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Symbiotic interactions between macrotermitine termites and their fungal symbionts have a moderate degree of specificity. Consistent with horizontal symbiont transmission, host switching has been frequent over evolutionary time so that single termite species can often be associated with several fungal symbionts. However, even in the few termite lineages that secondarily adopted vertical symbiont transmission, the fungal symbionts are not monophyletic. We addressed this paradox by studying differential transmission of fungal symbionts by alate male and female reproductives, and the genetic population structure of Termitomyces fungus gardens across 74 colonies of Macrotermes bellicosus in four west and central African countries. We confirm earlier, more limited, studies showing that the Termitomyces symbionts of M. bellicosus are normally transmitted vertically and clonally by dispersing males. We also document that the symbionts associated with this termite species belong to three main lineages that do not constitute a monophyletic group. The most common lineage occurs over the entire geographical region that we studied, including west, central and southern Africa, where it is also associated with the alternative termite hosts Macrotermes subhyalinus and Macrotermes natalensis. While Termitomyces associated with these alternative hosts are horizontally transmitted and recombine freely, the genetic population structure of the same Termitomyces associated with M. bellicosus is consistent with predominantly clonal reproduction and only occasional recombination. This implies that the genetic population structure of Termitomyces is controlled by the termite host and not by the Termitomyces symbiont., (© 2011 Blackwell Publishing Ltd.)

Chalkbrood (Ascosphaera apis) and stonebrood (Aspergillus flavus) are well known fungal brood diseases of honeybees (Apis mellifera), but they have hardly been systematically studied because the difficulty of rearing larvae in vitro has precluded controlled experimentation. Chalkbrood is a chronic honeybee-specific disease that can persist in colonies for years, reducing both brood and honey production, whereas stonebrood is a rare facultative pathogen that also affects hosts other than honeybees and can likely survive outside insect hosts. Hive infection trials have indicated that accidental drops in comb temperature increase the prevalence of chalkbrood, but it has remained unclear whether virulence is directly temperature-dependent. We used a newly established in vitro rearing technique for honeybee larvae to test whether there are systematic temperature effects on mortality induced by controlled infections, and whether such effects differed between the two fungal pathogens. We found that increasing spore dosage at infection had a more dramatic effect on mortality from stonebrood compared to chalkbrood. In addition, a 24h cooling period after inoculation increased larval mortality from chalkbrood infection, whereas such a cooling period decreased mortality after stonebrood infection. These results raise interesting questions about honeybee defenses against obligate and facultative pathogens and about the extent to which stress factors in the host (dis)favor pathogens with lesser degrees of specialization., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Ants have evolved very complex societies and are key ecosystem members. Some ants, such as the fire ant Solenopsis invicta, are also major pests. Here, we present a draft genome of S. invicta, assembled from Roche 454 and Illumina sequencing reads obtained from a focal haploid male and his brothers. We used comparative genomic methods to obtain insight into the unique features of the S. invicta genome. For example, we found that this genome harbors four adjacent copies of vitellogenin. A phylogenetic analysis revealed that an ancestral vitellogenin gene first underwent a duplication that was followed by possibly independent duplications of each of the daughter vitellogenins. The vitellogenin genes have undergone subfunctionalization with queen- and worker-specific expression, possibly reflecting differential selection acting on the queen and worker castes. Additionally, we identified more than 400 putative olfactory receptors of which at least 297 are intact. This represents the largest repertoire reported so far in insects. S. invicta also harbors an expansion of a specific family of lipid-processing genes, two putative orthologs to the transformer/feminizer sex differentiation gene, a functional DNA methylation system, and a single putative telomerase ortholog. EST data indicate that this S. invicta telomerase ortholog has at least four spliceforms that differ in their use of two sets of mutually exclusive exons. Some of these and other unique aspects of the fire ant genome are likely linked to the complex social behavior of this species.

The degradation of live plant biomass in fungus gardens of leaf-cutting ants is poorly characterised but fundamental for understanding the mutual advantages and efficiency of this obligate nutritional symbiosis. Controversies about the extent to which the garden-symbiont Leucocoprinus gongylophorus degrades cellulose have hampered our understanding of the selection forces that induced large scale herbivory and of the ensuing ecological footprint of these ants. Here we use a recently established technique, based on polysaccharide microarrays probed with antibodies and carbohydrate binding modules, to map the occurrence of cell wall polymers in consecutive sections of the fungus garden of the leaf-cutting ant Acromyrmex echinatior. We show that pectin, xyloglucan and some xylan epitopes are degraded, whereas more highly substituted xylan and cellulose epitopes remain as residuals in the waste material that the ants remove from their fungus garden. These results demonstrate that biomass entering leaf-cutting ant fungus gardens is only partially utilized and explain why disproportionally large amounts of plant material are needed to sustain colony growth. They also explain why substantial communities of microbial and invertebrate symbionts have evolved associations with the dump material from leaf-cutting ant nests, to exploit decomposition niches that the ant garden-fungus does not utilize. Our approach thus provides detailed insight into the nutritional benefits and shortcomings associated with fungus-farming in ants.

Background: Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved., Results: We determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian species of fungus-growing ants, representing eight genera. We mapped these activity profiles on an independently obtained molecular phylogeny of the symbionts and show that total proteinase activity in lower attine symbionts peaks at ca. pH 6. The higher attine symbionts that have no known free-living relatives had much higher proteinase activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH optimum of fungal proteinases may have changed after the irreversible domestication of evolutionary more derived fungal symbionts. This notion is also supported by buffering capacities of fungus gardens at pH 5.2 being remarkably high, and suggests that the fungal symbiont actively helps to maintain garden acidity at this specific level. Metalloproteinases dominated the activity profiles of lower attine gardens and may thus represent the ancestral type of proteinase production, whereas serine proteinase activity dominated the activity profiles of the higher attine gardens reared by Trachymyrmex and Sericomyrmex, suggesting that there may be trade-offs in the production of these enzyme classes. Remarkably, the single symbiont that is shared by species of the crown group of Atta and Acromyrmex leaf-cutting ants mostly showed metalloproteinase activity, suggesting that recurrent changes in enzyme production may have occurred throughout the domestication history of fungus-garden symbionts., Conclusions: Proteinase pH optima and buffering capacities of fungal symbionts appear to have evolved remarkable adaptations to living in obligate symbiosis with farming ants. Although the functional roles of serine and metalloproteinases in fungus gardens are unknown, the differential production of these classes of proteolytic enzymes suggest that substrate specificity may be important and that trade-offs may prevent the simultaneous upregulation of both classes of enzymes.

