Reproductive specialization in multiple-queen colonies of the ant Formica exsecta

In polygynous (multiple queens per nest) colonies of socialinsects, queens can increase their reproductive share by layingmore eggs or by increasing the proportion of eggs that developinto reproductive individuals instead of workers. We used polymorphicmicrosatellite loci to determine the genetically effective contributionof queens to the production of gynes (new queens), males, and2 different cohorts of workers in a polygynous population ofthe ant Formica exsecta. For this purpose, we developed a newmethod that can be used for diploid and haplodiploid organismsto quantify the degree of reproductive specialization amongbreeders in societies where there are too many breeders to ascertainparentage. Using this method, we found a high degree of reproductivespecialization among nest-mate queens in both female- and male-producingcolonies (sex ratio is bimodally distributed in the study population).For example, a high effective proportion of queens (25% and79%, respectively) were specialized in the production of malesin female- and male-producing colonies. Our analyses furtherrevealed that in female-producing colonies, significantly fewerqueens contributed to gyne production than to worker production.Finally, we found significant changes in the identity of queenscontributing to different cohorts of workers. Altogether, thesedata demonstrate that colonies of F. exsecta, and probably thoseof many other highly polygynous social insect species, are composedof reproductive individuals differing in their investment togynes, males, and workers. These findings demonstrate a newaspect of the highly dynamic social organization of complexanimal societies.     

Reproductive conflicts in polyandrous and polygynous ant Formica sanguinea

The occurrence of multiple reproductives within an ant colony changes the balance between indirect fitness benefits and reproductive competition. We test whether the number of matings by an ant queen (polyandry) correlates negatively with the number of reproductive queens in the colony (polygyny), whether the patrilines and matrilines differ in their contribution to the sexual and worker progeny and whether there is an overall reproductive skew. For these aims, we genotyped both worker and sexual offspring from colonies of the ant Formica sanguinea in three populations. Most colonies were monogynous, but eight (11%) were polygynous with closely related queens. Most queens in the monogynous colonies (86%) had mated with multiple males. The effective paternity was lower than the actual number of mates, and the paternity skew was significant. Furthermore, in some monogynous colonies, the patrilines were differently represented in the worker pupae and sexual pupae produced at the same time. Likewise, the matrilines in polygynous colonies were differently present in worker pupae and male offspring. The effective number of matings by a queen was significantly lower in polygynous colonies (mean m_e = 1.68) than in monogynous colonies (means 2.06–2.61). The results give support to the hypotheses that polyandry and polygyny are alternative breeding strategies and that reproductive competition can lead to different representation of patrilines and matrilines among the sexual and worker broods.     

Sex-ratio dependent execution of queens in polygynous colonies of the ant Formica exsecta

Formica exsecta has become an important model system for studying intraspecific variation in sex ratios. Patterns of sex allocation in polygynous (multiple queen per nest) populations of F. exsecta are generally consistent with the queen-replenishment hypothesis. This hypothesis states that colonies produce gynes (reproductive females) in order to increase queen number and enhance colony survival and/or productivity when the number of resident queens is low. However, the small proportion of colonies that raise gynes produce more than necessary for simple queen replenishment. It has been hypothesized that excess production of gynes may occur to reduce the frequency of accepting foreign unrelated gynes into the colony when workers cannot distinguish nestmate from non-nestmate queens. This explanation for excess gynes requires weak or no aggression between non-nestmates and is expected to lead to the selective execution of new queens by colonies that do not invest in the production of gynes. Experimental studies where gynes were introduced into natal and foreign colonies indeed suggested that polygynous populations of F. exsecta have a poor nestmate recognition system. Although gynes were significantly more likely to be accepted in their parental colony compared to another foreign female-producing colony, the difference was small. Moreover, encounters between workers from different colonies within the population showed very little aggression and were no more aggressive than encounters between nestmates, again suggesting a weak capacity for nestmate recognition. Our experiment also showed that colonies that produced only males executed most of the gynes that were experimentally introduced into the colony, whereas female-producing colonies accepted most gynes. This is consistent with ants using a simple rule of thumb to decrease parasitism by unrelated queens, whereby colonies selectively destroy gynes whenever gynes are not produced in the colonies.     

