Causes and consequences of a complex recombinational landscape in the ant Cardiocondyla obscurior.

Eusocial Hymenoptera have the highest recombination rates among all multicellular animals studied so far, but it is unclear why this is and how this affects the biology of individual species. A high-resolution linkage map for the ant Cardiocondyla obscurior corroborates genome-wide high recombination rates reported for ants (8.1 cM/Mb). However, recombination is locally suppressed in regions that are enriched with TEs, that have strong haplotype divergence, or that show signatures of epistatic selection in C. obscurior The results do not support the hypotheses that high recombination rates are linked to phenotypic plasticity or to modulating selection efficiency. Instead, genetic diversity and the frequency of structural variants correlate positively with local recombination rates, potentially compensating for the low levels of genetic variation expected in haplodiploid social Hymenoptera with low effective population size. Ultimately, the data show that recombination contributes to within-population polymorphism and to the divergence of the lineages within C. obscurior.

Sex- and caste-specific developmental responses to juvenile hormone in an ant with maternal caste determination.

Juvenile hormone is considered to be a master regulator of polyphenism in social insects. In the ant Cardiocondyla obscurior, whether a female egg develops into a queen or a worker is determined maternally and caste-specific differentiation occurs in embryos, so that queens and workers can be distinguished in a non-invasive manner from late embryogenesis onwards. This ant also exhibits two male morphs - winged and wingless males. Here, we used topical treatment with juvenile hormone III and its synthetic analogue methoprene, a method that influences caste determination and differentiation in some ant species, to investigate whether hormone manipulation affects the development and growth of male, queen- and worker-destined embryos and larvae. We found no effect of hormone treatment on female caste ratios or body sizes in any of the treated stages, even though individuals reacted to heightened hormone availability with increased expression of krüppel-homolog 1, a conserved JH first-response gene. In contrast, hormone treatment resulted in the emergence of significantly larger males, although male morph fate was not affected. These results show that in C. obscurior, maternal caste determination leads to irreversible and highly canalized caste-specific development and growth.

Queens control caste allocation in the ant Cardiocondyla obscurior.

Social insect queens and workers can engage in conflict over reproductive allocation when they have different fitness optima. Here, we show that queens have control over queen-worker caste allocation in the ant Cardiocondyla obscurior, a species in which workers lack reproductive organs. We describe crystalline deposits that distinguish castes from the egg stage onwards, providing the first report of a discrete trait that can be used to identify ant caste throughout pre-imaginal development. The comparison of queen and worker-destined eggs and larvae revealed size and weight differences in late development, but no discernible differences in traits that may be used in social interactions, including hair morphology and cuticular odours. In line with a lack of caste-specific traits, adult workers treated developing queens and workers indiscriminately. Together with previous studies demonstrating queen control over sex allocation, these results show that queens control reproductive allocation in C. obscurior and suggest that the fitness interests of colony members are aligned to optimize resource allocation in this ant.

Convergent evolution of a labile nutritional symbiosis in ants.

Ants are among the most successful organisms on Earth. It has been suggested that forming symbioses with nutrient-supplementing microbes may have contributed to their success, by allowing ants to invade otherwise inaccessible niches. However, it is unclear whether ants have evolved symbioses repeatedly to overcome the same nutrient limitations. Here, we address this question by comparing the independently evolved symbioses in Camponotus, Plagiolepis, Formica and Cardiocondyla ants. Our analysis reveals the only metabolic function consistently retained in all of the symbiont genomes is the capacity to synthesise tyrosine. We also show that in certain multi-queen lineages that have co-diversified with their symbiont for millions of years, only a fraction of queens carry the symbiont, suggesting ants differ in their colony-level reliance on symbiont-derived resources. Our results imply that symbioses can arise to solve common problems, but hosts may differ in their dependence on symbionts, highlighting the evolutionary forces influencing the persistence of long-term endosymbiotic mutualisms.

Late-life fitness gains and reproductive death in Cardiocondyla obscurior ants.