Altruism in social insects has evolved between closely related full-siblings. It is therefore of considerable interest why some groups have secondarily evolved low within-colony relatedness, which in turn affects the relatedness incentives of within-colony cooperation and conflict. The highest queen mating frequencies, and therefore among the lowest degrees of colony relatedness, occur in Apis honeybees and army ants of the subfamilies Aenictinae, Ecitoninae, and Dorylinae, suggesting that common life history features such as reproduction by colony fission and male biased numerical sex-ratios have convergently shaped these mating systems. Here we show that ponerine army ants of the genus Simopelta, which are distantly related but similar in general biology to other army ants, have strictly monandrous queens. Preliminary data suggest that workers reproduce in queenright colonies, which is in sharp contrast to other army ants. We hypothesize that differences in mature colony size and social complexity may explain these striking discrepancies., (© 2010 Blackwell Publishing Ltd.)

Ascosphaera apis causes chalkbrood in honeybees, a chronic disease that reduces the number of viable offspring in the nest. Although lethal for larvae, the disease normally has relatively low virulence at the colony level. A recent study showed that there is genetic variation for host susceptibility, but whether Ascosphaera apis strains differ in virulence is unknown. We exploited a recently modified in vitro rearing technique to infect honeybee larvae from three colonies with naturally mated queens under strictly controlled laboratory conditions, using four strains from two distinct A. apis clades. We found that both strain and colony of larval origin affected mortality rates. The strains from one clade caused 12-14% mortality while those from the other clade induced 71-92% mortality. Larvae from one colony showed significantly higher susceptibility to chalkbrood infection than larvae from the other two colonies, confirming the existence of genetic variation in susceptibility across colonies. Our results are consistent with antagonistic coevolution between a specialized fungal pathogen and its host, and suggest that beekeeping industries would benefit from more systematic monitoring of this chronic stress factor of their colonies.

Background: Leaf-cutting (attine) ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus Leucocoprinus gongylophorus that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth., Results: We tested this hypothesis by using proteomics methods to determine the gene sequences of fecal proteins in Acromyrmex echinatior leaf-cutting ants. Seven (21%) of the 33 identified proteins were pectinolytic enzymes that originated from the fungal symbiont and which were still active in the fecal droplets produced by the ants. We show that these enzymes are found in the fecal material only when the ants had access to fungus garden food, and we used quantitative polymerase chain reaction analysis to show that the expression of six of these enzyme genes was substantially upregulated in the fungal gongylidia. These unique structures serve as food for the ants and are produced only by the evolutionarily advanced garden symbionts of higher attine ants, but not by the fungi reared by the basal lineages of this ant clade., Conclusions: Pectinolytic enzymes produced in the gongylidia of the fungal symbiont are ingested but not digested by Acromyrmex leaf-cutting ants so that they end up in the fecal fluid and become mixed with new garden substrate. Substantial quantities of pectinolytic enzymes are typically found in pathogenic fungi that attack live plant tissue, where they are known to breach the cell walls to allow the fungal mycelium access to the cell contents. As the leaf-cutting ant symbionts are derived from fungal clades that decompose dead plant material, our results suggest that their pectinolytic enzymes represent secondarily evolved adaptations that are convergent to those normally found in phytopathogens.

Both developmental and environmental factors shape investment in costly immune defences. Social insect workers have different selection pressures on their innate immune system compared to non-social insects because workers do not reproduce and their longevity affects the fitness of relatives. Furthermore, hygienic behavioural defences found in social insects can result in increased survival after fungal infection, although it is not known if there is modulation in physiological immune defence associated with group living vs. solitary living. Here we investigated whether physiological immune defence is affected by both age and the short-term presence or absence of nestmates in the leaf-cutting ant Acromyrmex octospinosus. We predicted that older ants would show immune senescence and that group living may result in prophylactic differences in immune defence compared to solitarily kept ants. We kept old and young workers alone or in nestmate groups for 48h and assayed a key innate immune system enzyme, expressing phenoloxidase (PO) and its stored precursor (proPO), a defence that acts immediately, i.e. it is constitutive. Short-term solitary living did not affect PO or proPO levels relative to group living controls and we found no evidence for immunosenescence in proPO. However, we found a significant increase in active PO in older workers, which is consistent with two non-mutually exclusive explanations: it could be an adaptive response or indicative of immunosenescence. Our results suggest that future studies of immunosenescence should consider both active immune effectors in the body, such as PO, and the stored potential to express immune defences, such as proPO., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.

The evolution of sociality is facilitated by the recognition of close kin, but if kin recognition is too accurate, nepotistic behaviour within societies can dissolve social cohesion. In social insects, cuticular hydrocarbons act as nestmate recognition cues and are usually mixed among colony members to create a Gestalt odour. Although earlier studies have established that hydrocarbon profiles are influenced by heritable factors, transfer among nestmates and additional environmental factors, no studies have quantified these relative contributions for separate compounds. Here, we use the ant Formica rufibarbis in a cross-fostering design to test the degree to which hydrocarbons are heritably synthesized by young workers and transferred by their foster workers. Bioassays show that nestmate recognition has a significant heritable component. Multivariate quantitative analyses based on 38 hydrocarbons reveal that a subset of branched alkanes are heritably synthesized, but that these are also extensively transferred among nestmates. In contrast, especially linear alkanes are less heritable and little transferred; these are therefore unlikely to act as cues that allow within-colony nepotistic discrimination or as nestmate recognition cues. These results indicate that heritable compounds are suitable for establishing a genetic Gestalt for efficient nestmate recognition, but that recognition cues within colonies are insufficiently distinct to allow nepotistic kin discrimination.

