MICROSATELLITES REVEAL HIGH POPULATION VISCOSITY AND LIMITED DISPERSAL IN THE ANT FORMICA PARALUGUBRIS

We used microsatellites to study the fine-scale genetic structure of a highly polygynous and largely unicolonial population of the ant Formica paralugubris. Genetic data indicate that long-distance gene flow between established nests is limited and new queens are primarily recruited from within their natal nest. Most matings occur between nestmates and are random at this level. In the center of the study area, budding and permanent connections between nests result in strong population viscosity, with close nests being more similar genetically than distant nests. In contrast, nests located outside of this supercolony show no isolation by distance, suggesting that they have been initiated by queens that participated in mating flights rather than by budding from nearby nests in our sample population. Recruitment of nestmates as new reproductive individuals and population viscosity in the supercolony increase genetic differentiation between nests. This in turn inflates relatedness estimates among worker nestmates (r = 0.17) above what is due to close pedigree links. Local spatial genetic differentiation may favor the maintenance of altruism when workers raise queens that will disperse on foot and compete with less related queens from neighboring nests or disperse on the wing and compete with unrelated queens.     

Genetic clusters and sex-biased gene flow in a unicolonial Formica ant

Background  

Animal societies are diverse, ranging from small family-based groups to extraordinarily large social networks in which many unrelated individuals interact. At the extreme of this continuum, some ant species form unicolonial populations in which workers and queens can move among multiple interconnected nests without eliciting aggression. Although unicoloniality has been mostly studied in invasive ants, it also occurs in some native non-invasive species. Unicoloniality is commonly associated with very high queen number, which may result in levels of relatedness among nestmates being so low as to raise the question of the maintenance of altruism by kin selection in such systems. However, the actual relatedness among cooperating individuals critically depends on effective dispersal and the ensuing pattern of genetic structuring. In order to better understand the evolution of unicoloniality in native non-invasive ants, we investigated the fine-scale population genetic structure and gene flow in three unicolonial populations of the wood ant F. paralugubris.     

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.     

Multiple breeders, breeder shifts and inclusive fitness returns in an ant

In social insects, colonies may contain multiple reproductively active queens. This leads to potential conflicts over the apportionment of brood maternity, especially with respect to the production of reproductive offspring. We investigated reproductive partitioning in offspring females (gynes) and workers in the ant Formica fusca, and combined this information with data on the genetic returns gained by workers. Our results provide the first evidence that differential reproductive partitioning among breeders can enhance the inclusive fitness returns for sterile individuals that tend non-descendant offspring. Two aspects of reproductive partitioning contribute to this outcome. First, significantly fewer mother queens contribute to gyne (new reproductive females) than to worker brood, such that relatedness increases from worker to gyne brood. Second, and more importantly, adult workers were significantly more related to the reproductive brood raised by the colony, than to the contemporary worker brood. Thus, the observed breeder shift leads to genetic benefits for the adult workers that tend the brood. Our results also have repercussions for genetic population analyses. Given the observed pattern of reproductive partitioning, estimates of effective population size based on worker and gyne samples are not interchangeable.     

Inbreeding and kinship in the ant Plagiolepis pygmaea

In ants the presence of multiple reproductive queens (polygyny) decreases the relatedness among workers and the brood they rear, and subsequently dilutes their inclusive fitness benefits from helping. However, adoption of colony daughters, low male dispersal in conjunction with intranidal (within nest) mating and colony reproduction by budding may preserve local genetic differences, and slow down the erosion of relatedness. Reduced dispersal and intranidal mating may, however, also lead to detrimental effects owing to competition and inbreeding. We studied mating and dispersal patterns, and colony kinship in three populations of the polygynous ant Plagiolepis pygmaea using microsatellite markers. We found that the populations were genetically differentiated, but also a considerable degree of genetic structuring within populations. The genetic viscosity within populations can be attributed to few genetically homogeneous colony networks, which presumably have arisen through colony reproduction by budding. Hence, selection may act at different levels, the individuals, the colonies and colony networks. All populations were also significantly inbred (F=0.265) suggesting high frequencies of intranidal mating and low male dispersal. Consequently the mean regression relatedness among workers was significantly higher (r = 0.529-0.546) than would be expected under the typically reported number (5-35) of queens in nests of the species. Furthermore, new queens were mainly recruited from their natal or a neighbouring related colony. Finally, the effective number of queens coincided with that found upon excavation, suggesting low reproductive skew.     