A key hypothesis for the occurrence of senescence is the decrease in selection strength due to the decrease in the proportion of newborns from parents attaining an advanced age - the so-called selection shadow. Strikingly, queens of social insects have long lifespans and reproductive senescence seems to be negligible. By lifelong tracking of 99 Cardiocondyla obscurior (Formicidae: Myrmicinae) ant colonies, we find that queens shift to the production of sexuals in late life regardless of their absolute lifespan or the number of workers present. Furthermore, RNAseq analyses of old queens past their peak of reproductive performance showed the development of massive pathology while queens were still fertile, leading to rapid death. We conclude that the evolution of superorganismality is accompanied by 'continuusparity,' a life history strategy that is distinct from other iteroparous and semelparous strategies across the tree of life, in that it combines continuous reproduction with a fitness peak late in life.

Transposable elements and introgression introduce genetic variation in the invasive ant Cardiocondyla obscurior.

Introduced populations of invasive organisms have to cope with novel environmental challenges, while having reduced genetic variation caused by founder effects. The mechanisms associated with this "genetic paradox of invasive species" has received considerable attention, yet few studies have examined the genomic architecture of invasive species. Populations of the heart node ant Cardiocondyla obscurior belong to two distinct lineages, a New World lineage so far only found in Latin America and a more globally distributed Old World lineage. In the present study, we use population genomic approaches to compare populations of the two lineages with apparent divergent invasive potential. We find that the strong genetic differentiation of the two lineages began at least 40,000 generations ago and that activity of transposable elements (TEs) has contributed significantly to the divergence of both lineages, possibly linked to the very unusual genomic distribution of TEs in this species. Furthermore, we show that introgression from the Old World lineage is a dominant source of genetic diversity in the New World lineage, despite the lineages' strong genetic differentiation. Our study uncovers mechanisms underlying novel genetic variation in introduced populations of C. obscurior that could contribute to the species' adaptive potential.

Gene Coexpression Network Reveals Highly Conserved, Well-Regulated Anti-Ageing Mechanisms in Old Ant Queens.

Evolutionary theories of ageing predict a reduction in selection efficiency with age, a so-called "selection shadow," due to extrinsic mortality decreasing effective population size with age. Classic symptoms of ageing include a deterioration in transcriptional regulation and protein homeostasis. Understanding how ant queens defy the trade-off between fecundity and lifespan remains a major challenge for the evolutionary theory of ageing. It has often been discussed that the low extrinsic mortality of ant queens, that are generally well protected within the nest by workers and soldiers, should reduce the selection shadow acting on old queens. We tested this by comparing strength of selection acting on genes upregulated in young and old queens of the ant, Cardiocondyla obscurior. In support of a reduced selection shadow, we find old-biased genes to be under strong purifying selection. We also analyzed a gene coexpression network (GCN) with the aim to detect signs of ageing in the form of deteriorating regulation and proteostasis. We find no evidence for ageing. In fact, we detect higher connectivity in old queens indicating increased transcriptional regulation with age. Within the GCN, we discover five highly correlated modules that are upregulated with age. These old-biased modules regulate several antiageing mechanisms such as maintenance of proteostasis, transcriptional regulation, and stress response. We observe stronger purifying selection on central hub genes of these old-biased modules compared with young-biased modules. These results indicate a lack of transcriptional ageing in old C. obscurior queens, possibly facilitated by strong selection at old age and well-regulated antiageing mechanisms.

Cytoplasmic incompatibility between Old and New World populations of a tramp ant.

Reproductive manipulation by endosymbiotic Wolbachia can cause unequal inheritance, allowing the manipulator to spread and potentially impacting evolutionary dynamics in infected hosts. Tramp and invasive species are excellent models to study the dynamics of host-Wolbachia associations because introduced populations often diverge in their microbiomes after colonizing new habitats, resulting in infection polymorphisms between native and introduced populations. Ants are the most abundant group of insects on earth, and numerous ant species are classified as highly invasive. However, little is known about the role of Wolbachia in these ecologically dominant insects. Here, we provide the first description of reproductive manipulation by Wolbachia in an ant. We show that Old and New World populations of the cosmotropic tramp ant Cardiocondyla obscurior harbor distinct Wolbachia strains, and that only the Old World strain manipulates host reproduction by causing cytoplasmic incompatibility (CI) in hybrid crosses. By uncovering a symbiont-induced mechanism of reproductive isolation in a social insect, our study provides a novel perspective on the biology of tramp ants and introduces a new system for studying the evolutionary consequences of CI.

