Strong Purifying Selection in Haploid Tissue-Specific Genes of Scots Pine Supports the Masking Theory.

The masking theory states that genes expressed in a haploid stage will be under more efficient selection. In contrast, selection will be less efficient in genes expressed in a diploid stage, where the fitness effects of recessive deleterious or beneficial mutations can be hidden from selection in heterozygous form. This difference can influence several evolutionary processes such as the maintenance of genetic variation, adaptation rate, and genetic load. Masking theory expectations have been confirmed in single-cell haploid and diploid organisms. However, in multicellular organisms, such as plants, the effects of haploid selection are not clear-cut. In plants, the great majority of studies indicating haploid selection have been carried out using male haploid tissues in angiosperms. Hence, evidence in these systems is confounded with the effects of sexual selection and intraspecific competition. Evidence from other plant groups is scarce, and results show no support for the masking theory. Here, we have used a gymnosperm Scots pine megagametophyte, a maternally derived seed haploid tissue, and four diploid tissues to test the strength of purifying selection on a set of genes with tissue-specific expression. By using targeted resequencing data of those genes, we obtained estimates of genetic diversity, the site frequency spectrum of 0-fold and 4-fold sites, and inferred the distribution of fitness effects of new mutations in haploid and diploid tissue-specific genes. Our results show that purifying selection is stronger for tissue-specific genes expressed in the haploid megagametophyte tissue and that this signal of strong selection is not an artifact driven by high expression levels.

Positive selection has shaped the evolution of Argentine ant immune genes both in native and introduced supercolonies.

The highly invasive Argentine ant (Linepithema humile) started its colonisation from the species' native range in South America approximately 150 years ago and has since become one of the major pests in the world. We investigated how the shifts into new ranges have affected the evolution of Argentine ants' immune genes. To the best of our knowledge, this is the first broadscale population genetic study focusing on ants' immune genes. We analysed comprehensive targeted-seq data of immune and non-immune genes containing 174 genes from 18 Argentine ant supercolonies covering the species' native and introduced ranges. We predicted that the immune gene evolution of introduced supercolonies differs from that of the native supercolonies and proposed two different, non-mutually exclusive hypotheses for this: 1) the enemy release hypothesis and 2) the higher pathogen pressure hypothesis - both of which seem to explain the observed evolutionary patterns on their behalf. Our results show that the introduced supercolonies were targeted by weaker selection than natives, but positive selection was evident among supercolonies of both ranges. Moreover, in some cases, such as the antiviral RNAi genes, introduced range supercolonies harboured a higher proportion of positively selected genes than natives. This observation was striking, knowing the recent demographic history and the detected generally lower selection efficacy of introduced supercolonies. In conclusion, it is evident that pathogen pressure is ubiquitous and strongly affects the immune gene evolution in Argentine ants.

Queen fecundity, worker entourage and cuticular chemistry in the ant Formica fusca.

Cooperative breeding entails conflicts over reproductive shares that may be settled in different ways. In ants, where several queens simultaneously reproduce in a colony, both queens and workers may influence the reproductive apportionment and offspring quality. Queens may vary in their intrinsic fecundity, which may influence the size of the worker entourage attending individual queens, and this may eventually dictate the reproductive output of a queen. We tested whether the reproductive success of queens is affected by the size of their worker entourage, their fecundity at the onset of the reproductive season, and whether the queen cuticular hydrocarbon profile carries information on fecundity. We show that in the ant Formica fusca both queen fecundity and egg hatching success increase with the size of their entourage, and that newly hatched larvae produced by initially highly fecund queens are smaller. Furthermore, higher relatedness among workers increased queen fecundity. Finally, the queens that received a large worker entourage differed in the cuticular chemistry from those that received a small worker entourage. Our results thus show that workers play a pivotal role in determining queen fitness, that high intracolony relatedness among workers enhances the overall reproductive output in the colony, and that queen fecundity is reflected in their cuticular hydrocarbon profile.

Formica fusca ants use aphid supplemented foods to alleviate effects during the acute phase of a fungal infection.