Colony structure of a slavemaking ant. I. Intracolony relatedness, worker reproduction, and polydomy

Colony and population structure of the obligate slavemaker ant Protomognathus americanus was analyzed via four nuclear microsatellite loci and mitochondrial DNA (mtDNA) markers. Colonies of P. americanus usually contain a single queen, and here we show that she is singly inseminated. Nestmate workers are generally full sisters and their relatedness does not deviate from the expected value of 0.75. Even though colonies were strictly monogynous, we were able to infer that colony takeover by related queens was common and queen replacement by unrelated queens was rare. Polydomy is widespread, with neighboring nests having the same genetic composition. Although we found no evidence of population viscosity or inbreeding from nuclear markers, mtDNA markers provided evidence for small-scale genetic structuring. Haplotype structuring and takeover by related queens suggest philopatry of newly mated queens. In this species, workers reproduce in queenright and queenless nests and worker reproduction accounts for more than 70% of all males. Although sex-ratio theory points to slavemaking ants as important systems for studying queen-worker conflict, our results indicate no basis for such conflict in P. americanus, because extensive worker reproduction generates shifts in relatedness values. Rather, the dual effects of independent polydomous nest units and local resource competition among queens produce male-biased allocation ratios in this species.     

Kin structure and queen execution in the Argentine ant Linepithema humile

Every spring, workers of the Argentine Ant Linepithema humile kill a large proportion of queens within their nests. Although this behaviour inflicts a high energetic cost on the colonies, its biological significance has remained elusive so far. An earlier study showed that the probability of a queen being executed is not related to her weight, fecundity, or age. Here we test the hypothesis that workers collectively eliminate queens to which they are less related, thereby increasing their inclusive fitness. We found no evidence for this hypothesis. Workers of a nest were on average not significantly less related to executed queens than to surviving ones. Moreover, a population genetic analysis revealed that workers were not genetically differentiated between nests. This means that workers of a given nest are equally related to any queen in the population and that there can be no increase in average worker–queen relatedness by selective elimination of queens. Finally, our genetic analyses also showed that, in contrast to workers, queens were significantly genetically differentiated between nests and that there was significant isolation by distance for queens.     

Mating frequency and genetic structure of the Argentine ant Linepithema humile

The nest and population genetic structures of the Argentine ant, Linepithema humile were investigated using eight microsatellite loci. Genotypes of the sperm from spermathecae of 87 queens were consistent with all queens being singly inseminated. The probability of a double mating remaining undetected was low (0.012) suggesting that no queens or only a very low proportion mate multiply. The relatedness between the queens and their mates was negative (R = -0.164 +/- 0.044) and significantly different to zero (P = 0.020). However, the high negative relatedness value was caused by a significant allele frequency difference between the sexes at a single locus (Lhum-28). When this locus was removed from the analyses, the relatedness was not significantly different from zero (R = 0.013 +/- 0.050, P = 0.812). Analysis of 10 nests revealed that the genetic differentiation among nests was weak (FST = 0.003) and not distinguishable from zero (P = 0.468). Similarly, the overall relatedness among nestmate females was not significantly different from zero (R = 0.007 +/- 0.018, P = 0.706). These results are consistent with the lack of distinct nest boundaries and the large number of queens per nest in the population studied. Although mating takes place inside the nest, the inbreeding coefficient was close to zero (F = 0.007 +/- 0.025, P = 0.786). Overall, these data indicate substantial local gene flow mediated by movement of reproductives among colonies.     

