Direct and indirect genetic effects of a social supergene.

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 laboratory-reared workers and compared these with previously published data from field-collected prereproductive 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.

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.

Positive selection on sociobiological traits in invasive fire ants.

The fire ant Solenopsis invicta and its close relatives are highly invasive. Enhanced social cooperation may facilitate invasiveness in these and other invasive ant species. We investigated whether invasiveness in Solenopsis fire ants was accompanied by positive selection on sociobiological traits by applying a phylogenomics approach to infer ancient selection, and a population genomics approach to infer recent and ongoing selection in both native and introduced S. invicta populations. A combination of whole-genome sequencing of 40 haploid males and reduced-representation genomic sequencing of 112 diploid workers identified 1,758,116 and 169,682 polymorphic markers, respectively. The resulting high-resolution maps of genomic polymorphism provide high inference power to test for positive selection. Our analyses provide evidence of positive selection on putative ion channel genes, which are implicated in neurological functions, and on vitellogenin, which is a key regulator of development and caste determination. Furthermore, molecular functions implicated in pheromonal signalling have experienced recent positive selection. Genes with signatures of positive selection were significantly more often those overexpressed in workers compared with queens and males, suggesting that worker traits are under stronger selection than queen and male traits. These results provide insights into selection pressures and ongoing adaptation in an invasive social insect and support the hypothesis that sociobiological traits are under more positive selection than nonsocial traits in such invasive species.

Vitellogenin underwent subfunctionalization to acquire caste and behavioral specific expression in the harvester ant Pogonomyrmex barbatus.

The reproductive ground plan hypothesis (RGPH) proposes that the physiological pathways regulating reproduction were co-opted to regulate worker division of labor. Support for this hypothesis in honeybees is provided by studies demonstrating that the reproductive potential of workers, assessed by the levels of vitellogenin (Vg), is linked to task performance. Interestingly, contrary to honeybees that have a single Vg ortholog and potentially fertile nurses, the genome of the harvester ant Pogonomyrmex barbatus harbors two Vg genes (Pb_Vg1 and Pb_Vg2) and nurses produce infertile trophic eggs. P. barbatus, thus, provides a unique model to investigate whether Vg duplication in ants was followed by subfunctionalization to acquire reproductive and non-reproductive functions and whether Vg reproductive function was co-opted to regulate behavior in sterile workers. To investigate these questions, we compared the expression patterns of P. barbatus Vg genes and analyzed the phylogenetic relationships and molecular evolution of Vg genes in ants. qRT-PCRs revealed that Pb_Vg1 is more highly expressed in queens compared to workers and in nurses compared to foragers. By contrast, the level of expression of Pb_Vg2 was higher in foragers than in nurses and queens. Phylogenetic analyses show that a first duplication of the ancestral Vg gene occurred after the divergence between the poneroid and formicoid clades and subsequent duplications occurred in the lineages leading to Solenopsis invicta, Linepithema humile and Acromyrmex echinatior. The initial duplication resulted in two Vg gene subfamilies preferentially expressed in queens and nurses (subfamily A) or in foraging workers (subfamily B). Finally, molecular evolution analyses show that the subfamily A experienced positive selection, while the subfamily B showed overall relaxation of purifying selection. Our results suggest that in P. barbatus the Vg gene underwent subfunctionalization after duplication to acquire caste- and behavior- specific expression associated with reproductive and non-reproductive functions, supporting the validity of the RGPH in ants.

Sociogenomics of cooperation and conflict during colony founding in the fire ant Solenopsis invicta.

