Species-specific genes under selection characterize the co-evolution of slavemaker and host lifestyles.

BACKGROUND: The transition to a parasitic lifestyle entails comprehensive changes to the selective regime. In parasites, genes encoding for traits that facilitate host detection, exploitation and transmission should be under selection. Slavemaking ants are social parasites that exploit the altruistic behaviour of their hosts by stealing heterospecific host brood during raids, which afterwards serve as slaves in slavemaker nests. Here we search for evidence of selection in the transcriptomes of three slavemaker species and three closely related hosts. We expected selection on genes underlying recognition and raiding or defense behaviour. Analyses of selective forces in species with a slavemaker or host lifestyle allowed investigation into whether or not repeated instances of slavemaker evolution share the same genetic basis. To investigate the genetic basis of host-slavemaker co-evolution, we created orthologous clusters from transcriptome sequences of six Temnothorax ant species - three slavemakers and three hosts - to identify genes with signatures of selection. We further tested for functional enrichment in selected genes from slavemakers and hosts respectively and investigated which pathways the according genes belong to. RESULTS: Our phylogenetic analysis, based on more than 5000 ortholog sequences, revealed sister species status for two slavemakers as well as two hosts, contradicting a previous phylogeny based on mtDNA. We identified 309 genes with signs of positive selection on branches leading to slavemakers and 161 leading to hosts. Among these were genes potentially involved in cuticular hydrocarbon synthesis, thus species recognition, and circadian clock functionality possibly explaining the different activity patterns of slavemakers and hosts. There was little overlap of genes with signatures of positive selection among species, which are involved in numerous different functions and different pathways. CONCLUSIONS: We identified different genes, functions and pathways under positive selection in each species. These results point to species-specific adaptations rather than convergent trajectories during the evolution of the slavemaker and host lifestyles suggesting that the evolution of parasitism, even in closely related species, may be achieved in diverse ways.

The influence of space and time on the evolution of altruistic defence: the case of ant slave rebellion.

How can antiparasite defence traits evolve even if they do not directly benefit their carriers? An example of such an indirect defence is rebellion of enslaved Temnothorax longispinosus ant workers against their social parasite Temnothorax americanus, a slavemaking ant. Ant slaves have been observed to kill their oppressors' offspring, a behaviour from which the sterile slaves cannot profit directly. Parasite brood killing could, however, reduce raiding pressure on related host colonies nearby. We analyse with extensive computer simulations for the Temnothorax slavemaker system under what conditions a hypothetical rebel allele could invade a host population, and in particular, how host-parasite dynamics and population structure influence the rebel allele's success. Exploring a wide range of model parameters, we only found a small number of parameter combinations for which kin selection or multilevel selection could allow a slave rebellion allele to spread in the host population. Furthermore, we did not detect any cases in which the reduction of raiding pressure in the close vicinity of the slavemaker nest would substantially contribute to the inclusive fitness of rebels. This suggests that slave rebellion is not costly and perhaps a side-effect of some other beneficial trait. In some of our simulations, however, even a costly rebellion allele could spread in the population. This was possible when host-parasite interactions led to a metapopulation dynamic with frequent local extinctions and recolonizations of demes by the offspring of few immigrants.

The ecological success of a social parasite increases with manipulation of collective host behaviour.

Many parasites alter the behaviour of their host to their own advantage, yet hosts often vary in their susceptibility to manipulation. The ecological and evolutionary implications of such variation can be profound, as resistant host populations may suffer lower parasite pressures than those susceptible to manipulation. To test this prediction, we assessed parasite-induced aggressive behaviours across 16 populations of two Temnothorax ant species, many of which harbour the slavemaker ant Protomognathus americanus. This social parasite uses its Dufour's gland secretions to manipulate its hosts into attacking nestmates, which may deter defenders away from itself during invasion. We indeed find that colonies that were manipulated into attacking their Dufour-treated nestmates were less aggressive towards the slavemaker than those that did not show slavemaker-induced nestmate attack. Slavemakers benefited from altering their hosts' aggression, as both the likelihood that slavemakers survived host encounters and slavemaker prevalence in ant communities increased with slavemaker-induced nestmate attack. Finally, we show that Temnothorax longispinosus colonies were more susceptible to manipulation than Temnothorax curvispinosus colonies. This explains why T. curvispinosus colonies responded with more aggression towards invading slavemakers, why they were less likely to let slavemakers escape and why they were less frequently parasitized by the slavemaker than T. longispinosus. Our findings highlight that large-scale geographic variation in resistance to manipulation can have important implications for the prevalence and host preference of parasites.