Highly variable social organisation of colonies in the ant Formica cinerea

Social organisation of colonies was examined in the ant Formica cinerea by estimating the coefficient of genetic relatedness among worker nest mates. The estimates based on microsatellite genotypes at three loci ranged from values close to zero to 0.61 across the populations studied in Finland. These results showed that a fundamental feature of colonies, the number of reproductive queens, varied greatly among the populations. Colonies in some populations had a single queen, whereas the nests could have a high number number of queens in other populations. There was a weak but non-significant correlation between the genetic and metric distance of nests within two populations with intermediate level of relatedness. Differentiation among nearby populations (within the dispersal distance of individuals) in one locality indicated limited dispersal or founder effects. This could occur when females are philopatric and stay in the natal polygynous colony which expands by building a network of nest galleries within a single habitat patch.     

A TEST OF QUEEN RECRUITMENT MODELS USING NUCLEAR AND MITOCHONDRIAL MARKERS IN THE FIRE ANT SOLENOPSIS INVICTA

We assess nestmate queen relatedness and the genetic similarity of neighboring nests in the polygyne (multiple-queen) social form of the introduced fire ant Solenopsis invicta using both nuclear and mitochondrial markers. We find that estimates of queen relatedness calculated with both types of markers do not differ statistically from zero. Furthermore, there is no significant relationship between the genetic similarity and geographic proximity of nests in each of six study sites. In contrast to these findings, sites show strong mitochondrial, but no nuclear, genetic differentiation. Our results suggest that nonnestmate queen recruitment occurs at a high frequency in introduced populations of this species. Moreover, queens within nests seem to represent a random sample of the queens within the site in which they reside. Therefore, kin selection models that rely on the recruitment of only nestmate queens to explain the persistence of polygyny in ants do not apply to polygyne S. invicta in its introduced range.     

Convergent development of low-relatedness supercolonies in Myrmica ants

Many ant species have independently evolved colony structures with multiple queens and very low relatedness among nestmate workers, but it has remained unclear whether low-relatedness kin structures can repeatedly arise in populations of the same species. Here we report a study of Danish island populations of the red ant Myrmica sulcinodis and show that it is likely that such repeated developments occur. Two microsatellite loci were used to estimate genetic differentiation (F(ST)) among three populations and nestmate relatedness within these populations. The F(ST) values were highly significant due to very different allele frequencies among the three populations with relatively few common alleles and relatively many rare alleles, possibly caused by single queen foundation and rare subsequent immigration. Given the isolation of the islands and the low investment in reproduction, we infer that each of the populations was most likely established by a single queen, even though all three extant populations now have within-colony relatedness 95%), and the genetic differentiation of nests showed a significantly positive correlation with the distance between them. Both male-biased sex-ratio and genetic viscosity are expected characteristics of populations where queens have very local dispersal and where new colonies are initiated through nest-budding. Based on a comparison with other M. sulcinodis populations we hypothesise a distinct succession of population types and suggest that this may be a possible pathway to unicoloniality, ie, development towards a complete lack of colony kin structure and unrelated nestmate workers.     

Breeding system and reproductive skew in a highly polygynous ant population

Factors affecting relatedness among nest members in ant colonies with high queen number are still poorly understood. In order to identify the major determinants of nest kin structure, we conducted a detailed analysis of the breeding system of the ant Formica exsecta. We estimated the number of mature queens by mark-release-recapture in 29 nests and dissected a sub-sample of queens to assess their reproductive status. We also used microsatellites to estimate relatedness within and between all classes of nestmates (queens, their mates, worker brood, queen brood and male brood). Queen number was very high, with an arithmetic mean of 253 per nest. Most queens (90%) were reproductively active, consistent with the genetic analyses revealing that there was only a minimal reproductive skew among nestmate queens. Despite the high queen number and low reproductive skew, almost all classes of individuals were significantly related to each other. Interestingly, the number of resident queens was a poor predictor of kin structure at the nest level, consistent with the observation that new queens are produced in bursts leading to highly fluctuating queen number across years. Queen number also varied tremendously across nests, with estimates ranging from five to several hundred queens. Accordingly, the harmonic mean queen number (40.5) was six times lower than the arithmetic mean. The variation in queen number was the most important factor of the breeding system contributing to a significant relatedness between almost all classes of nestmates despite a high average number of queens per nest. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 12 November 2007; revised 4 May 2008; accepted 8 May 2008.     