We characterized evolutionary patterns of host plant use across about 2500 species of British Lepidoptera, using character optimization and independent phylogenetic contrasts among 95 operational taxa, and evaluated the extent to which caterpillars are monophagous, use woody host plants, and feed concealed. We also analyzed the use of different Angiosperm superorders and related these associations to other key variables. The Nepticulidae, Pterophoridae, and Gracillariidae allowed explicit comparisons between the British fauna and the Lepidoptera worldwide, which indicated that our broad categorizations for Britain are accurate predictors for the global fauna. The first (lower glossatan) radiation of the Lepidoptera started with monophagous, internal feeding on woody Eurosids I. Polyphagy on nonwoody Eurosids I evolved together with the ability to feed externally, but did initially not produce significant radiations. Exposed feeding became associated with radiations in the lower Ditrysia and Apoditrysia and remained correlated with more polyphagy, fewer woody host plants, and increasing use of other Angiosperm superorders. The macrolepidopteran radiation has frequent reversals to monophagy on woody Eurosids I, particularly in taxa that lost concealed feeding. We discuss the general implications of these results and address several key adaptations and constraints that have characterized the major transitions in lepidopteran life histories.

Queens of ants and bees normally obtain a lifetime supply of sperm on a single day of sexual activity, and sperm competition is expected to occur in lineages where queens receive sperm from multiple males. We compared singly mated (monandrous) and multiply mated (polyandrous) sister groups of ants and bees and show that seminal fluid of polyandrous species has a more positive effect on the survival of a male's own sperm than on other males' sperm. This difference was not observed in the monandrous species, suggesting that incapacitation of competing sperm may have independently evolved in both bees and ants. In Atta leafcutter ants, the negative effect of the seminal fluid of other males was negated by secretion from the queen sperm-storage organ, suggesting that queens may control ejaculate competition after sperm storage.

Theory predicts that altruism is only evolutionarily stable if it is preferentially directed towards relatives, so that any such behaviour towards seemingly unrelated individuals requires scrutiny. Queenless army ant colonies, which have anecdotally been reported to fuse with queenright foreign colonies, are such an enigmatic case. Here we combine experimental queen removal with population genetics and cuticular chemistry analyses to show that colonies of the African army ant Dorylus molestus frequently merge with neighbouring colonies after queen loss. Merging colonies often have no direct co-ancestry, but are on average probably distantly related because of overall population viscosity. The alternative of male production by orphaned workers appears to be so inefficient that residual inclusive fitness of orphaned workers might be maximized by indiscriminately merging with neighbouring colonies to increase their reproductive success. We show that worker chemical recognition profiles remain similar after queen loss, but rapidly change into a mixed colony Gestalt odour after fusion, consistent with indiscriminate acceptance of alien workers that are no longer aggressive. We hypothesize that colony fusion after queen loss might be more widespread, especially in spatially structured populations of social insects where worker reproduction is not profitable.

Social insect castes represent some of the most spectacular examples of phenotypic plasticity, with each caste being associated with different environmental conditions during their life. Here we examine the level of genetic variation in different castes of two polyandrous species of Acromyrmex leaf-cutting ant for the antibiotic-producing metapleural gland, which has a major role in defence against parasites. Gland size increases allometrically. The small workers that play the main role in disease defence have relatively large glands compared with larger workers, while the glands of gynes are substantially larger than those of any workers, for their body size. The gland size of large workers varies significantly between patrilines in both Acromyrmex echinatior and Acromyrmex octospinosus. We also examined small workers and gynes in A. echinatior, again finding genetic variation in gland size in these castes. There were significant positive relationships between the gland sizes of patrilines in the different castes, indicating that the genetic mechanism underpinning the patriline variation has remained similar across phenotypes. The level of expressed genetic variation decreased from small workers to large workers to gynes. This is consistent with the hypothesis that there is individual selection on disease defence in founding queens and colony-level selection on disease defence in the worker castes.

In many species, females store sperm between copulation and egg fertilization, but the consequences of sperm storage and patterns of sperm use for female life history and reproductive success have not been investigated in great detail. In hymenopteran insect societies (ants, bees, wasps), reproduction is usually monopolized by one or relatively few queens, who mate only during a brief period early in life and store sperm for later use. The queens of some ants are particularly long-lived and have the potential to produce millions of offspring during their life. To do so, queens store many sperm cells, and this sperm must remain viable throughout the years of storage. Queens should also be under strong selection to use stored sperm prudently when fertilizing eggs. We used the leaf-cutter ant Atta colombica to investigate the dynamics of sperm use during egg fertilization. We show that queens are able to fertilize close to 100 per cent of the eggs and that the average sperm use per egg is very low, but increases with queen age. The robustness of stored sperm was found to decrease with years of storage, signifying that senescence affects sperm either directly or indirectly via the declining glandular secretions or deteriorating sperm-storage organs. We evaluate our findings with a heuristic model, which suggests that the average queen has sperm for almost 9 years of normal colony development. We discuss the extent to which leaf-cutter ant queens have been able to optimize their sperm expenditure and infer that our observed averages of sperm number, sperm robustness and sperm use are consistent with sperm depletion being a significant cause of mortality of mature colonies of Atta leaf-cutter ants.

It is unclear how mutualistic relationships can be stable when partners disperse freely and have the possibility of forming associations with many alternative genotypes. Theory predicts that high symbiont relatedness should resolve this problem, but the mechanisms to enforce this have rarely been studied. We show that African fungus-growing termites propagate single variants of their Termitomyces symbiont, despite initiating cultures from genetically variable spores from the habitat. High inoculation density in the substrate followed by fusion among clonally related mycelia enhances the efficiency of spore production in proportion to strain frequency. This positive reinforcement results in an exclusive lifetime association of each host colony with a single fungal symbiont and hinders the evolution of cheating. Our findings explain why vertical symbiont transmission in fungus-growing termites is rare and evolutionarily derived.