Queen recruitment and split sex ratios in polygynous colonies of the ant Formica exsecta

Sex ratios in social insects have become a general model for tests of inclusive fitness theory, sex ratio theory and parent–offspring conflict. In populations of Formica exsecta with multiple queens per colony , sex ratios vary greatly among colonies and the dry-weight sex ratio is extremely male-biased, with 89% of the colonies producing males but no gynes (reproductive females). Here we test the queen-replenishment hypothesis, which was proposed to explain sex ratio specialization in this and other highly polygynous ants (i.e. those with many queens per nest). This hypothesis proposes that, in such ants, colonies produce gynes to recruit them back into the colony when the number of resident queens falls below a given threshold limiting colony productivity or survival. We tested predictions of the queen-replenishment hypothesis by following F. exsecta colonies across two breeding seasons and relating the change in effective queen number with changes in sex ratio, colony size and brood production. As predicted by the queen-replenishment hypothesis, we found that colonies that specialized in producing females increased their effective queen number and were significantly more likely to specialize in male production the following year. The switch to male production also coincided with a drop in productivity per queen as predicted. However, adoption of new queens did not result in a significant increase in total colony productivity the following year. We suggest that this is because queen production comes at the expense of worker production and thus queen production leads to resource limitation the following year, buffering the effect of greater queen number on total productivity.     

Restricted effective queen dispersal at a microgeographic scale in polygynous populations of the ant Formica exsecta

Ecological constraints on effective dispersal have been suggested to be a key factor influencing social evolution in animal societies as well as the shift from single queen colonies (monogyny) to multiple queen colonies (polygyny) in ants. However, little is known about the effective dispersal patterns of ant queens. Here we investigate the microgeographic genetic structure of mitochondrial haplotypes in polygynous populations of the ant Formica exsecta, both between pastures and among nests within pastures. An analysis of molecular variance revealed a very high genetic differentiation (phiST = 0.72) between pastures, indicating that queens rarely disperse successfully between pastures, despite the fact that pastures were sometimes as close as 1 km. Most of the pastures contained only a single haplotype, and haplotypes were frequently distinct between nearby pastures and even between groups of nests within the same pasture. In the three pastures that contained several haplotypes, haplotypes were not randomly distributed, the genetic differentiation between nests being phiST = 0.17, 0.52, and 0.69. This indicates that most queens are recruited within their parental colonies. However, a large proportion of nests contained more than one haplotype, demonstrating that colonies will sometimes accept foreign queens. The relatedness of mitochondrial genes among nestmates varied between 0.62 and 0.75 when relatedness was measured within each pasture and ranged between 0.72 and 1.0 when relatedness was assessed with all pastures as a reference population. Neighboring nests were more genetically similar than distant ones, and there was significant isolation by distance. This pattern may be due to new nests being formed by budding or by limited effective queen dispersal, probably on foot between neighboring nests. These results show that effective queen dispersal is extremely restricted even at a small geographical scale, a pattern consistent with the idea that ecological constraints are an important selective force leading to the evolution and maintenance of polygyny.     

Colony sex ratios vary with queen number but not relatedness asymmetry in the ant Formica exsecta

Split-sex-ratio theory assumes that conflict over whether to produce predominately males or female reproductives (gynes) is won by the workers in haplodiploid insect societies and the outcome is determined by colony kin structure. Tests of the theory have the potential to provide support for kin-selection theory and evidence of social conflict. We use natural variation in kinship among polygynous (multiple-queen) colonies of the ant Formica exsecta to study the associations between sex ratios and the relatedness of workers to female versus male brood (relatedness asymmetry). The population showed split sex ratios with about 89% of the colonies producing only males, resulting in an extremely male-biased investment ratio in the population. We make two important points with our data. First, we show that queen number may affect sex ratio independently of relatedness asymmetry. Colonies producing only males had greater genetic effective queen number but did not have greater relatedness asymmetry from the perspective of the adult workers that rear the brood. This lack of a difference in relatedness asymmetry between colonies producing females and those producing only males was associated with a generally low relatedness between workers and brood. Second, studies that suggest support for the relatedness-asymmetry hypothesis based on indirect measures of relatedness asymmetry (e.g. queen number estimated from relatedness data taken from the brood only) should be considered with caution. We propose a new hypothesis that explains split sex ratios in polygynous social insects based on the value of producing replacement queens.     