Oh sister, where art thou? Spatial population structure and the evolution of an altruistic defence trait.

The evolution of parasite virulence and host defences is affected by population structure. This effect has been confirmed in studies focusing on large spatial scales, whereas the importance of local structure is not well understood. Slavemaking ants are social parasites that exploit workers of another species to rear their offspring. Enslaved workers of the host species Temnothorax longispinosus have been found to exhibit an effective post-enslavement defence behaviour: enslaved workers were observed killing a large proportion of the parasites' offspring. As enslaved workers do not reproduce, they gain no direct fitness benefit from this 'rebellion' behaviour. However, there may be an indirect benefit: neighbouring host nests that are related to 'rebel' nests can benefit from a reduced raiding pressure, as a result of the reduction in parasite nest size due to the enslaved workers' killing behaviour. We use a simple mathematical model to examine whether the small-scale population structure of the host species could explain the evolution of this potentially altruistic defence trait against slavemaking ants. We find that this is the case if enslaved host workers are related to nearby host nests. In a population genetic study, we confirm that enslaved workers are, indeed, more closely related to host nests within the raiding range of their resident slavemaker nest, than to host nests outside the raiding range. This small-scale population structure seems to be a result of polydomy (e.g. the occupation of several nests in close proximity by a single colony) and could have enabled the evolution of 'rebellion' by kin selection.

Gene expression patterns associated with caste and reproductive status in ants: worker-specific genes are more derived than queen-specific ones.

Variation in gene expression leads to phenotypic diversity and plays a central role in caste differentiation of eusocial insect species. In social Hymenoptera, females with the same genetic background can develop into queens or workers, which are characterized by divergent morphologies, behaviours and lifespan. Moreover, many social insects exhibit behaviourally distinct worker castes, such as brood-tenders and foragers. Researchers have just started to explore which genes are differentially expressed to achieve this remarkable phenotypic plasticity. Although the queen is normally the only reproductive individual in the nest, following her removal, young brood-tending workers often develop ovaries and start to reproduce. Here, we make use of this ability in the ant Temnothorax longispinosus and compare gene expression patterns in the queens and three worker castes along a reproductive gradient. We found the largest expression differences between the queen and the worker castes (~2500 genes) and the smallest differences between infertile brood-tenders and foragers (~300 genes). The expression profile of fertile workers is more worker-like, but to a certain extent intermediate between the queen and the infertile worker castes. In contrast to the queen, a high number of differentially expressed genes in the worker castes are of unknown function, pointing to the derived status of hymenopteran workers within insects.

Macro- and microgeographic genetic structure in an ant species with alternative reproductive tactics in sexuals.

The genetic structure of social insect populations is influenced by their social organization and dispersal modes. The ant Hypoponera opacior shows diverse reproductive behaviours with regular cycles of outbreeding via winged sexuals and inbreeding via within-nest mating wingless sexuals that reproduce by budding. This unusual life cycle should be reflected in the genetic population structure, and we studied this on different scales using microsatellites. On a macrogeographic scale, populations were considerably structured and migration rates within the Chiricahuas were higher than those in between mountain ranges. On a local scale, our analyses revealed population viscosity through dependent colony foundation and a high genetic diversity with a multicolonial structure. The latter was also evident from recognition trials revealing consistent aggression between non-nestmates. Within-nest matings led to high inbreeding coefficients. Finally, the observed seasonal changes in relatedness can be explained by variation in queen number and differential dispersal of the two reproductive morphs.

Similar evolutionary potentials in an obligate ant parasite and its two host species.