All evidence currently available indicates that obligatory sterile eusocial castes only arose via the association of lifetime monogamous parents and offspring. This is consistent with Hamilton's rule (br(s) > r(o)c), but implies that relatedness cancels out of the equation because average relatedness to siblings (r(s)) and offspring (r(o)) are both predictably 0.5. This equality implies that any infinitesimally small benefit of helping at the maternal nest (b), relative to the cost in personal reproduction (c) that persists throughout the lifespan of entire cohorts of helpers suffices to establish permanent eusociality, so that group benefits can increase gradually during, but mostly after the transition. The monogamy window can be conceptualized as a singularity comparable with the single zygote commitment of gametes in eukaryotes. The increase of colony size in ants, bees, wasps and termites is thus analogous to the evolution of multicellularity. Focusing on lifetime monogamy as a universal precondition for the evolution of obligate eusociality simplifies the theory and may help to resolve controversies about levels of selection and targets of adaptation. The monogamy window underlines that cooperative breeding and eusociality are different domains of social evolution, characterized by different sectors of parameter space for Hamilton's rule.

Evolutionary theory predicts that hosts are selected to prevent mixing of genetically different symbionts when competition among lineages reduces the productivity of a mutualism. The symbionts themselves may also defend their interests: recent studies of Acromyrmex leaf-cutting ants showed that somatic incompatibility enforces single-clone gardens within mature colonies, thereby constraining horizontal transmission of fungal symbionts. However, phylogenetic analyses indicate that symbiont switches occur frequently enough to remove most signs of host-symbiont cocladogenesis. Here we resolve this paradox by showing that transmission among newly founded Acromyrmex colonies is not constrained. All tested queens of sympatric A. octospinosus and A. echinatior offered a novel fragment of fungus garden accepted the new symbiont. The outcome was unaffected by genetic distance between the novel and the original symbiont, and by the ant species the novel symbiont came from. The colony founding stage may thus provide an efficient but transient window for horizontal transmission, in which the fungus is unable to actively defend its partnership position before the host feeds on it, so that host fecal droplets remain compatible with alternative strains during the early stage of colony founding. We discuss how brief stages of low commitment between partners may increase the evolutionary stability of ancient coevolved mutualisms.

Specialized parasites are expected to express complex adaptations to their hosts. Manipulation of host behavior is such an adaptation. We studied the fungus Ophiocordyceps unilateralis, a locally specialized parasite of arboreal Camponotus leonardi ants. Ant-infecting Ophiocordyceps are known to make hosts bite onto vegetation before killing them. We show that this represents a fine-tuned fungal adaptation: an extended phenotype. Dead ants were found under leaves, attached by their mandibles, on the northern side of saplings approximately 25 cm above the soil, where temperature and humidity conditions were optimal for fungal growth. Experimental relocation confirmed that parasite fitness was lower outside this manipulative zone. Host resources were rapidly colonized and further secured by extensive internal structuring. Nutritional composition analysis indicated that such structuring allows the parasite to produce a large fruiting body for spore production. Our findings suggest that the osmotrophic lifestyle of fungi may have facilitated novel exploitation strategies.

To combat disease, most fungus-growing ants (Attini) use antibiotics from mutualistic bacteria (Pseudonocardia) that are cultured on the ants' exoskeletons and chemical cocktails from exocrine glands, especially the metapleural glands (MG). Previous work has hypothesized that (i) Pseudonocardia antibiotics are narrow-spectrum and control a fungus (Escovopsis) that parasitizes the ants' fungal symbiont, and (ii) MG secretions have broad-spectrum activity and protect ants and brood. We assessed the relative importance of these lines of defence, and their activity spectra, by scoring abundance of visible Pseudonocardia for nine species from five genera and measuring rates of MG grooming after challenging ants with disease agents of differing virulence. Atta and Sericomyrmex have lost or greatly reduced the abundance of visible bacteria. When challenged with diverse disease agents, including Escovopsis, they significantly increased MG grooming rates and expanded the range of targets. By contrast, species of Acromyrmex and Trachymyrmex maintain abundant Pseudonocardia. When challenged, these species had lower MG grooming rates, targeted primarily to brood. More elaborate MG defences and reduced reliance on mutualistic Pseudonocardia are correlated with larger colony size among attine genera, raising questions about the efficacy of managing disease in large societies with chemical cocktails versus bacterial antimicrobial metabolites.

Internal fertilization requires live sperm to be transferred from male to female before egg fertilization. Both males and females assist the insemination process by providing sperm with glandular secretions, which have been inferred to contain subsets of proteins that maintain sperm viability. Here we show that in the honeybee (Apis mellifera) secretions of the male accessory glands, the major contributors towards seminal fluid, enhance sperm survival. We further demonstrate that the protein fraction of the male accessory gland secretion is indeed important for achieving the maximal effect on sperm survival. After sperm storage, the queens also provide sperm with secretions from spermathecal glands and we show that these secretions have a comparable positive effect on sperm viability. SDS gels show that the proteomic profiles of accessory gland secretion and spermathecal fluid secretion hardly overlap, which suggests that males and females use different proteins to enhance sperm viability during, respectively, ejaculation and final sperm storage.

