, the workers that cull SB/SB queens, are overrepresented in comparison to SB/SB workers. This demonstrates that supergenes such as the social chromosome can have complex effects on phenotypes at various stages of development.

Ant genomics sheds light on the molecular regulation of social organization.

Ants are powerful model systems for the study of cooperation and sociality. In this review, we discuss how recent advances in ant genomics have contributed to our understanding of the evolution and organization of insect societies at the molecular level.

A simple genetic basis for complex social behaviour mediates widespread gene expression differences.

A remarkable social polymorphism is controlled by a single Mendelian factor in the fire ant Solenopsis invicta. A genomic element marked by the gene Gp-9 determines whether workers tolerate one or many fertile queens in their colony. Gp-9 was recently shown to be part of a supergene with two nonrecombining variants, SB and Sb. SB/SB and SB/Sb queens differ in how they initiate new colonies, and in many physiological traits, for example odour and maturation rate. To understand how a single genetic element can affect all these traits, we used a microarray to compare gene expression patterns between SB/SB and SB/Sb queens of three different age classes: 1-day-old unmated queens, 11-day-old unmated queens and mated, fully reproductive queens collected from mature field colonies. The number of genes that were differentially expressed between SB/SB and SB/Sb queens of the same age class was smallest in 1-day-old queens, maximal in 11-day-old queens and intermediate in reproductive queens. Gene ontology analysis showed that SB/SB queens upregulate reproductive genes faster than SB/Sb queens. For all age classes, genes inside the supergene were overrepresented among the differentially expressed genes. Consistent with the hypothesized greater number of transposons in the Sb supergene, 13 transposon genes were upregulated in SB/Sb queens. Viral genes were also upregulated in SB/Sb mature queens, consistent with the known greater parasite load in colonies headed by SB/Sb queens compared with colonies headed by SB/SB queens. Eighteen differentially expressed genes between reproductive queens were involved in chemical signalling. Our results suggest that many genes in the supergene are involved in regulating social organization and queen phenotypes in fire ants.

Social insect genomes exhibit dramatic evolution in gene composition and regulation while preserving regulatory features linked to sociality.

Genomes of eusocial insects code for dramatic examples of phenotypic plasticity and social organization. We compared the genomes of seven ants, the honeybee, and various solitary insects to examine whether eusocial lineages share distinct features of genomic organization. Each ant lineage contains ∼4000 novel genes, but only 64 of these genes are conserved among all seven ants. Many gene families have been expanded in ants, notably those involved in chemical communication (e.g., desaturases and odorant receptors). Alignment of the ant genomes revealed reduced purifying selection compared with Drosophila without significantly reduced synteny. Correspondingly, ant genomes exhibit dramatic divergence of noncoding regulatory elements; however, extant conserved regions are enriched for novel noncoding RNAs and transcription factor-binding sites. Comparison of orthologous gene promoters between eusocial and solitary species revealed significant regulatory evolution in both cis (e.g., Creb) and trans (e.g., fork head) for nearly 2000 genes, many of which exhibit phenotypic plasticity. Our results emphasize that genomic changes can occur remarkably fast in ants, because two recently diverged leaf-cutter ant species exhibit faster accumulation of species-specific genes and greater divergence in regulatory elements compared with other ants or Drosophila. Thus, while the "socio-genomes" of ants and the honeybee are broadly characterized by a pervasive pattern of divergence in gene composition and regulation, they preserve lineage-specific regulatory features linked to eusociality. We propose that changes in gene regulation played a key role in the origins of insect eusociality, whereas changes in gene composition were more relevant for lineage-specific eusocial adaptations.

Tracking individuals shows spatial fidelity is a key regulator of ant social organization.

Ants live in organized societies with a marked division of labor among workers, but little is known about how this division of labor is generated. We used a tracking system to continuously monitor individually tagged workers in six colonies of the ant Camponotus fellah over 41 days. Network analyses of more than 9 million interactions revealed three distinct groups that differ in behavioral repertoires. Each group represents a functional behavioral unit with workers moving from one group to the next as they age. The rate of interactions was much higher within groups than between groups. The precise information on spatial and temporal distribution of all individuals allowed us to calculate the expected rates of within- and between-group interactions. These values suggest that the network of interaction within colonies is primarily mediated by age-induced changes in the spatial location of workers.

A Y-like social chromosome causes alternative colony organization in fire ants.

Intraspecific variability in social organization is common, yet the underlying causes are rarely known. In the fire ant Solenopsis invicta, the existence of two divergent forms of social organization is under the control of a single Mendelian genomic element marked by two variants of an odorant-binding protein gene. Here we characterize the genomic region responsible for this important social polymorphism, and show that it is part of a pair of heteromorphic chromosomes that have many of the key properties of sex chromosomes. The two variants, hereafter referred to as the social B and social b (SB and Sb) chromosomes, are characterized by a large region of approximately 13 megabases (55% of the chromosome) in which recombination is completely suppressed between SB and Sb. Recombination seems to occur normally between the SB chromosomes but not between Sb chromosomes because Sb/Sb individuals are non-viable. Genomic comparisons revealed limited differentiation between SB and Sb, and the vast majority of the 616 genes identified in the non-recombining region are present in the two variants. The lack of recombination over more than half of the two heteromorphic social chromosomes can be explained by at least one large inversion of around 9 megabases, and this absence of recombination has led to the accumulation of deleterious mutations, including repetitive elements in the non-recombining region of Sb compared with the homologous region of SB. Importantly, most of the genes with demonstrated expression differences between individuals of the two social forms reside in the non-recombining region. These findings highlight how genomic rearrangements can maintain divergent adaptive social phenotypes involving many genes acting together by locally limiting recombination.