The spatial structure of host-parasite coevolution is shaped by population structure and genetic diversity of the interacting species. We analysed these population genetic parameters in three related ant species: the parasitic slavemaking ant Protomognathus americanus and its two host species Temnothorax longispinosus and T. curvispinosus. We sampled throughout their range, genotyped ants on six to eight microsatellite loci and an MtDNA sequence and found high levels of genetic variation and strong population structure in all three species. Interestingly, the most abundant species and primary host, T. longispinosus, is characterized by less structure, but lower local genetic diversity. Generally, differences between the species were small, and we conclude that they have similar evolutionary potentials. The coevolutionary interaction between this social parasite and its hosts may therefore be less influenced by divergent evolutionary potentials, but rather by varying selection pressures. We employed different methods to quantify and compare genetic diversity and structure between species and genetic markers. We found that Jost D is well suited for these comparisons, as long as mutation rates between markers and species are similar. If this is not the case, for example, when using MtDNA and microsatellites to study sex-specific dispersal, model-based inference should be used instead of descriptive statistics (such as D or G(ST) ). Using coalescent-based methods, we indeed found that males disperse much more than females, but this sex bias in dispersal differed between species. The findings of the different approaches with regard to genetic diversity and structure were in good accordance with each other.

Genetic diversity, population structure and sex-biased dispersal in three co-evolving species.

Genetic diversity and spatial structure of populations are important for antagonistic coevolution. We investigated genetic variation and population structure of three closely related European ant species: the social parasite Harpagoxenus sublaevis and its two host species Leptothorax acervorum and Leptothorax muscorum. We sampled populations in 12 countries and analysed eight microsatellite loci and an mtDNA sequence. We found high levels of genetic variation in all three species, only slightly less variation in the host L. muscorum. Using a newly introduced measure of differentiation (Jost's D(est)), we detected strong population structuring in all species and less male-biased dispersal than previously thought. We found no phylogeographic patterns that could give information on post-glacial colonization routes - northern populations are as variable as more southern populations. We conclude that conditions for Thompson's geographic mosaic of coevolution are ideal in this system: all three species show ample genetic variation and strong population structure.

A chemical level in the coevolutionary arms race between an ant social parasite and its hosts.

Here we investigate the coevolutionary interactions between the slavemaking ant Protomognathus americanus and its Temnothorax hosts on a chemical level. We show that, although this social parasite is principally well-adapted to its hosts' cuticular hydrocarbon profile, there are pronounced differences in the fine-tuning of this adaptation. Between populations, chemical adaptation varies with host community composition, as the parasite faces a trade-off when confronted with more than one host species. In addition to adaptation of its own chemical signature, the slavemaker causes a reciprocal adjustment in its slaves' cuticular profile, the degree of which depends on the slave species. On the host side, successful parasite defence requires efficient enemy recognition, and in behavioural aggression trials, host colonies could indeed discriminate between invading slaves, which commonly accompany slavemakers on raids, and free-living conspecifics. Furthermore, hosts shifted their acceptance threshold over the seasons, presumably to reduce the costs of defence.

Local co-adaptation leading to a geographical mosaic of coevolution in a social parasite system.

The geographical mosaic theory of coevolution predicts differences in the advance or trajectory of the coevolutionary process between local communities due to their composition and the strength of ecological selection pressures through competition and resource availability. In this study, we investigate local co-adaptation in different populations of a social parasite. We conducted cross-fostering experiments to test for interpopulational differences in raiding efficiency between various populations of a slave-making ant and the defence abilities of local hosts. Here, we demonstrate that the success of raids strongly depends on the combination of populations of the parasite Harpagoxenus sublaevis and its host Leptothorax acervorum, indicating very localized coevolution. We found no absolute differences between slave-maker populations; the outcome of an encounter depended more on whether the two opponents occur in sympatry or allopatry. Furthermore, this study supports the results of our earlier work, that the unparasitized English L. acervorum population is most aggressive against the parasite.

Ecology, life history and resource allocation in the ant, Leptothorax nylanderi.

We aimed at identifying the causal basis of previously shown interrelations between demographic and genetic colony structure, ecological factors and split sex ratios in the ant, Leptothorax nylanderi. Colony-level variation in sex allocation was only partly explained by annual fluctuations during eight study years and by resource availability as indicated by sexual production of colonies. Allocation ratios were highly male-biased in dense populations with ephemeral nest sites and high frequencies of colonies containing several unrelated matrilines. Field observations and experimental manipulations showed that nest site limitation leads to such heterogeneous colonies. Laboratory experiments demonstrated that genetic heterogeneity directly causes male-biased investment, although relatedness asymmetry is not influenced by invasion of unrelated queens. The influence of genetic composition on allocation strategies might either be explained by negative feedback mechanisms connected with habitat saturation or by a lower efficiency of heterogeneous colonies. Our results thus demonstrate which factors other than variation in relatedness asymmetry can explain split sex ratios in ants. An empirical test of a model on reproductive allocation revealed on-going queen-worker conflict over colony growth and sexual reproduction. Workers controlled reproductive allocation, but queen-worker conflict ceased in large colonies with a high survival rate.