Parasites are likely to play an important role in structuring host populations. Many adaptively manipulate host behaviour, so that the extended phenotypes of these parasites and their distributions in space and time are potentially important ecological variables. The fungus Ophiocordyceps unilateralis, which is pan-tropical in distribution, causes infected worker ants to leave their nest and die under leaves in the understory of tropical rainforests. Working in a forest dynamic plot in Southern Thailand we mapped the occurrence of these dead ants by examining every leaf in 1,360 m(2) of primary rainforest. We established that high density aggregations exist (up to 26 dead ants/m(2)), which we coined graveyards. We further established that graveyards are patchily distributed in a landscape with no or very few O. unilateralis-killed ants. At some, but not all, spatial scales of analysis the density of dead ants correlated with temperature, humidity and vegetation cover. Remarkably, having found 2243 dead ants inside graveyards we only found 2 live ants of the principal host, ant Camponotus leonardi, suggesting that foraging host ants actively avoid graveyards. We discovered that the principal host ant builds nests in high canopy and its trails only occasionally descend to the forest floor where infection occurs. We advance the hypothesis that rare descents may be a function of limited canopy access to tree crowns and that resource profitability of such trees is potentially traded off against the risk of losing workers due to infection when forest floor trails are the only access routes. Our work underscores the need for an integrative approach that recognises multiple facets of parasitism, such as their extended phenotypes.

Background: Experiments under controlled laboratory conditions can produce decisive evidence for testing biological hypotheses, provided they are representative of the more complex natural conditions. However, whether this requirement is fulfilled is seldom tested explicitly. Here we provide a lab/field comparison to investigate the identity of an egg-marking signal of ant queens. Our study was based on ant workers resolving conflict over male production by destroying each other's eggs, but leaving queen eggs unharmed. For this, the workers need a proximate cue to discriminate between the two egg types. Earlier correlative evidence indicated that, in the ant Pachycondyla inversa, the hydrocarbon 3,11-dimethylheptacosane (3,11-diMeC(27)) is more abundant on the surface of queen-laid eggs., Methodology: We first tested the hypothesis that 3,11-diMeC(27) functions as a queen egg-marking pheromone using laboratory-maintained colonies. We treated worker-laid eggs with synthetic 3,11-diMeC(27) and found that they were significantly more accepted than sham-treated worker-laid eggs. However, we repeated the experiment with freshly collected field colonies and observed no effect of treating worker-laid eggs with 3,11-diMeC(27), showing that this compound by itself is not the natural queen egg-marking pheromone. We subsequently investigated the overall differences of entire chemical profiles of eggs, and found that queen-laid eggs in field colonies are more distinct from worker-laid eggs than in lab colonies, have more variation in profiles, and have an excess of longer-chain hydrocarbons., Conclusions: Our results suggest that queen egg-marking signals are significantly affected by transfer to the laboratory, and that this change is possibly connected to reduced queen fertility as predicted by honest signaling theory. This change is reflected in the worker egg policing response under field and laboratory conditions.

The massive environmentally buffered nests of some social insects can contain millions of individuals and a wide variety of parasites, commensals and mutualists. We suggest that the ways in which these homeostatic fortress environments affect the evolution of social insect symbionts are relevant for epidemiology, evolutionary biology and macroecology. We contend that specialized parasites will tend to become less virulent and mutualists less cooperative, compared to those associated with solitary or small-colony hosts. These processes are expected to contribute to the very high symbiont diversity observed in these nests. We hypothesize that biodiversity gradients in these hotspots might be less affected by abiotic latitudinal clines than gradients in neighboring 'control' habitats. We suggest several research lines to test these ideas.

Understanding the relative evolutionary importance of parasites to different host taxa is problematic because the expression of disease and resistance are often confounded by factors such as host age and condition. The antibiotic-producing metapleural glands of ants are a potentially useful exception to this rule because they are a key first-line defense that are fixed in size in adults. Here we conduct a comparative analysis of the size of the gland reservoir across the fungus-growing ants (tribe Attini). Most attines have singly mated queens, but in two derived genera, the leaf-cutting ants, the queens are multiply mated, which is hypothesized to have evolved to improve colony-level disease resistance. We found that, relative to body size, the gland reservoirs of most attines are similar in size but that those of the leaf-cutting ants are significantly larger. In contrast, the size of the reservoir did not relate with the evolutionary transition from lower to higher attines and correlated at most only slightly with colony size. The results thus suggest that the relationship between leaf-cutting ants and their parasites is distinctly different from that for other attine ants, in accord with the hypothesis that multiple mating by queens evolved to improve colony-level disease resistance.

A recent study has discovered a novel extended phenotype of a nematode which alters its ant host to resemble ripe fruit. The infected ants are in turn eaten by frugivorous birds that disperse the nematode's eggs.

Social groups are vulnerable to cheating because the reproductive interests of group members are rarely identical. All cooperative systems are therefore predicted to involve a mix of cooperative and cheating genotypes, with the frequency of the latter being constrained by the suppressive abilities of the former. The most significant potential conflict in social insect colonies is over which individuals become reproductive queens rather than sterile workers. This reproductive division of labor is a defining characteristic of eusocial societies, but individual larvae will maximize their fitness by becoming queens whereas their nestmates will generally maximize fitness by forcing larvae to become workers. However, evolutionary constraints are thought to prevent cheating by removing genetic variation in caste propensity. Here, we show that one-fifth of leaf-cutting ant patrilines cheat their nestmates by biasing their larval development toward becoming queens rather than workers. Two distinct mechanisms appear to be involved, one most probably involving a general tendency to become a larger adult and the other relating specifically to the queen-worker developmental switch. Just as evolutionary theory predicts, these "royal" genotypes are rare both in the population and within individual colonies. The rarity of royal cheats is best explained as an evolutionary strategy to avoid suppression by cooperative genotypes, the efficiency of which is frequency-dependent. The results demonstrate that cheating can be widespread in even the most cooperative of societies and illustrate that identical principles govern social evolution in highly diverse systems.