Venom alkaloid and cuticular hydrocarbon profiles are associated with social organization, queen fertility status, and queen genotype in the fire ant Solenopsis invicta.

Queens in social insect colonies advertise their presence in the colony to: a) attract workers' attention and care; b) gain acceptance by workers as replacement or supplemental reproductives; c) prevent reproductive development in nestmates. We analyzed the chemical content of whole body surface extracts of adult queens of different developmental and reproductive stages, and of adult workers from monogyne (single colony queen) and polygyne (multiple colony queens) forms of the fire ant Solenopsis invicta. We found that the composition of the most abundant components, venom alkaloids, differed between queens and workers, as well as between reproductive and non-reproductive queens. Additionally, workers of the two forms could be distinguished by alkaloid composition. Finally, sexually mature, non-reproductive queens from polygyne colonies differed in their proportions of cis-piperidine alkaloids, depending on their Gp-9 genotype, although the difference disappeared once they became functional reproductives. Among the unsaturated cuticular hydrocarbons characteristic of queens, there were differences in amounts of alkenes/alkadienes between non-reproductive polygyne queens of different Gp-9 genotypes, between non-reproductive and reproductive queens, and between polygyne and monogyne reproductive queens, with the amounts increasing at a relatively higher rate through reproductive ontogeny in queens bearing the Gp-9 b allele. Given that the genotype-specific piperidine differences reflect differences in rates of reproductive maturation between queens, we speculate that these abundant and unique compounds have been co-opted to serve in fertility signaling, while the cuticular hydrocarbons now play a complementary role in regulation of social organization by signaling queen Gp-9 genotype.

Visualization and quality assessment of de novo genome assemblies.

SUMMARY: Recent technological progress has greatly facilitated de novo genome sequencing. However, de novo assemblies consist in many pieces of contiguous sequence (contigs) arranged in thousands of scaffolds instead of small numbers of chromosomes. Confirming and improving the quality of such assemblies is critical for subsequent analysis. We present a method to evaluate genome scaffolding by aligning independently obtained transcriptome sequences to the genome and visually summarizing the alignments using the Cytoscape software. Applying this method to the genome of the red fire ant Solenopsis invicta allowed us to identify inconsistencies in 7%, confirm contig order in 20% and extend 16% of scaffolds. CONTACT: oksana.ribagrognuz@unil.ch; yannick.wurm@unil.ch AVAILABILITY: Scripts that generate tables for visualization in Cytoscape from FASTA sequence and scaffolding information files are publicly available at https://github.com/ksanao/TGNet. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Polymorphic social organization in an ant.

Identifying species exhibiting variation in social organization is an important step towards explaining the genetic and environmental factors underlying social evolution. In most studied populations of the ant Leptothorax acervorum, reproduction is shared among queens in multiple queen colonies (polygyny). By contrast, reports from other populations, but based on weaker evidence, suggest a single queen may monopolize all reproduction in multiple queen colonies (functional monogyny). Here we identify a marked polymorphism in social organization in this species, by conclusively showing that functional monogyny is exhibited in a Spanish population, showing that the social organization is stable and not purely a consequence of daughter queens overwintering, that daughter queen re-adoption is frequent and queen turnover is low. Importantly, we show that polygynous and functionally monogynous populations are not genetically distinct from one another based on mtDNA and nDNA. This suggests a recent evolutionary divergence between social phenotypes. Finally, when functionally monogynous and polygynous colonies were kept under identical laboratory conditions, social organization did not change, suggesting a genetic basis for the polymorphism. We discuss the implications of these findings to the study of reproductive skew.

Adaptation and the genetics of social behaviour.

In recent years much progress has been made towards understanding the selective forces involved in the evolution of social behaviour including conflicts over reproduction among group members. Here, I argue that an important additional step necessary for advancing our understanding of the resolution of potential conflicts within insect societies is to consider the genetics of the behaviours involved. First, I discuss how epigenetic modifications of behaviour may affect conflict resolution within groups. Second, I review known natural polymorphisms of social organization to demonstrate that a lack of consideration of the genetic mechanisms involved may lead to erroneous explanations of the adaptive significance of behaviour. Third, I suggest that, on the basis of recent genetic studies of sexual conflict in Drosophila, it is necessary to reconsider the possibility of within-group manipulation by means of chemical substances (i.e. pheromones). Fourth, I address the issue of direct versus indirect genetic effects, which is of particular importance for the study of behaviour in social groups. Fifth, I discuss the issue of how a genetic influence on dominance hierarchies and reproductive division of labour can have secondary effects, for example in the evolution of promiscuity. Finally, because the same sets of genes (e.g. those implicated in chemical signalling and the responses that are triggered) may be used even in species as divergent as ants, cooperative breeding birds and primates, an integration of genetic mechanisms into the field of social evolution may also provide unifying ideas.