Coevolution in host-parasite systems: behavioural strategies of slave-making ants and their hosts.

Recently, avian brood parasites and their hosts have emerged as model systems for the study of host-parasite coevolution. However, empirical studies of the highly analogous social parasites, which use the workers of another eusocial species to raise their own young, have never explicitly examined the dynamics of these systems from a coevolutionary perspective. Here, we demonstrate interpopulational variation in behavioural interactions between a socially parasitic slave-maker ant and its host that is consistent with the expectations of host-parasite coevolution. Parasite pressure, as inferred by the size, abundance and raiding frequency of Protomognathus americanus colonies, was highest in a New York population of the host Leptothorax longispinosus and lowest in a West Virginia population. As host-parasite coevolutionary theory would predict, we found that the slave-makers and the hosts from New York were more effective at raiding and defending against raiders, respectively, than were conspecifics from the West Virginia population. Some of these variations in efficacy were brought about by apparently simple shifts in behaviour. These results demonstrate that defence mechanisms against social parasites can evolve, and they give the first indications of the existence of a coevolutionary arms race between a social parasite and its host.

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.

Colony structure of a slavemaking ant. II. Frequency of slave raids and impact on the host population.

The parasite pressure exerted by the slavemaker ant Protomognathus americanus on its host species Leptothorax longispinosus was analyzed demographically and genetically. The origin of slaves found in colonies of the obligate slavemaker was examined with nuclear and mitochondrial DNA markers to make inferences about the frequency and severity of slave raids. Relatedness of enslaved L. longispinosus workers in the same nest was very low, and our data suggest that, on average, each slavemaker nest raids six host colonies per season. Therefore, the influence of slavemaker species on their hosts is much stronger than simple numerical ratios suggest. We also found that slave relatedness was higher in small than in large slavemaker nests; thus, larger nests wield a much stronger influence on the host. We estimated that in the study population, on average, a host nest has a 50% chance of being attacked by a slavemaker colony per year. Free-living Leptothorax colonies in the vicinity of slavemaker nests did not represent the source of slaves working in P. americanus colonies, which suggests that raided nests either do not survive or migrate after being raided. Colony composition and intranest relatedness of free-living L. longispinosus colonies differed markedly between areas with slavemakers and those that are parasite-free. In the presence of slavemakers, host colonies were less likely to be polygynous and had fewer workers and a higher relatedness among worker brood. Host nests with slavemaker neighbors allocated more resources into sexuals, possibly caused by these shifts in nest demography. Finally, enslaved Leptothorax workers in P. americanus nests appeared to be less efficient than their counterparts in free-living colonies. Thus, slavemakers exert a much stronger impact on their hosts than had previously been suspected and represent an unique system to study parasite-host coevolution.

Conflict over sex allocation drives conflict over reproductive allocation in perennial social insect colonies.

Queen-worker conflict in the social Hymenoptera has become a cornerstone of sex-ratio theory. Extending that theory to conflict over life-history decisions, however, has proven controversial. Pamilo first proposed that queen-worker conflict over reproductive allocation should be important in perennial, social insect colonies, but Bourke and Chan have questioned the generality of that claim. Here, we reexamine this problem for the simplest case of a monogynous and monandrous hymenopteran society by relaxing assumptions of Pamilo's model. In populations with monomorphic sex ratios, queens and workers agree on allocation to growth versus reproduction. However, variation in sex allocation across colonies can induce queen-worker conflict over reproductive allocation; the former is a necessary condition for the latter. We explore how conflict over reproductive allocation depends on the population-wide sex ratio, the survivorship probabilities for existing colonies, and the likelihood of establishing new colonies. We then test our theory for two ant species, each with two years of data. We find considerable support for our contention of queen-worker conflict over reproductive allocation and suggest how future studies should be structured to explore this conflict further.