Background: Leaf-cutting ants live in symbiosis with a fungus that they rear for food by providing it with live plant material. Until recently the fungus' main inferred function was to make otherwise inaccessible cell wall degradation products available to the ants, but new studies have shed doubt on this idea. To provide evidence for the cell wall degrading capacity of the attine ant symbiont, we designed PCR primers from conserved regions of known xylanase genes, to be used in PCR with genomic DNA from the symbiont as template. We also measured xylanase, cellulase and proteinase activities in the fungus gardens in order to investigate the dynamics of degradation activities., Results: We cloned a xylanase gene from the mutualistic fungus of Acromyrmex echinatior, determined its protein sequence, and inserted it in a yeast expression vector to confirm its substrate specificity. Our results show that the fungus has a functional xylanase gene. We also show by lab experiments in vivo that the activity of fungal xylanase and cellulase is not evenly distributed, but concentrated in the lower layer of fungus gardens, with only modest activity in the middle layer where gongylidia are produced and intermediate activity in the newly established top layer. This vertical distribution appears to be negatively correlated with the concentration of glucose, which indicates a directly regulating role of glucose, as has been found in other fungi and has been previously suggested for the ant fungal symbiont., Conclusion: The mutualistic fungus of Acromyrmex echinatior has a functional xylanase gene and is thus presumably able to at least partially degrade the cell walls of leaves. This finding supports a saprotrophic origin of the fungal symbiont. The observed distribution of enzyme activity leads us to propose that leaf-substrate degradation in fungus gardens is a multi-step process comparable to normal biodegradation of organic matter in soil ecosystems, but with the crucial difference that a single fungal symbiont realizes most of the steps that are normally provided by a series of microorganisms that colonize fallen leaves in a distinct succession.

Background: The invasive garden ant, Lasius neglectus, is the most recently detected pest ant and the first known invasive ant able to become established and thrive in the temperate regions of Eurasia. In this study, we aim to reconstruct the invasion history of this ant in Europe analysing 14 populations with three complementary approaches: genetic microsatellite analysis, chemical analysis of cuticular hydrocarbon profiles and behavioural observations of aggression behaviour. We evaluate the relative informative power of the three methodological approaches and estimate both the number of independent introduction events from a yet unknown native range somewhere in the Black Sea area, and the invasive potential of the existing introduced populations., Results: Three clusters of genetically similar populations were detected, and all but one population had a similar chemical profile. Aggression between populations could be predicted from their genetic and chemical distance, and two major clusters of non-aggressive groups of populations were found. However, populations of L. neglectus did not separate into clear supercolonial associations, as is typical for other invasive ants., Conclusion: The three methodological approaches gave consistent and complementary results. All joint evidence supports the inference that the 14 introduced populations of L. neglectus in Europe likely arose from only very few independent introductions from the native range, and that new infestations were typically started through introductions from other invasive populations. This indicates that existing introduced populations have a very high invasive potential when the ants are inadvertently spread by human transport.

Mechanisms of recognition are essential to the evolution of mutualistic and parasitic interactions between species. One such example is the larval mimicry that Maculinea butterfly caterpillars use to parasitize Myrmica ant colonies. We found that the greater the match between the surface chemistry of Maculinea alcon and two of its host Myrmica species, the more easily ant colonies were exploited. The geographic patterns of surface chemistry indicate an ongoing coevolutionary arms race between the butterflies and Myrmica rubra, which has significant genetic differentiation between populations, but not between the butterflies and a second, sympatric host, Myrmica ruginodis, which has panmictic populations. Alternative hosts may therefore provide an evolutionary refuge for a parasite during periods of counteradaptation by their preferred hosts.

It is unclear why some species become successful invaders whilst others fail, and whether invasive success depends on pre-adaptations already present in the native range or on characters evolving de-novo after introduction. Ants are among the worst invasive pests, with Lasius neglectus and its rapid spread through Europe and Asia as the most recent example of a pest ant that may become a global problem. Here, we present the first integrated study on behavior, morphology, population genetics, chemical recognition and parasite load of L. neglectus and its non-invasive sister species L. turcicus. We find that L. neglectus expresses the same supercolonial syndrome as other invasive ants, a social system that is characterized by mating without dispersal and large networks of cooperating nests rather than smaller mutually hostile colonies. We conclude that the invasive success of L. neglectus relies on a combination of parasite-release following introduction and pre-adaptations in mating system, body-size, queen number and recognition efficiency that evolved long before introduction. Our results challenge the notion that supercolonial organization is an inevitable consequence of low genetic variation for chemical recognition cues in small invasive founder populations. We infer that low variation and limited volatility in cuticular hydrocarbon profiles already existed in the native range in combination with low dispersal and a highly viscous population structure. Human transport to relatively disturbed urban areas thus became the decisive factor to induce parasite release, a well established general promoter of invasiveness in non-social animals and plants, but understudied in invasive social insects.

Neotropical attine ants live in obligatory symbiosis with a fungus that they grow for food on a substrate of primarily plant material harvested by workers. Nestmate recognition is likely based on chemical cues as in most other social insects, but recent studies have indicated that both the ants and their mutualistic fungi may contribute to the recognition templates. To investigate the within-colony variation in chemical profiles, we extracted and identified compounds from the cuticle of workers, the postpharyngeal gland of workers, ant pupae and larvae, and the fungal symbiont of three species of higher attine ants: Atta colombica, Acromyrmex echinatior, and Sericomyrmex amabilis. The relative proportions of identified compounds were compared and represented 11 classes: n-alkanes, alkenes, branched methylalkanes, branched dimethylalkanes, trimethylalkanes, branched alkenes, aldehydes, alcohols, acetates, acids, and esters. The chemical profiles in all three species are likely to be sufficiently different to allow discrimination at the species and colony level and sufficiently similar within colonies to generate a relatively constant colony-specific chemical gestalt. The relative likelihood of individual compounds being derived from the ants, the ant brood, or the fungal symbiont are discussed. We hypothesize that hydrocarbons are particularly important as recognition cues because they appear to simultaneously allow the assessment of developmental stages and the identification of symbiont, colony, and species.

Elaborate division of labour has contributed significantly to the ecological success of social insects. Division of labour is achieved either by behavioural task specialization or by morphological specialization of colony members. In physical caste systems, the diet and rearing environment of developing larvae is known to determine the phenotype of adult individuals, but recent studies have shown that genetic components also contribute to the determination of worker caste. One of the most extreme cases of worker caste differentiation occurs in the army ant genus Eciton, where queens mate with many males and colonies are therefore composed of numerous full-sister subfamilies. This high intracolonial genetic diversity, in combination with the extreme caste polymorphism, provides an excellent test system for studying the extent to which caste determination is genetically controlled. Here we show that genetic effects contribute significantly to worker caste fate in Eciton burchellii. We conclude that the combination of polyandry and genetic variation for caste determination may have facilitated the evolution of worker caste diversity in some lineages of social insects.