Inbreeding and reproductive investment in the ant Formica exsecta

In social animals, inbreeding depression may manifest by compromising care or resources individuals receive from inbred group members. We studied the effects of worker inbreeding on colony productivity and investment in the ant Formica exsecta. The production of biomass decreased with increasing inbreeding, as did biomass produced per worker. Inbred colonies produced fewer gynes (unmated reproductive females), whereas the numbers of males remained unchanged. As a result, sex ratios showed increased male bias, and the fraction of workers increased among the diploid brood. Males raised in inbred colonies were smaller, whereas the weight of gynes remained unchanged. The results probably reflect a trade-off between number and quality of offspring, which is expected if the reproductive success of gynes is more dependent on their weight or condition than it is for males. As males are haploid (with the exception of abnormal diploid males produced in very low frequencies in this population), and therefore cannot be inbred themselves, the effect on their size must be mediated through the workers of the colony. We suggest the effects are caused by the inbred workers being less proficient in feeding the growing larvae. This represents a new kind of social inbreeding depression that may affect sex ratios as well as caste fate in social insects.     

Experimental manipulation of queen number affects colony sex ratio investment in the highly polygynous ant Formica exsecta

In polygynous (multiple queens per nest) ants, queen dispersal is often limited with young queens being recruited within the parental colony. This mode of dispersal leads to local resource competition between nestmate queens and is frequently associated with extremely male-biased sex ratios at the population level. The queen-replenishment hypothesis has been recently proposed to explain colony sex ratio investment under such conditions. It predicts that colonies containing many queens (subject to high local resource competition) should only produce males, whereas colonies hosting few queens (reduced or no local resource competition) should produce new queens in addition to males. We experimentally tested this hypothesis in the ant Formica exsecta by manipulating queen number over three consecutive years in 120 colonies of a highly polygynous population. Queens were transferred from 40 colonies into another 40 colonies while queen number was not manipulated in 40 control colonies. Genetic analyses of worker offspring revealed that our treatment significantly changed the number of reproductive queens. The sex ratio of colonies was significantly different between treatments in the third breeding season following the experiment initiation. We found that, as predicted by the queen-replenishment hypothesis, queen removal resulted in a significant increase in the proportion of colonies that produced new queens. These results provide the first experimental evidence for the queen-replenishment hypothesis, which might account for sex ratio specialization in many highly polygynous ant species.     

Coexistence of the social types: genetic population structure in the ant Formica exsecta

The ant Formica exsecta has two types of colonies that exist in sympatry but usually as separate subpopulations: colonies with simple social organization and single queens (M type) or colonial networks with multiple queens (P type). We used both nuclear (DNA microsatellites) and mitochondrial markers to study the transition between the social types, and the contribution of males and females in gene flow within and between the types. Our results showed that the social types had different spatial genetic structures. The M subpopulations formed a fairly uniform population, whereas the P subpopulations were, on average, more differentiated from each other than from the nearby M subpopulations and could have been locally established from the M-type colonies, followed by philopatric behavior and restricted emigration of females. Thus, the relationship between the two social types resembles that of source (M type) and sink (P type) populations. The comparison of mitochondrial (phiST) and nuclear (FST) differentiation indicates that the dispersal rate of males is four to five times larger than that of females both among the P-type subpopulations and between the social types. Our results suggest that evolution toward complex social organization can have an important effect on genetic population structure through changes in dispersal behavior associated with different sociogenetic organizations.     

Polymorphic microsatellite DNA markers in the ant Formica exsecta

Highly polymorphic genetic markers provide a useful tool for estimating important genetic parameters in studies of the evolution of sociality in insects. Here we report 14 polymorphic microsatellite markers developed in the ant Formica exsecta. The number of alleles found ranged between 3 and 18 per locus. These markers were developed for studying genetic population structure and mating structure in F. exsecta populations with varying social organizations (monogyne and polygyne types of societies). Cross‐species amplification indicated that some of the markers might be usable even in species belonging to different subfamilies.     

Inbreeding and sex-biased gene flow in the ant Formica exsecta

Abstract.— The objective of this study was to assess breeding and dispersal patterns of both males and females in a monogyne (a single queen per colony) population of ants. Monogyny is commonly associated with extensive nuptial flights, presumably leading to considerable gene flow over large areas. Opposite to these expectations we found evidence of both inbreeding and sex-biased gene flow in a monogyne population of Formica exsecta . We found a significant degree of population subdivision at a local scale (within islands) for queens (females heading established colonies) and workers, but not for colony fathers (the males mated to the colony queens). However, we found little evidence of population subdivision at a larger scale (among islands). More conclusive support for sex-biased gene flow comes from the analysis of isolation by distance on the largest island, and from assignment tests revealing differences in female and male philopatry. The genetic similarity between pairs of queens decreased significantly when geographical distance increased, demonstrating limited dispersal and isolation by distance in queens. By contrast, we found no such pattern for colony fathers. Furthermore, a significantly greater fraction of colony queens were assigned as having originated from the population of residence, as compared to colony fathers. Inbreeding coefficients were significantly positive for workers, but not for mother queens. The queen-male relatedness coefficient of 0.23 (regression relatedness) indicates that mating occurs between fairly close relatives. These results suggest that some monogyne species of ants have complex dispersal and mating systems that can result in genetic isolation by distance over small geographical scales. More generally, this study also highlights the importance of identifying the relevant scale in analyses of population structure and dispersal.     