The modulation of nutritional intake by animals to combat pathogens is a behaviour that is receiving increasing attention. Ant studies using isolated compounds or nutrients in artificial diets have revealed a lot of the dynamics of the behaviour, but natural sources of medicine are yet to be confirmed. Here we explored whether Formica fusca ants exposed to a fungal pathogen can use an artificial diet containing foods spiked with different concentrations of crushed aphids for a medicinal benefit. We show that pathogen exposed colonies adjusted their diet to include more aphid supplemented foods during the acute phase of the infection, reducing the mortality caused by the disease. However, the benefit was only attained when having access to a varied diet, suggesting that while aphids contain nutrients or compounds beneficial against infection, it is a part of a complex nutritional system where costs and benefits of compounds and nutrients need to be moderated.

Whole-genome analysis of multiple wood ant population pairs supports similar speciation histories, but different degrees of gene flow, across their European ranges.

The application of demographic history modelling and inference to the study of divergence between species has become a cornerstone of speciation genomics. Speciation histories are usually reconstructed by analysing single populations from each species, assuming that the inferred population history represents the actual speciation history. However, this assumption may not be met when species diverge with gene flow, for example, when secondary contact may be confined to specific geographic regions. Here, we tested whether divergence histories inferred from heterospecific populations may vary depending on their geographic locations, using the two wood ant species Formica polyctena and F. aquilonia. We performed whole-genome resequencing of 20 individuals sampled in multiple locations across the European ranges of both species. Then, we reconstructed the histories of distinct heterospecific population pairs using a coalescent-based approach. Our analyses always supported a scenario of divergence with gene flow, suggesting that divergence started in the Pleistocene (c. 500 kya) and occurred with continuous asymmetrical gene flow from F. aquilonia to F. polyctena until a recent time, when migration became negligible (2-19 kya). However, we found support for contemporary gene flow in a sympatric pair from Finland, where the species hybridise, but no signature of recent bidirectional gene flow elsewhere. Overall, our results suggest that divergence histories reconstructed from a few individuals may be applicable at the species level. Nonetheless, the geographical context of populations chosen to represent their species should be taken into account, as it may affect estimates of migration rates between species when gene flow is spatially heterogeneous.

Superorganismal anisogamy: queen-male dimorphism in eusocial insects.

Colonies of insects such as ants and honeybees are commonly viewed as 'superorganisms', with division of labour between reproductive 'germline-like' queens and males and 'somatic' workers. On this view, properties of the superorganismal colony are comparable with those of solitary organisms to such an extent that the colony itself can be viewed as a unit analogous to an organism. Thus, the concept of a superorganism can be useful as a guide to thinking about life history and allocation traits of colonies as a whole. A pattern that seems to reoccur in insects with superorganismal societies is size dimorphism between queens and males, where queens tend to be larger than males. It has been proposed that this is analogous to the phenomenon of anisogamy at the level of gametes in organisms with separate sexes; more specifically, it is suggested that this caste dimorphism may have evolved via similar selection pressures as gamete dimorphism arises in the 'gamete competition' theory for the evolution of anisogamy. In this analogy, queens are analogous to female gametes, males are analogous to male gametes, and colony survival is analogous to zygote survival in gamete competition theory. Here, we explore if this question can be taken beyond an analogy, and whether a mathematical model at the superorganism level, analogous to gamete competition at the organism level, may explain the caste dimorphism seen in superorganismal insects. We find that the central theoretical idea holds, but that there are also significant differences between the way this generalized 'propagule competition' theory operates at the levels of solitary organisms and superorganisms. In particular, we find that the theory can explain superorganismal caste dimorphism, but compared with anisogamy evolution, a central coevolutionary link is broken, making the requirements for the theory to work less stringent than those found for the evolution of anisogamy.

The possible role of ant larvae in the defence against social parasites.