Habitat fragmentation may interrupt trophic interactions if herbivores and their specific parasitoids respond differently to decreasing connectivity of populations. Theoretical models predict that species at higher trophic levels are more negatively affected by isolation than lower trophic level species. By combining ecological data with genetic information from microsatellite markers we tested this hypothesis on the butterfly Maculinea nausithous and its specialist hymenopteran parasitoid Neotypus melanocephalus. We assessed the susceptibility of both species to habitat fragmentation by measuring population density, rate of parasitism, overall genetic differentiation (theta(ST)) and allelic richness in a large metapopulation. We also simulated the dynamics of genetic differentiation among local populations to asses the relative effects of migration rate, population size, and haplodiploid (parasitoid) and diploid (host) inheritance on metapopulation persistence. We show that parasitism by N. melanocephalus is less frequent at larger distances to the nearest neighbouring population of M. nausithous hosts, but that host density itself is not affected by isolation. Allelic richness was independent of isolation, but the mean genetic differentiation among local parasitoid populations increased with the distance between these populations. Overall, genetic differentiation in the parasitoid wasp was much greater than in the butterfly host and our simulations indicate that this difference is due to a combination of haplodiploidy and small local population sizes. Our results thus support the hypothesis that Neotypus parasitoid wasps are more sensitive to habitat fragmentation than their Maculinea butterfly hosts.

I redevelop the hypothesis that lifetime monogamy is a fundamental condition for the evolution of eusocial lineages with permanent non-reproductive castes, and that later elaborations--such as multiply-mated queens and multi-queen colonies--arose without the re-mating promiscuity that characterizes non-social and cooperative breeding. Sexually selected traits in eusocial lineages are therefore peculiar, and their evolution constrained. Indirect (inclusive) fitness benefits in cooperatively breeding vertebrates appear to be negatively correlated with promiscuity, corroborating that kin selection and sexual selection tend to generally exclude each other. The monogamy window required for transitions from solitary and cooperative breeding towards eusociality implies that the relatedness and benefit-cost variables of Hamilton's rule do not vary at random, but occur in distinct and only partly overlapping combinations in cooperative, eusocial, and derived eusocial breeding systems.

Background: Termites of the subfamily Macrotermitinae live in a mutualistic symbiosis with basidiomycete fungi of the genus Termitomyces. Here, we explored interaction specificity in fungus-growing termites using samples from 101 colonies in South-Africa and Senegal, belonging to eight species divided over three genera. Knowledge of interaction specificity is important to test the hypothesis that inhabitants (symbionts) are taxonomically less diverse than 'exhabitants' (hosts) and to test the hypothesis that transmission mode is an important determinant for interaction specificity., Results: Analysis of Molecular Variance among symbiont ITS sequences across termite hosts at three hierarchical levels showed that 47 % of the variation occurred between genera, 18 % between species, and the remaining 35 % between colonies within species. Different patterns of specificity were evident. High mutual specificity was found for the single Macrotermes species studied, as M. natalensis was associated with a single unique fungal haplotype. The three species of the genus Odontotermes showed low symbiont specificity: they were all associated with a genetically diverse set of fungal symbionts, but their fungal symbionts showed some host specificity, as none of the fungal haplotypes were shared between the studied Odontotermes species. Finally, bilaterally low specificity was found for the four tentatively recognized species of the genus Microtermes, which shared and apparently freely exchanged a common pool of divergent fungal symbionts., Conclusion: Interaction specificity was high at the genus level and generally much lower at the species level. A comparison of the observed diversity among fungal symbionts with the diversity among termite hosts, indicated that the fungal symbiont does not follow the general pattern of an endosymbiont, as we found either similar diversity at both sides or higher diversity in the symbiont. Our results further challenge the hypothesis that transmission-mode is a general key-determinant of interaction specificity in fungus-growing termites.

Advanced societies owe their success to an efficient division of labour that, in some social insects, is based on specialized worker phenotypes. The system of caste determination in such species is therefore critical. Here, we examine in a leaf-cutting ant (Acromyrmex echinatior) how a recently discovered genetic influence on caste determination interacts with the social environment. By removing most of one phenotype (large workers; LW) from test colonies, we increased the stimulus for larvae to develop into this caste, while for control colonies we removed a representative sample of all workers so that the stimulus was unchanged. We established the relative tendencies of genotypes to develop into LW by genotyping workers before and after the manipulation. In the control colonies, genotypes were similarly represented in the large worker caste before and after worker removal. In the test colonies, however, this relationship was significantly weaker, demonstrating that the change in environmental stimuli had altered the caste propensity of at least some genotypes. The results indicate that the genetic influence on worker caste determination acts via genotypes differing in their response thresholds to environmental cues and can be conceptualized as a set of overlapping reaction norms. A plastic genetic influence on division of labour has thus evolved convergently in two distantly related polyandrous taxa, the leaf-cutting ants and the honeybees, suggesting that it may be a common, potentially adaptive, property of complex, genetically diverse societies.