Genetic determination of female castes in a hybridogenetic desert ant

In most social insects, the brood is totipotent and environmental factors determine whether a female egg will develop into a reproductive queen or a functionally sterile worker. However, genetic factors have been shown to affect the female's caste fate in a few ant species. The desert ant Cataglyphis hispanica reproduces by social hybridogenesis. All populations are characterized by the coexistence of two distinct genetic lineages. Queens are almost always found mated with a male of the alternate lineage than their own. Workers develop from hybrid crosses between the genetic lineages, whereas daughter queens are produced asexually via parthenogenesis. Here, we show that the association between genotype and caste in this species is maintained by a ‘hard‐wired’ genetic caste determination system, whereby nonhybrid genomes have lost the ability to develop as workers. Genetic analyses reveal that, in a rare population with multiple‐queen colonies, a significant proportion of nestmate queens are mated with males of their own lineage. These queens fail to produce worker offspring; they produce only purebred daughter queens by sexual reproduction. We discuss how the production of reproductive queens through sexual, intralineage crosses may favour the stability of social hybridogenesis in this species.     

Resource allocation in a social wasp: effects of breeding system and life cycle on reproductive decisions

Organisms must make important decisions on how to allocate resources to reproduction. We investigated allocation decisions in the social wasp Vespula maculifrons to understand how social insects make reproductive choices. We first determined how annual colonies apportioned resources to growth and reproduction by analysing developing brood. In contrast to expectations, colonies invested in both growth (workers) and reproduction (males) simultaneously. In addition, colonies showed evidence of producing males in pulses and reversing their reproductive choices by decreasing investment in males late in the season. This reversal is consistent with theory suggesting that colonies decrease production in males if fitness of late emerging males is low. To further investigate reproductive decisions within colonies, we determined if the male mates of multiply-mated queens varied in their reproductive success over time. Sperm use by queens did vary over time suggesting that male success may depend on sperm clumping within the female reproductive tract. Finally, we tested if colony sex ratio conformed to expectations under kin selection theory that nestmate relatedness would positively correlate with investment in new queens if workers controlled sex allocation. Surprisingly, the proportion of queens produced by colonies was negatively correlated with nestmate relatedness, suggesting that allocation may be shaped by advantages arising from increased genetic diversity resulting from multiple mating by queens. Overall, our study suggests that the reproductive decisions of colonies are flexible and may depend both on environmental cues arising from energetic needs of the colony and genetic cues arising from mating behaviours of queens.     

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.     

Sib‐mating in the ant Plagiolepis pygmaea: adaptative inbreeding?

Multiple functional queens in a colony (polygyny) and multiple mating by queens (polyandry) in social insects challenge kin selection, because they dilute inclusive fitness benefits from helping. Colonies of the ant Plagiolepis pygmaea brash contain several hundreds of multiply mated queens. Yet, within‐colony relatedness remains unexpectedly high. This stems from low male dispersal, extensive mating among relatives and adoption of young queens in the natal colony. We investigated whether inbreeding results from workers expelling foreign males, and/or from preferential mating between related partners. Our data show that workers actively repel unrelated males entering their colony, and that queens preferentially mate with related males. These results are consistent with inclusive fitness being a driving force for inbreeding: by preventing outbreeding, workers reduce erosion of relatedness within colonies due to polygyny and polyandry. That virgin queens mate preferentially with related males could result from a long history of inbreeding, which is expected to reduce depression in species with regular sibmating.     