Temporary social parasite ant queens initiate new colonies by entering colonies of host species, where they begin laying eggs. As the resident queen can be killed during this process, host colonies may lose their entire future reproductive output. Selection thus favours the evolution of defence mechanisms, before and after parasite intrusion. Most studies on social parasites focus on host worker discrimination of parasite queens and their offspring. However, ant larvae can also influence brood composition by consuming eggs. This raises the question whether host larvae can aid in preventing colony takeover by consuming eggs laid by parasite queens. To test whether larvae could play a role in anti-parasite defence, we compared the rates at which larvae of a common host species, Formica fusca, consumed eggs laid by social parasite, non-parasite, nest-mate, or conspecific non-nest-mate queens. Larvae consumed social parasite eggs more than eggs laid by a heterospecific non-parasite queen, irrespective of the chemical distance between the egg cuticular profiles. Also, larvae consumed eggs laid by conspecific non-nest-mate queens more than those laid by nest-mate queens. Our study suggests that larvae may act as players in colony defence against social parasitism, and that social parasitism is a key factor shaping discrimination behaviour in ants.

Changes in gene DNA methylation and expression networks accompany caste specialization and age-related physiological changes in a social insect.

Social insects provide systems for studying epigenetic regulation of phenotypes, particularly with respect to differentiation of reproductive and worker castes, which typically arise from a common genetic background. The role of gene expression in caste specialization has been extensively studied, but the role of DNA methylation remains controversial. Here, we perform well replicated, integrated analyses of DNA methylation and gene expression in brains of an ant (Formica exsecta) with distinct female castes using traditional approaches (tests of differential methylation) combined with a novel approach (analysis of co-expression and co-methylation networks). We found differences in expression and methylation profiles between workers and queens at different life stages, as well as some overlap between DNA methylation and expression at the functional level. Large portions of the transcriptome and methylome are organized into "modules" of genes, some significantly associated with phenotypic traits of castes and developmental stages. Several gene co-expression modules are preserved in co-methylation networks, consistent with possible regulation of caste-specific gene expression by DNA methylation. Surprisingly, brain co-expression modules were highly preserved when compared with a previous study that examined whole-body co-expression patterns in 16 ant species, suggesting that these modules are evolutionarily conserved and for specific functions in various tissues. Altogether, these results suggest that DNA methylation participates in regulation of caste specialization and age-related physiological changes in social insects.

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.

Genetic structure of native ant supercolonies varies in space and time.

Ant supercolonies are the largest cooperative units known in nature. They consist of networks of interconnected nests with hundreds of reproductive queens, where individuals move freely between nests, cooperate across nest boundaries and show little aggression towards non-nestmates. The combination of high queen numbers and free mixing of workers, queens and brood between nests results in extremely low nestmate relatedness. In such low-relatedness societies, cooperative worker behaviour appears maladaptive because it may aid random individuals instead of relatives. Here, we provide a comprehensive picture of genetic substructure in supercolonies of the native wood ant Formica aquilonia using traditional population genetic as well as network analysis methods. Specifically, we test for spatial and temporal variation in genetic structure of different classes of individuals within supercolonies and analyse the role of worker movement in determining supercolony genetic networks. We find that relatedness within supercolonies is low but positive when viewed on a population level, which may be due to limited dispersal of individuals and/or ecological factors such as nest site limitation and competition against conspecifics. Genetic structure of supercolonies varied with both sample class and sampling time point, which indicates that mobility of individuals varies according to both caste and season and suggests that generalizing has to be carried out with caution in studies of supercolonial species. Overall, our analysis provides novel evidence that native wood ant supercolonies exhibit fine-scale genetic substructure, which may explain the maintenance of cooperation in these low-relatedness societies.

Queen pheromones modulate DNA methyltransferase activity in bee and ant workers.

DNA methylation is emerging as an important regulator of polyphenism in the social insects. Research has concentrated on differences in methylation between queens and workers, though we hypothesized that methylation is involved in mediating other flexible phenotypes, including pheromone-dependent changes in worker behaviour and physiology. Here, we find that exposure to queen pheromone affects the expression of two DNA methyltransferase genes in Apis mellifera honeybees and in two species of Lasius ants, but not in Bombus terrestris bumblebees. These results suggest that queen pheromones influence the worker methylome, pointing to a novel proximate mechanism for these key social signals.