Inbreeding-related trade-offs in stress resistance in the ant Formica exsecta

Inbred individuals and populations are predicted to suffer from inbreeding depression, especially in times of stress. Under natural conditions, organisms are exposed to more than one stressor at any one time, highlighting the importance of stress resistance traits. We studied how inbreeding- and immunity-related traits are correlated under different dietary conditions in the ant Formica exsecta. Its natural diet varies in the amount and nature of plant secondary compounds and the level of free radicals, all of which require detoxification to maintain organismal homeostasis. We found that inbreeding decreased general antibacterial activity under dietary stress, suggesting inbreeding-related physiological trade-offs.     

Chemical basis of nest-mate discrimination in the ant Formica exsecta.

Distinguishing nest-mates from non-nest-mates underlies key animal behaviours, such as territoriality, altruism and the evolution of sociality. Despite its importance, there is very little empirical support for such a mechanism in nature. Here we provide data that the nest-mate recognition mechanism in an ant is based on a colony-specific Z9-alkene signature, proving that surface chemicals are indeed used in ant nest-mate recognition as was suggested 100 years ago. We investigated the cuticular hydrocarbon profiles of 10 Formica exsecta colonies that are composed almost entirely of a Z9-alkene and alkane component. Then we showed that worker aggression is only elicited by the Z9-alkene part. This was confirmed using synthetic Z9-alkene and alkane blends matched to the individual colony profiles of the two most different chemical colonies. In both colonies, only glass beads with 'nest-mate' alkene profiles received reduced aggression. Finally, changing the abundance of a single Z9-alkene on live ants was shown to significantly increase the aggression they received from nest-mates in all five colonies tested. Our data suggest that nest-mate discrimination in the social insects has evolved to rely upon highly sensitive responses to relatively few compounds.     

High levels of multiple Wolbachia infection and recombination in the ant Formica exsecta

Wolbachia bacteria are intracellular symbionts of many arthropod species. Their spread through host populations is promoted by drastic alterations imposed on their hosts' reproductive physiology. In the present study, we analyzed the association between Wolbachia strains and host mitochondrial haplotypes in a Swiss population of the ant Formica exsecta. In this species, female dispersal is extremely limited and the mitochondrial haplotypes are strongly differentiated between and within subpopulations. Our study revealed exceptionally high levels of multiple infection, with all ants harboring four or five distinct Wolbachia strains. Four of these strains were present in all ants analyzed. A fifth strain was associated with only three of the five mitochondrial haplotypes. An analysis of the Wolbachia gene wsp further revealed an unexpected high rate of recombination, with three of the five Wolbachia strains appearing to have arisen by homologous recombination.     

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.     

Reproductive sharing among queens in the ant Formica fusca

Reproductive sharing among cobreeders, in which reproductiveshares may vary from equal contribution (low reproductive skew)to reproductive dominance by one individual (high reproductiveskew), is a fundamental feature of animal societies. Recenttheoretical work, the reproductive skew models, has focusedon factors affecting the degree to which reproduction is skewedwithin a society. We used the parameters provided by skew modelsas a guideline to study determinants of reproductive sharingin polygyne ants. As a model system we used two-queen laboratorycolonies of the ant Formica fusca in which the reproductiveshares of each queen was assessed from offspring by using allozymesand DNA microsatellites. We tested how the different variablesincluded in reproductive skew models (queen-queen relatedness,potential fighting ability, productivity, and worker relatednessreflected by queen number in the colony of origin) affect reproductivesharing among queens. The results showed that the relatednessamong queens explained 26% of the variation in reproductiveskew. The size difference between queens (reflecting potentialfighting ability), colony productivity, and worker relatednessdid not have an effect on reproductive partitioning among cobreeders.To our knowledge, this is the first study to test for the effectsof various determinants of skew in an experimental setting.     