Polygyny reduces rather than increases nestmate discrimination cue diversity in Formica exsecta ants

Although the majority of social insect colonies are headed by a single queen, some species possess nests that contain numerous reproductive queens (polygyny), a trait that is particularly widespread amongst the ants. Polygyny is often associated with a lack of conspecific inter-nest aggression between workers. This is hypothesised to result from increased nestmate cue diversity within nests, since polygynous nests are more genetically diverse than monogynous nests. Alternatively, it may reflect the common origin of polygynous nests that form polydomous networks. We exploit the recent discovery that the nestmate discrimination system in the ant Formica exsecta is based on cuticular hydrocarbons to investigate cue (Z9-alkenes) diversity in several monogynous and polygynous populations. Contrary to previous predictions, in all polygynous populations, the variation between nests in the Z9-alkene profiles was reduced relative to that found in monogynous populations. However, nest-specific Z9-alkene profiles with little variation amongst nestmate workers were still maintained irrespective of nest type or population. This suggests a very effective gestalt mechanism that homogenises the chemical discrimination cues, despite genetic diversity within colonies. Although the reduction in variation between nests was associated with reduced worker aggression on the population level, it cannot totally explain the weak aggression associated with polygynous populations.     

Low relatedness among cooperatively breeding workers of the greenhead ant Rhytidoponera metallica

The greenhead ant Rhytidoponera metallica has long been recognized as posing a potential challenge to kin selection theory, because it has large queenless colonies where apparently many of the morphological workers are mated and reproducing. However, this species has never been studied genetically and important elements of its breeding system and kin structure remain uncertain. We used microsatellite markers to measure the relatedness among nestmates, unravel the fine‐scale population genetic structure, and infer the breeding system of R. metallica. The genetic relatedness among worker nestmates is very low but significantly greater than zero (r=0.082 ± 0.015), which demonstrates that nests contain many distantly related breeders. The inbreeding coefficient is very close to and not significantly different from zero, indicating random mating and lack of microgeographic genetic differentiation. On average, closely located nests are not more similar genetically than distant nests, which is surprising, as new colonies form by budding and female dispersal is restricted. Lack of inbreeding and absence of population viscosity indicates high gene flow mediated by males. Overall, the genetic pattern detected in R. metallica suggests that a high number of moderately related workers mate with unrelated males from distant nests. This breeding system results in the lowest relatedness among nestmates reported for social insect species where breeders and helpers are not morphologically differentiated.     

Genetic components to caste allocation in a multiple-queen ant species

Reproductive division of labor and the coexistence of distinct castes are hallmarks of insect societies. In social insect species with multiple queens per colony, the fitness of nestmate queens directly depends on the process of caste allocation (i.e., the relative investment in queen, sterile worker and male production). The aim of this study is to investigate the genetic components to the process of caste allocation in a multiple-queen ant species. We conducted controlled crosses in the Argentine ant Linepithema humile and established single-queen colonies to identify maternal and paternal family effects on the relative production of new queens, workers, and males. There were significant effects of parental genetic backgrounds on various aspects of caste allocation: the paternal lineage affected the proportion of queens and workers produced whereas the proportions of queens and males, and females and males were influenced by the interaction between parental lineages. In addition to revealing nonadditive genetic effects on female caste determination in a multiple-queen ant species, this study reveals strong genetic compatibility effects between parental genomes on caste allocation components.     

Stay or drift? Queen acceptance in the ant <Emphasis Type="Italic">Formica paralugubris</Emphasis>

The acceptance of new queens in ant colonies has profound effects on colony kin structure and inclusive fitness of workers. Therefore, it is important to study the recognition and discrimination behaviour of workers towards reproductive individuals entering established colonies. We examined the acceptance rate of queens in populations of the highly polygynous ant F. paralugubris, where the genetic differentiation among nests and discrimination ability among workers suggest that workers might reject foreign queens. We experimentally introduced young queens in their natal nest and in foreign nests. Surprisingly, the survival rate of mated queens did not differ significantly when introduced in a foreign male-producing nest, a foreign female-producing nest, or the natal nest. Moreover, the survival of virgin queens in their natal nest was twice the one of mated queens, suggesting that mating status plays an important role for acceptance. The results indicate that other factors than queen discrimination by workers are implicated in the limited longdistance gene flow between nests in these populations. Received 8 April 2008; revised 16 June 2008; accepted 1 July 2008.