Not only for egg yolk--functional and evolutionary insights from expression, selection, and structural analyses of Formica ant vitellogenins.

Vitellogenin (Vg), a storage protein, has been extensively studied for its egg-yolk precursor role, and it has been suggested to be fundamentally involved in caste differences in social insects. More than one Vg copy has been reported in several oviparous species, including ants. However, the number and function of different Vgs, their phylogenetic relatedness, and their role in reproductive queens and nonreproductive workers have been studied in few species only. We studied caste-biased expression of Vgs in seven Formica ant species. Only one copy of conventional Vg was identified in Formica species, and three Vg homologs, derived from ancient duplications, which represent yet undiscovered Vg-like genes. We show that each of these Vg-like genes is present in all studied Hymenoptera and some of them in other insects as well. We show that after each major duplication event, at least one of the Vg-like genes has experienced a period of positive selection. This, combined with the observation that the Vg-like genes have acquired or lost specific protein domains suggests sub- or neofunctionalization between Vg and the duplicated genes. In contrast to earlier studies, Vg was not consistently queen biased in its expression, and the caste bias of the three Vg-like genes was highly variable among species. Furthermore, a truncated and Hymenoptera-specific Vg-like gene, Vg-like-C, was consistently worker biased. Multispecies comparisons are essential for Vg expression studies, and for gene expression studies in general, as we show that expression and also, putative functions cannot be generalized even among closely related species.

Unmatedness promotes the evolution of helping more in diplodiploids than in haplodiploids.

The predominance of haplodiploidy (where males develop from unfertilized haploid eggs and females from fertilized diploid eggs) among eusocial species has inspired a body of research that focuses on the possible role of relatedness asymmetries in the evolution of helping and eusociality. Previous theory has shown that in order for relatedness asymmetries to favor the evolution of helping, there needs to be variation in sex ratios among nests in the population (i.e., split sex ratios). In haplodiploid species, unmated females can produce a brood of all males, and this is considered the most likely mechanism for split sex ratios at the origin of helping. In contrast, in diploidiploids unmatedness means total reproductive failure. We compare the effect of unmatedness on selection for male and female helping in haplodiploids and diplodiploids. We show that in haplodiploids, unmatedness promotes helping in females but not in males within the empirical range. In diplodiploids, unmatedness promotes helping by both sexes, and the effect is stronger than in haplodiploids, all else being equal. Our study highlights the need to consider interactions between ecological and genetic factors in the evolution of helping and eusociality.

Ant larvae as players in social conflict: relatedness and individual identity mediate cannibalism intensity.

Conflict arises among social organisms when individuals differ in their inclusive-fitness interests. Ant societies are excellent models for understanding how genetic relatedness mediates conflict intensity. However, although conflicts within colonies typically arise over offspring production, the role of larvae as actors in social conflict has received little attention. We develop and empirically test kin-selection theory of larval egg cannibalism in ant societies. Specifically, we investigate how selection for cannibalism is mediated by nestmate relatedness and larval sex in a mathematical model and then test the model's predictions by measuring cannibalism levels in eight ant species with varying nestmate relatedness. In line with our theoretical predictions, cannibalism levels in larvae were significantly influenced by relatedness and sex. Increased relatedness was associated with reduced levels of cannibalism, indicating that larval behavior is mediated by inclusive-fitness considerations. Levels of cannibalism were significantly higher in male larvae, and our model suggests that this is due to sex differences in the benefits of cannibalism. By examining the selfish interests of larvae and the constraints they face in a social environment, our study presents a novel perspective on conflict in ants and on the evolution of selfish elements in social systems in general.

The value of oviposition timing, queen presence and kinship in a social insect.