Inhibition of HSP90 causes morphological variation in the invasive ant Cardiocondyla obscurior.

Canalization underlies the expression of steady phenotypes in the face of unsteady environmental conditions or varying genetic backgrounds. The chaperone HSP90 has been identified as a key component of the molecular machinery regulating canalization and a growing body of research suggests that HSP90 could act as a general capacitator in evolution. However, empirical data about HSP90-dependent phenotypic variation and its evolutionary impact is still scarce, particularly for non-model species. Here we report how pharmacological suppression of HSP90 increases morphological variation up to 87% in the invasive ant Cardiocondyla obscurior. We show that workers treated with the HSP90 inhibitor 17-DMAG are significantly more diverse compared to untreated workers in two of four measured traits: maximal eye distance and maximal propodeal spine distance. We further find morphological differentiation between natural populations of C. obscurior in the same traits that responded to our pharmacological treatment. These findings add support for the putative impact of HSP90 on canalization, the modularity of phenotypic traits, and its potential role in morphological evolution of ants.

Interruption points in the wing gene regulatory network underlying wing polyphenism evolved independently in male and female morphs in Cardiocondyla ants.

Wing polyphenism in ants, which produces a winged female queen caste and a wingless female worker caste, evolved approximately 150 million years ago and has been key to the remarkable success of ants. Approximately 20 million years ago, the myrmicine ant genus Cardiocondyla evolved an additional wing polyphenism among males producing two male morphs: wingless males that fight to enhance mating success and winged males that disperse. Here we show that interruption of rudimentary wing-disc development in larvae of the ant Cardiocondyla obscurior occurs further downstream in the network in wingless males as compared with wingless female workers. This pattern is corroborated in C. kagutsuchi, a species from a different clade within the genus, indicating that late interruption of wing development in males is conserved across Cardiocondyla. Therefore, our results show that the novel male wing polyphenism was not developmentally constrained by the pre-existing female wing polyphenism and evolved through independent alteration of interruption points in the wing gene network. Furthermore, a comparison of adult morphological characters in C. obscurior reveals that developmental trajectories lead to similar morphological trait integration between winged and wingless females, but dramatically different integration between winged and wingless males. This suggests that the alternative sex-specific developmental routes to achieve winglessness in the genus Cardiocondyla may have evolved through different selection regimes acting on wingless males and females.

The Role of Brood in Eusocial Hymenoptera.

Study of social traits in offspring traditionally reflects on interactions in simple family groups, with famous examples including parent-offspring conflict and sibling rivalry in birds and mammals. In contrast, studies of complex social groups such as the societies of ants, bees, and wasps focus mainly on adults and, in particular, on traits and interests of queens and workers. The social role of developing individuals in complex societies remains poorly understood. We attempt to fill this gap by illustrating that development in social Hymenoptera constitutes a crucial life stage with important consequences for the individual as well as the colony. We begin by describing the complex social regulatory network that modulates development in Hymenoptera societies. By highlighting the inclusive fitness interests of developing individuals, we show that they may differ from those of other colony members. We then demonstrate that offspring have evolved specialized traits that allow them to play a functional, cooperative role within colonies and give them the potential power to act toward increasing their inclusive fitness. We conclude by providing testable predictions for investigating the role of brood in colony interactions and giving a general outlook on what can be learned from studying offspring traits in hymenopteran societies.

Accelerated Evolution of Developmentally Biased Genes in the Tetraphenic Ant Cardiocondyla obscurior.

Plastic gene expression underlies phenotypic plasticity and plastically expressed genes evolve under different selection regimes compared with ubiquitously expressed genes. Social insects are well-suited models to elucidate the evolutionary dynamics of plastic genes for their genetically and environmentally induced discrete polymorphisms. Here, we study the evolution of plastically expressed genes in the ant Cardiocondyla obscurior-a species that produces two discrete male morphs in addition to the typical female polymorphism of workers and queens. Based on individual-level gene expression data from 28 early third instar larvae, we test whether the same evolutionary dynamics that pertain to plastically expressed genes in adults also pertain to genes with plastic expression during development. In order to quantify plasticity of gene expression over multiple contrasts, we develop a novel geometric measure. For genes expressed during development, we show that plasticity of expression is positively correlated with evolutionary rates. We furthermore find a strong correlation between expression plasticity and expression variation within morphs, suggesting a close link between active and passive plasticity of gene expression. Our results support the notion of relaxed selection and neutral processes as important drivers in the evolution of adaptive plasticity.