Caste‐biases in gene expression are specific to developmental stage in the ant Formica exsecta

Understanding how a single genome creates and maintains distinct phenotypes is a central goal in evolutionary biology. Social insects are a striking example of co‐opted genetic backgrounds giving rise to dramatically different phenotypes, such as queen and worker castes. A conserved set of molecular pathways, previously envisioned as a set of ‘toolkit’ genes, has been hypothesized to underlie queen and worker phenotypes in independently evolved social insect lineages. Here, we investigated the toolkit from a developmental point of view, using RNA‐Seq to compare caste‐biased gene expression patterns across three life stages (pupae, emerging adult and old adult) and two female castes (queens and workers) in the ant Formica exsecta. We found that the number of genes with caste‐biased expression increases dramatically from pupal to old adult stages. This result suggests that phenotypic differences between queens and workers at the pupal stage may derive from a relatively low number of caste‐biased genes, compared to higher number of genes required to maintain caste differences at the adult stage. Gene expression patterns were more similar among castes within developmental stages than within castes despite the extensive phenotypic differences between queens and workers. Caste‐biased expression was highly variable among life stages at the level of single genes, but more consistent when gene functions (gene ontology terms) were investigated. Finally, we found that a large part of putative toolkit genes were caste‐biased at least in some life stages in F. exsecta, and the caste‐biases, but not their direction, were more often shared between F. exsecta and other ant species than between F. exsecta and bees. Our results indicate that gene expression should be examined across several developmental stages to fully reveal the genetic basis of polyphenisms.     

Size matters: mole (Talpa europaea) hills and nest-site selection of the ant Formica exsecta

Moles are fossorial mammals that can act both as zoogeomorphic agents and species diversity drivers. These popular animals regularly push heaps of earth from their subterranean tunnel systems to the surface. Thereby they rearrange and improve the local microtopography for ant nesting. Here we use a strongly molehill (Talpa europaea) mediated nest system of the unicolonial wood ant Formica (Coptoformica) exsecta to test for ecological factors influencing nest-site selection at the microhabitat scale. Our results show that the size of molehills plays an important role in the multifactorial process of the ant’s nest-site choice with solar insolation as a paramount factor. The ants clearly favored larger and better sun-exposed molehills, suggesting that the coaction of a zoogeomorphic modified microrelief and solar insolation can drive the spatial colonization of F. exsecta.     

Individual variation in immune function in the ant Formica exsecta; effects of the nest, body size and sex

It has been generally thought that sex differences in the immune system are the result of the differing life history strategies of the sexes, although the available data are not entirely consistent with the hypothesis. In this study, we studied the variation in the immune function in the mound-building wood ant Formica exsecta. F. exsecta has two forms of males, distinguished by size: the small males (micraners) and the large males (macraners), which die after the mating period, whereas females live tens of years laying their eggs. We found that in general males have a lower encapsulation response against nylon monofilament (i.e. lower immune function) than queens. Among males, the micraners had a lower encapsulation rate than the macraners. However, in queens, there was no correlation between size and encapsulation rate. The origin nest had an effect on the encapsulation rate of males: males from the large nests had a stronger encapsulation rate than males from small nests. However, in queens, nest size did not have any effect on encapsulation response. The observed variation between sexes and individuals in the encapsulation rate is discussed in the context of reproductive strategies and parasite-mediated sexual selection.     

Male aggressiveness in a polygynous ungulate varies with social and ecological context

Aggression is a social behaviour which can be affected by numerous factors. The quality and quantity of food resources may play an important role in the aggressiveness of territorial ungulates as the defence of these resources influences female choice and mating opportunities. However, the relationship between food resources and aggression remains poorly understood. We assessed the ecological and social factors that influence aggression in Lama guanicoe, a territorial ungulate exhibiting resource‐defence polygyny, during three periods (group‐formation, mating and post‐mating) in the reproductive seasons of 2014 and 2016. We recorded 460 focal observations of territorial (family groups, solitary) and non‐territorial (mixed and bachelor groups) males. We performed analyses at the population level (including all focal observations) and at the group level (each social unit separately), to test whether the factors that influence aggression differ at these different scales. We also identified proxies of vegetation quality as potential predictors of aggression. At the population level, we found that the presence of aggressive behaviour peaked during the mating season and that post‐mating aggression may have been driven by inter‐annual environmental variations. For family groups and solitary males, variables reflecting high vegetation quality/quantity were predictors of aggressive behaviour, reflecting the resource‐defence strategy of this species. Conversely, for mixed‐group males, aggression may be more associated with social instability and group size, although this hypothesis has yet to be tested. Our research reinforces the idea that aggression can occur in multiple contexts depending on male status (e.g. territorial or non‐territorial) and contributes to our understanding of how ecological (i.e. availability of food resources) and social factors influence aggression in a territorial ungulate.