Dynamics and genetic structure of Argentine ant supercolonies in their native range.

Some introduced ant populations have an extraordinary social organization, called unicoloniality, whereby individuals mix freely within large supercolonies. We investigated whether this mode of social organization also exists in native populations of the Argentine ant Linepithema humile. Behavioral analyses revealed the presence of 11 supercolonies (width 1 to 515 m) over a 3-km transect. As in the introduced range, there was always strong aggression between but never within supercolonies. The genetic data were in perfect agreement with the behavioral tests, all nests being assigned to identical supercolonies with the different methods. There was strong genetic differentiation between supercolonies but no genetic differentiation among nests within supercolonies. We never found more than a single mitochondrial haplotype per supercolony, further supporting the view that supercolonies are closed breeding units. Genetic and chemical distances between supercolonies were positively correlated, but there were no other significant associations between geographic, genetic, chemical, and behavioral distances. A comparison of supercolonies sampled in 1999 and 2005 revealed a very high turnover, with about one-third of the supercolonies being replaced yearly. This dynamic is likely to involve strong competition between supercolonies and thus act as a potent selective force maintaining unicoloniality over evolutionary time.

Genome-wide expression patterns and the genetic architecture of a fundamental social trait.

Explaining how interactions between genes and the environment influence social behavior is a fundamental research goal, yet there is limited relevant information for species exhibiting natural variation in social organization. The fire ant Solenopsis invicta is characterized by a remarkable form of social polymorphism, with the presence of one or several queens per colony and the expression of other phenotypic and behavioral differences being completely associated with allelic variation at a single Mendelian factor marked by the gene Gp-9. Microarray analyses of adult workers revealed that differences in the Gp-9 genotype are associated with the differential expression of an unexpectedly small number of genes, many of which have predicted functions, implying a role in chemical communication relevant to the regulation of colony queen number. Even more surprisingly, worker gene expression profiles are more strongly influenced by indirect effects associated with the Gp-9 genotypic composition within their colony than by the direct effect of their own Gp-9 genotype. This constitutes an unusual example of an "extended phenotype" and suggests a complex genetic architecture with a single Mendelian factor, directly and indirectly influencing the individual behaviors that, in aggregate, produce an emergent colony-level phenotype.

Contrasting population genetic structure for workers and queens in the putatively unicolonial ant Formica exsecta.

The theory of inclusive fitness provides a powerful explanation for reproductive altruism in social insects, whereby workers gain inclusive fitness benefit by rearing the brood of related queens. Some ant species, however, have unicolonial population structures where multiple nests, each containing numerous queens, are interconnected and individuals move freely between nests. In such cases, nestmate relatedness values may often be indistinguishable from zero, which is problematic for inclusive fitness-based explanations of reproductive altruism. We conducted a detailed population genetic study in the polygynous ant Formica exsecta, which has been suggested to form unicolonial populations in its native habitat. Analyses based on adult workers indeed confirmed a genetic structuring consistent with a unicolonial population structure. However, at the population level the genetic structuring inferred from worker pupae was not consistent with a unicolonial population structure, but rather suggested a multicolonial population structure of extended family-based nests. These contrasting patterns suggest limited queen dispersal and free adult worker dispersal. That workers indeed disperse as adults was confirmed by mark-recapture measures showing consistent worker movement between nests. Together, these findings describe a new form of social organization, which possibly also characterizes other ant species forming unicolonial populations in their native habitats. Moreover, the genetic analyses also revealed that while worker nestmate relatedness was indistinguishable from zero at a small geographical scale, it was significantly positive at the population level. This highlights the need to consider the relevant geographical scale when investigating the role of inclusive fitness as a selective force maintaining reproductive altruism.

Uncovering the biodiversity of genetic and reproductive systems: time for a more open approach. American Society of Naturalists E. O. Wilson Award winner address.

Important scientific findings frequently arise from serendipitous findings. Unfortunately, many scientists are not prepared to take advantage of unexpected results and to question established paradigms, and this prevents them from capitalizing on their good fortune. In this essay, I first explain how pure serendipity led us to discover unusual modes of reproduction such as clonal reproduction by males and a green-beard gene. Next, I argue that the reproductive systems of ants and other organisms are probably much more diverse than is generally appreciated. This leads me to advocate for a new "molecular naturalist" approach to reproductive systems and a more "naturalistic" approach in population and evolutionary genetics. Finally, I make two further points. The first is that our current funding and education systems tend to hinder originality and curiosity. The other is that the field of ecology and evolution, and more generally all of science, would benefit from a shift in values from scientific productivity to scientific creativity. A few suggestions are made to this effect.