Fitness and aging in Cardiocondyla obscurior ant queens.

Easy maintenance, controlled mating and short generation time make Cardiocondyla obscurior an interesting model for social insect aging research. Using this ant we have begun to study the proximate genomic relationship between mating and aging. Although mating in general has a positive effect and results in fertile queens with long life but drastically reduced metabolic rates, mating can also dramatically reduce queen fitness. Here we review a decade of research on factors affecting queen aging rate and contrast these findings with studies on honeybees and solitary aging models. We conclude by giving a brief outlook of what is to be expected from this model in coming years.

Evolution of Social Insect Polyphenism Facilitated by the Sex Differentiation Cascade.

The major transition to eusociality required the evolution of a switch to canalize development into either a reproductive or a helper, the nature of which is currently unknown. Following predictions from the 'theory of facilitated variation', we identify sex differentiation pathways as promising candidates because of their pre-adaptation to regulating development of complex phenotypes. We show that conserved core genes, including the juvenile hormone-sensitive master sex differentiation gene doublesex (dsx) and a krüppel homolog 2 (kr-h2) with putative regulatory function, exhibit both sex and morph-specific expression across life stages in the ant Cardiocondyla obscurior. We hypothesize that genes in the sex differentiation cascade evolved perception of alternative input signals for caste differentiation (i.e. environmental or genetic cues), and that their inherent switch-like and epistatic behavior facilitated signal transfer to downstream targets, thus allowing them to control differential development into morphological castes.

A novel intracellular mutualistic bacterium in the invasive ant Cardiocondyla obscurior.

The evolution of eukaryotic organisms is often strongly influenced by microbial symbionts that confer novel traits to their hosts. Here we describe the intracellular Enterobacteriaceae symbiont of the invasive ant Cardiocondyla obscurior, 'Candidatus Westeberhardia cardiocondylae'. Upon metamorphosis, Westeberhardia is found in gut-associated bacteriomes that deteriorate following eclosion. Only queens maintain Westeberhardia in the ovarian nurse cells from where the symbionts are transmitted to late-stage oocytes during nurse cell depletion. Functional analyses of the streamlined genome of Westeberhardia (533 kb, 23.41% GC content) indicate that neither vitamins nor essential amino acids are provided for the host. However, the genome encodes for an almost complete shikimate pathway leading to 4-hydroxyphenylpyruvate, which could be converted into tyrosine by the host. Taken together with increasing titers of Westeberhardia during pupal stage, this suggests a contribution of Westeberhardia to cuticle formation. Despite a widespread occurrence of Westeberhardia across host populations, one ant lineage was found to be naturally symbiont-free, pointing to the loss of an otherwise prevalent endosymbiont. This study yields insights into a novel intracellular mutualist that could play a role in the invasive success of C. obscurior.

Expression of the Foraging Gene Is Associated with Age Polyethism, Not Task Preference, in the Ant Cardiocondyla obscurior.

One of the fundamental principles of social organization, age polyethism, describes behavioral maturation of workers leading to switches in task preference. Here we present a system that allows for studying division of labor (DOL) by taking advantage of the relative short life of Cardiocondyla obscurior workers and thereby the pace of behavioral transitions. By challenging same-age young and older age cohorts to de novo establish DOL into nurse and foraging tasks and by forcing nurses to precociously become foragers and vice versa we studied expression patterns of one of the best known candidates for social insect worker behavior, the foraging gene. Contrary to our expectations we found that foraging gene expression correlates with age, but not with the task foraging per se. This suggests that this nutrition-related gene, and the pathways it is embedded in, correlates with physiological changes over time and potentially primes, but not determines task preference of individual workers.

Transcriptomic Signatures Mirror the Lack of the Fecundity/Longevity Trade-Off in Ant Queens.