One of the fundamental questions in biology is how cooperative and altruistic behaviors evolved. The majority of studies seeking to identify the genes regulating these behaviors have been performed in systems where behavioral and physiological differences are relatively fixed, such as in the honey bee. During colony founding in the monogyne (one queen per colony) social form of the fire ant Solenopsis invicta, newly-mated queens may start new colonies either individually (haplometrosis) or in groups (pleometrosis). However, only one queen (the "winner") in pleometrotic associations survives and takes the lead of the young colony while the others (the "losers") are executed. Thus, colony founding in fire ants provides an excellent system in which to examine the genes underpinning cooperative behavior and how the social environment shapes the expression of these genes. We developed a new whole genome microarray platform for S. invicta to characterize the gene expression patterns associated with colony founding behavior. First, we compared haplometrotic queens, pleometrotic winners and pleometrotic losers. Second, we manipulated pleometrotic couples in order to switch or maintain the social ranks of the two cofoundresses. Haplometrotic and pleometrotic queens differed in the expression of genes involved in stress response, aging, immunity, reproduction and lipid biosynthesis. Smaller sets of genes were differentially expressed between winners and losers. In the second experiment, switching social rank had a much greater impact on gene expression patterns than the initial/final rank. Expression differences for several candidate genes involved in key biological processes were confirmed using qRT-PCR. Our findings indicate that, in S. invicta, social environment plays a major role in the determination of the patterns of gene expression, while the queen's physiological state is secondary. These results highlight the powerful influence of social environment on regulation of the genomic state, physiology and ultimately, social behavior of animals.

Effects of ploidy and sex-locus genotype on gene expression patterns in the fire ant Solenopsis invicta.

Males in many animal species differ greatly from females in morphology, physiology and behaviour. Ants, bees and wasps have a haplodiploid mechanism of sex determination whereby unfertilized eggs become males while fertilized eggs become females. However, many species also have a low frequency of diploid males, which are thought to develop from diploid eggs when individuals are homozygous at one or more sex determination loci. Diploid males are morphologically similar to haploids, though often larger and typically sterile. To determine how ploidy level and sex-locus genotype affect gene expression during development, we compared expression patterns between diploid males, haploid males and females (queens) at three developmental timepoints in Solenopsis invicta. In pupae, gene expression profiles of diploid males were very different from those of haploid males but nearly identical to those of queens. An unexpected shift in expression patterns emerged soon after adult eclosion, with diploid male patterns diverging from those of queens to resemble those of haploid males, a pattern retained in older adults. The finding that ploidy level effects on early gene expression override sex effects (including genes implicated in sperm production and pheromone production/perception) may explain diploid male sterility and lack of worker discrimination against them during development.

The genome of the fire ant Solenopsis invicta.

Ants have evolved very complex societies and are key ecosystem members. Some ants, such as the fire ant Solenopsis invicta, are also major pests. Here, we present a draft genome of S. invicta, assembled from Roche 454 and Illumina sequencing reads obtained from a focal haploid male and his brothers. We used comparative genomic methods to obtain insight into the unique features of the S. invicta genome. For example, we found that this genome harbors four adjacent copies of vitellogenin. A phylogenetic analysis revealed that an ancestral vitellogenin gene first underwent a duplication that was followed by possibly independent duplications of each of the daughter vitellogenins. The vitellogenin genes have undergone subfunctionalization with queen- and worker-specific expression, possibly reflecting differential selection acting on the queen and worker castes. Additionally, we identified more than 400 putative olfactory receptors of which at least 297 are intact. This represents the largest repertoire reported so far in insects. S. invicta also harbors an expansion of a specific family of lipid-processing genes, two putative orthologs to the transformer/feminizer sex differentiation gene, a functional DNA methylation system, and a single putative telomerase ortholog. EST data indicate that this S. invicta telomerase ortholog has at least four spliceforms that differ in their use of two sets of mutually exclusive exons. Some of these and other unique aspects of the fire ant genome are likely linked to the complex social behavior of this species.

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.

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.

Oral transfer of chemical cues, growth proteins and hormones in social insects.

Social insects frequently engage in oral fluid exchange - trophallaxis - between adults, and between adults and larvae. Although trophallaxis is widely considered a food-sharing mechanism, we hypothesized that endogenous components of this fluid might underlie a novel means of chemical communication between colony members. Through protein and small-molecule mass spectrometry and RNA sequencing, we found that trophallactic fluid in the ant Camponotus floridanus contains a set of specific digestion- and non-digestion related proteins, as well as hydrocarbons, microRNAs, and a key developmental regulator, juvenile hormone. When C. floridanus workers' food was supplemented with this hormone, the larvae they reared via trophallaxis were twice as likely to complete metamorphosis and became larger workers. Comparison of trophallactic fluid proteins across social insect species revealed that many are regulators of growth, development and behavioral maturation. These results suggest that trophallaxis plays previously unsuspected roles in communication and enables communal control of colony phenotypes.