Reproductive cooperation confers benefits, but simultaneously creates conflicts among cooperators. Queens in multi-queen colonies of ants share a nest and its resources, but reproductive competition among queens often results in unequal reproduction. Two mutually non-exclusive factors may produce such inequality in reproduction: worker intervention or queen traits. Workers may intervene by favouring some queens over others, owing to either kinship or queen signals. Queens may differ in their intrinsic fecundity at the onset of oviposition or in their timing of the onset of oviposition, leading to their unequal representation in the brood. Here, we test the role of queen kin value (relatedness) to workers, timing of the onset of oviposition and signals of presence by queens in determining the maternity of offspring. We show that queens of the ant Formica fusca gained a significantly higher proportion of sexuals in the brood when ovipositing early, and that the presence of a caged queen resulted in a significant increase in both her share of sexual brood and her overall reproductive share. Moreover, the lower the kin value of the queen, the more the workers invested in their own reproduction by producing males. Our results show that both kinship and breeding phenology influence the outcome of reproductive conflicts, and the balance of direct and indirect fitness benefits in the multi-queen colonies of F. fusca.

Family-based guilds in the ant Pachycondyla inversa.

High relatedness promotes the evolution of sociality because potentially costly cooperative behaviours are directed towards kin. However, societies, such as those of social insects, also benefit from genetic diversity, e.g. through enhanced disease resistance and division of labour. Effects of genetic diversity have been investigated in a few complex eusocial species. Here, we show that genetically based division of labour may also be important in 'simple societies', with fewer individuals and limited morphological caste differentiation. The ponerine ant Pachycondyla inversa has small colonies, headed by several unrelated queens. We show that nest-mate workers from different matrilines engage in different tasks, have distinct chemical profiles and associate preferentially with kin in the nest, while queens and brood stay together. This suggests that genetically based division of labour may precede the evolution of complex eusociality and facilitate the existence of low relatedness societies functioning as associations of distinct families that mutually benefit from group living.

Towards greater realism in inclusive fitness models: the case of worker reproduction in insect societies.

The conflicts over sex allocation and male production in insect societies have long served as an important test bed for Hamilton's theory of inclusive fitness, but have for the most part been considered separately. Here, we develop new coevolutionary models to examine the interaction between these two conflicts and demonstrate that sex ratio and colony productivity costs of worker reproduction can lead to vastly different outcomes even in species that show no variation in their relatedness structure. Empirical data on worker-produced males in eight species of Melipona bees support the predictions from a model that takes into account the demographic details of colony growth and reproduction. Overall, these models contribute significantly to explaining behavioural variation that previous theories could not account for.

Relatedness modulates reproductive competition among queens in ant societies with multiple queens.

Reproductive sharing in animal groups with multiple breeders, insects and vertebrates alike, contains elements of both conflict and cooperation, and depends on both relatedness between co-breeders, as well as their internal and external conditions. We studied how queens of the ant Formica fusca adjust their reproductive efforts in response to experimental manipulations of the kin competition regime in their nest. Queens respond to the presence of competitors by increasing their egg laying efforts, but only if the competitors are highly fecund and distantly related. Such a mechanism is likely to decrease harmful competition among close relatives. We demonstrate that queens of Formica fusca fine-tune their cooperative breeding behaviors in response to kinship and fecundity of others in a remarkably precise and flexible manner.

The role of cognition in nesting.

For many animals, nests are essential for reproductive success. Nesting individuals need to carry out a range of potentially challenging tasks, from selecting an appropriate site and choosing suitable materials to constructing the nest and defending it against competitors, parasites and predators. Given the high fitness stakes involved, and the diverse impacts both the abiotic and social environment can have on nesting success, we might expect cognition to facilitate nesting efforts. This should be especially true under variable environmental conditions, including those changing due to anthropogenic impacts. Here, we review, across a wide range of taxa, evidence linking cognition to nesting behaviours, including selection of nesting sites and materials, nest construction, and nest defence. We also discuss how different cognitive abilities may increase an individual's nesting success. Finally, we highlight how combining experimental and comparative research can uncover the links between cognitive abilities, nesting behaviours and the evolutionary pathways that may have led to the associations between them. In so doing, the review highlights current knowledge gaps and provides suggestions for future research. This article is part of the theme issue 'The evolutionary ecology of nests: a cross-taxon approach'.