Life-history theory predicts a trade-off between reproductive investment and self-maintenance. The negative association between fertility and longevity found throughout multicellular organisms supports this prediction. As an important exception, the reproductives of many eusocial insects (ants, bees, and termites) are simultaneously very long-lived and highly fertile. Here, we examine the proximate basis for this exceptional relationship by comparing whole-body transcriptomes of differently aged queens of the ant Cardiocondyla obscurior. We show that the sets of genes differentially expressed with age significantly overlap with age-related expression changes previously found in female Drosophila melanogaster. We identified several developmental processes, such as the generation of neurons, as common signatures of aging. More generally, however, gene expression in ant queens and flies changes with age mainly in opposite directions. In contrast to flies, reproduction-associated genes were upregulated and genes associated with metabolic processes and muscle contraction were downregulated in old relative to young ant queens. Furthermore, we searched for putative C. obscurior longevity candidates associated with the previously reported lifespan-prolonging effect of mating by comparing the transcriptomes of queens that differed in mating and reproductive status. We found 21 genes, including the putative aging candidate NLaz (an insect homolog of APOD), which were consistently more highly expressed in short-lived, unmated queens than in long-lived, mated queens. Our study provides clear evidence that the alternative regulation of conserved molecular pathways that mediate the interplay among mating, egg laying, and aging underlies the lack of the fecundity/longevity trade-off in ant queens.

Sphingolipids, Transcription Factors, and Conserved Toolkit Genes: Developmental Plasticity in the Ant Cardiocondyla obscurior.

Developmental plasticity allows for the remarkable morphological specialization of individuals into castes in eusocial species of Hymenoptera. Developmental trajectories that lead to alternative caste fates are typically determined by specific environmental stimuli that induce larvae to express and maintain distinct gene expression patterns. Although most eusocial species express two castes, queens and workers, the ant Cardiocondyla obscurior expresses diphenic females and males; this provides a unique system with four discrete phenotypes to study the genomic basis of developmental plasticity in ants. We sequenced and analyzed the transcriptomes of 28 individual C. obscurior larvae of known developmental trajectory, providing the first in-depth analysis of gene expression in eusocial insect larvae. Clustering and transcription factor binding site analyses revealed that different transcription factors and functionally distinct sets of genes are recruited during larval development to induce the four alternative trajectories. In particular, we found complex patterns of gene regulation pertaining to sphingolipid metabolism, a conserved molecular pathway involved in development, obesity, and aging.

Transposable element islands facilitate adaptation to novel environments in an invasive species.

Adaptation requires genetic variation, but founder populations are generally genetically depleted. Here we sequence two populations of an inbred ant that diverge in phenotype to determine how variability is generated. Cardiocondyla obscurior has the smallest of the sequenced ant genomes and its structure suggests a fundamental role of transposable elements (TEs) in adaptive evolution. Accumulations of TEs (TE islands) comprising 7.18% of the genome evolve faster than other regions with regard to single-nucleotide variants, gene/exon duplications and deletions and gene homology. A non-random distribution of gene families, larvae/adult specific gene expression and signs of differential methylation in TE islands indicate intragenomic differences in regulation, evolutionary rates and coalescent effective population size. Our study reveals a tripartite interplay between TEs, life history and adaptation in an invasive species.

A map of brain neuropils and fiber systems in the ant Cardiocondyla obscurior.

A wide spectrum of occupied ecological niches and spectacular morphological adaptations make social insects a prime object for comparative neuroanatomical studies. Eusocial insects have evolved complex societies based on caste polyphenism. A diverse behavioral repertoire of morphologically distinct castes of the same species requires a high degree of plasticity in the central nervous system. We have analyzed the central brain neuropils and fiber tract systems of the worker of the ant Cardiocondyla obscurior, a model for the study of social traits. Our analysis is based on whole mount preparations of adult brains labeled with an antibody against Drosophila-Synapsin, which cross-reacts strongly with synapses in Cardiocondyla. Neuropil compartments stand out as domains with a certain texture and intensity of the anti-Synapsin signal. By contrast, fiber tracts, which are composed of bundles of axons accompanied by glia and are devoid of synapses, appear as channels or sheaths with low anti-Synapsin signal. We have generated a digital 3D atlas of the Cardiocondyla brain neuropil. The atlas provides a reference for future studies of brain polymorphisms in distinct castes, brain development or localization of neurotransmitter systems.