Background: Army ants are the prime arthropod predators in tropical forests, with huge colonies and an evolutionary derived nomadic life style. Five of the six recognized subgenera of Old World Dorylus army ants forage in the soil, whereas some species of the sixth subgenus (Anomma) forage in the leaf-litter and some as conspicuous swarm raiders on the forest floor and in the lower vegetation (the infamous driver ants). Here we use a combination of nuclear and mitochondrial DNA sequences to reconstruct the phylogeny of the Dorylus s.l. army ants and to infer the evolutionary transitions in foraging niche and associated morphological adaptations., Results: Underground foraging is basal and gave rise to leaf-litter foraging. Leaf-litter foraging in turn gave rise to two derived conditions: true surface foraging (the driver ants) and a reversal to subterranean foraging (a clade with most of the extant Dorylus s.s. species). This means that neither the subgenus Anomma nor Dorylus s.s. is monophyletic, and that one of the Dorylus s.s. lineages adopted subterranean foraging secondarily. We show that this latter group evolved a series of morphological adaptations to underground foraging that are remarkably convergent to the basal state., Conclusion: The evolutionary transitions in foraging niche were more complex than previously thought, but our comparative analysis of worker morphology lends strong support to the contention that particular foraging niches have selected for very specific worker morphologies. The surprising reversal to underground foraging is therefore a striking example of convergent morphological evolution.

The evolution of mating systems in eusocial Hymenoptera is constrained because females mate only during a brief period early in life, whereas inseminated queens and their stored sperm may live for decades. Considerable research effort during recent years has firmly established that obligate multiple mating has evolved only a few times: in Apis honeybees, Vespula wasps, Pogonomyrmex harvester ants, Atta and Acromyrmex leaf-cutting ants, the ant Cataglyphis cursor, and in at least some army ants. Here we provide estimates of queen-mating frequency for New World Neivamyrmex and Old World Aenictus species, which, compared to other army ants, have relatively small colonies and little size polymorphism among workers. To provide the first overall comparative analysis of the evolution of army ant mating systems, we combine these new results with previous estimates for African Dorylus and New World Eciton army ants, which have very large colonies and considerable worker polymorphism. We show that queens of Neivamyrmex and Aenictus mate with the same high numbers of males (usually ca. 10-20) as do queens of army ant species with very large colony sizes. We infer that multiple queen mating is ancestral in army ants and has evolved over 100 million years ago as part of the army ant adaptive syndrome. A comparison of army ants and honeybees suggests that mating systems in these two distantly related groups may have been convergently shaped by strikingly similar selective pressures.

Switching of symbiotic partners pervades most mutualisms, despite mechanisms that appear to enforce partner fidelity. To investigate the interplay of forces binding and dissolving mutualistic pairings, we investigated partner fidelity at the population level in the attine ant-fungal cultivar mutualism. The ants and their cultivars exhibit both broad-scale co-evolution, as well as cultivar switching, with short-term symbiont fidelity maintained by vertical transmission of maternal garden inoculates via dispersing queens and by the elimination of alien cultivar strains. Using microsatellite markers, we genotyped cultivar fungi associated with five co-occurring Panamanian attine ant species, representing the two most derived genera, leaf-cutters Atta and Acromyrmex. Despite the presence of mechanisms apparently ensuring the cotransmission of symbiont genotypes, different species and genera of ants sometimes shared identical fungus garden genotypes, indicating widespread cultivar exchange. The cultivar population was largely unstructured with respect to host ant species, with only 10% of the structure in genetic variance being attributable to partitioning among ant species and genera. Furthermore, despite significant genetic and ecological dissimilarity between Atta and Acromyrmex, generic difference accounted for little, if any, variance in cultivar population structure, suggesting that cultivar exchange dwarfs selective forces that may act to create co-adaptive ant-cultivar combinations. Thus, binding forces that appear to enforce host fidelity are relatively weak and pairwise associations between cultivar lineages and ant species have little opportunity for evolutionary persistence. This implicates that mechanisms other than partner fidelity feedback play important roles in stabilizing the leafcutter ant-fungus mutualism over evolutionary time.

Copulation behavior has often been shaped by sexually selected sperm competition or cryptic female choice. However, manipulation of previously deposited ejaculates is unknown in the social Hymenoptera and the degree to which sperm competes after insemination or is actively selected by females has remained ambiguous. We studied the mating process in the leaf-cutting ants Atta colombica and A. cephalotes, which belong to one of the few derived social insect lineages where obligate multiple mating has evolved. As copulations often occur at night and in remote places, direct observations were impossible, so we had to reconstruct the sequential copulation events by morphological analysis of the male and female genitalia and by tracking the process of sperm transfer and sperm storage. We show that Atta male genitalia have two external rows of spiny teeth, which fit into a specialized pouch organ in the female sexual tract. Reconstruction of the sperm storage process indicated that sperm is transferred to the spermatheca during or immediately after ejaculation and without being mixed with sperm and seminal fluids from other males. A convergent mechanism of direct sperm transfer to the spermatheca of queens is known from two species of dwarf honeybees. Direct sperm transfer may restrict female control over the sperm storage process and the number of males that contribute to the stored sperm., (Copyright (c) 2006 Wiley-Liss, Inc.)

All colonies of the fungus-growing termite Macrotermes natalensis studied so far are associated with a single genetically variable lineage of Termitomyces symbionts. Such limited genetic variation of symbionts and the absence of sexual fruiting bodies (mushrooms) on M. natalensis mounds would be compatible with clonal vertical transmission, as is known to occur in Macrotermes bellicosus. We investigated this hypothesis by analysing DNA sequence polymorphisms as codominant SNP markers of four single-copy gene fragments of Termitomyces isolates from 31 colonies of M. natalensis. A signature of free recombination was found, indicative of frequent sexual horizontal transmission. First, all 31 strains had unique multilocus genotypes. Second, SNP markers (n = 55) were largely in Hardy-Weinberg equilibrium (90.9%) and almost all possible pairs of SNPs between genetically unlinked loci were in linkage equilibrium (96.7%). Finally, extensive intragenic recombination was found, especially in the EF1alpha fragment. Substantial genetic variation and a freely recombining population structure can only be explained by frequent horizontal and sexual transmission of Termitomyces. The apparent variation in symbiont transmission mode among Macrotermes species implies that vertical symbiont transmission can evolve rapidly. The unexpected finding of horizontal transmission makes the apparent absence of Termitomyces mushrooms on M. natalensis mounds puzzling. To our knowledge, this is the first detailed study of the genetic population structure of a single lineage of Termitomyces.