The loss of flight in ant workers enabled an evolutionary redesign of the thorax for ground labour.

BACKGROUND: Explanations for the ecological dominance of ants generally focus on the benefits of division of labour and cooperation during foraging. However, the principal innovation of ants relative to their wasp ancestors was the evolution of a new phenotype: a wingless worker caste optimized for ground labour. Ant workers are famous for their ability to lift and carry heavy loads, but we know surprisingly little about the morphological basis of their strength. Here we examine the consequences of the universal loss of flight in ant workers on skeletomuscular adaptations in the thorax for enhanced foraging on six legs. RESULTS: Using X-ray microcomputed tomography and 3D segmentation, we compared winged queens and wingless workers in Euponera sikorae (subfamily Ponerinae) and Cataglyphis savignyi (subfamily Formicinae). Workers are characterized by five major changes to their thorax: i) fusion of the articulated flight thorax (queens) into a rigid box optimized to support the muscles that operate the head, legs and abdomen, ii) redesign of internal cuticular structures for better bracing and muscle attachment, iii) substantial enlargement of the neck muscles for suspending and moving the head, iv) lengthening of the external trochanter muscles, predominant for the leg actions that lift the body off the ground, v) modified angle of the petiole muscles that are key for flexion of the abdomen. We measured volumes and pennation angles for a few key muscles to assess their increased efficacy. Our comparisons of additional workers across five genera in subfamilies Dorylinae and Myrmicinae show these modifications in the wingless thorax to be consistent. In contrast, a mutillid wasp showed a different pattern of muscle adaptations resulting from the lack of wing muscles. CONCLUSIONS: Rather than simply a subtraction of costly flight muscles, we propose the ant worker thorax evolved into a power core underlying stronger mandibles, legs, and sting. This contrasts with solitary flightless insects where the lack of central place foraging generated distinct selective pressures for rearranging the thorax. Stronger emphasis is needed on morphological innovations of social insects to further our understanding of the evolution of social behaviours.

Skeletomuscular adaptations of head and legs of Melissotarsus ants for tunnelling through living wood.

BACKGROUND: While thousands of ant species are arboreal, very few are able to chew and tunnel through living wood. Ants of the genus Melissotarsus (subfamily Myrmicinae) inhabit tunnel systems excavated under the bark of living trees, where they keep large numbers of symbiotic armoured scale insects (family Diaspididae). Construction of these tunnels by chewing through healthy wood requires tremendous power, but the adaptations that give Melissotarsus these abilities are unclear. Here, we investigate the morphology of the musculoskeletal system of Melissotarsus using histology, scanning electron microscopy, X-ray spectrometry, X-ray microcomputed tomography (micro-CT), and 3D modelling. RESULTS: Both the head and legs of Melissotarsus workers contain novel skeletomuscular adaptations to increase their ability to tunnel through living wood. The head is greatly enlarged dorsoventrally, with large mandibular closer muscles occupying most of the dorsal half of the head cavity, while ventrally-located opener muscles are also exceptionally large. This differs from the strong closing: opening asymmetry typical of most mandibulated animals, where closing the mandibles requires more force than opening. Furthermore, the mandibles are short and cone-shaped with a wide articulatory base that concentrates the force generated by the muscles towards the tips. The increased distance between the axis of mandibular rotation and the points of muscle insertion provides a mechanical advantage that amplifies the force from the closer and opener muscles. We suggest that the uncommonly strong opening action is required to move away crushed plant tissues during tunnelling and allow a steady forward motion. X-ray spectrometry showed that the tip of the mandibles is reinforced with zinc. Workers in this genus have aberrant legs, including mid- and hindlegs with hypertrophied coxae and stout basitarsi equipped with peg-like setae, and midleg femura pointed upward and close to the body. This unusual design famously prevents them from standing and walking on a normal two-dimensional surface. We reinterpret these unique traits as modifications to brace the body during tunnelling rather than locomotion per se. CONCLUSIONS: Melissotarsus represents an extraordinary case study of how the adaptation to - and indeed engineering of - a novel ecological niche can lead to the evolutionary redesign of core biomechanical systems.

Uncoupling Flight and Reproduction in Ants: Evolution of Ergatoid Queens in Two Lineages of Megalomyrmex (Hymenoptera: Formicidae).

Megalomyrmex Forel (Myrmicinae: Solenopsidini) consists of 44 species with diverse life history strategies. Most species are predatory and may also tend honeydew-producing insects. A morphologically derived group of species are social parasites that consume the brood and fungus garden within fungus-growing ant nests. The reproductive strategies of Megalomyrmex queens are somewhat aligned with these life-style patterns. Predatory species in the leoninus species group are large in body size and have ergatoid (i.e., permanently wingless) queens whereas the social parasitic species are smaller and typically have winged queens. We examined two ergatoid phenotypes of Megalomyrmex foreli Emery and Megalomyrmex wallacei Mann and compared them to winged species, one a social lestobiotic or "thief ant" parasite (Megalomyrmex mondabora Brandão) and the other a predator (Megalomyrmex modestus Emery). Megalomyrmex foreli colonies have a single queen with an enlarged gaster that is morphologically distinct from workers. Megalomyrmex wallacei colonies have several queens that are similar in body size to workers. Queens in both species showed a simplification of the thorax, but there was a dramatic difference in the number of ovarioles. Megalomyrmex foreli had 60-80 ovarioles compared to eight in M. wallacei and M. mondabora and M. modestus had 22-28. Along with flight loss in queens, there is an obligate shift to dependent colony founding (also called budding or fission) consequently influencing dispersal patterns. These constraints in life history traits may help explain the variation in nesting biology among Megalomyrmex species.

Recurrent evolution of dependent colony foundation across eusocial insects.

The spectacular success of eusocial insects can be attributed to their sophisticated cooperation, yet cooperation is conspicuously absent during colony foundation when queens are alone. Selection against this solitary stage has led to a dramatically different strategy in thousands of eusocial insect species in which colonies are started by groups of nestmates and the benefits of sociality are retained continuously. Dependent colony foundation (DCF) evolved recurrently multiple times across the ants, bees, and wasps, though its prevalence in termites remains unclear. We review adaptations at both the colony level (reproductive investment shifts from sexuals to workers) and the individual level (wingless queens evolve in ants), and other consequences for life history (invasiveness, parasite transmission). Although few studies have focused on DCF, the accumulated data from anecdotal reports, supported by indirect information including morphology, population genetics, and colony demographics, make it clear that this strategy is more diverse and widespread than is usually recognized.

Evolution of novel mosaic castes in ants: modularity, phenotypic plasticity, and colonial buffering.

Many ants have independently evolved castes with novel morphology as well as function, such as soldiers and permanently wingless (ergatoid) queens. We present a conceptual model, based on modularity in morphology and development, in which evolutionary innovation is facilitated by the ancestral ant polyphenism of winged queens and wingless workers. We suggest that novel castes evolved from rare intercastes, anomalous mosaics of winged queens and workers, erratically produced by colonies through environmental or genetic perturbations. The colonial environment is highly accommodating and buffers viable intercastes from individual selection. Their cost is limited because they are diluted by the large number of nestmates, yet some can bring disproportionate benefits to their colonies in the context of defense or reproduction (e.g., wingless intercastes able to mate). Useful intercastes will increase in frequency as their morphology is stabilized through genetic accommodation. We show that both soldiers and ergatoid queens are mosaics of winged queens and workers, and they are strikingly similar to some intercastes. Modularity and developmental plasticity together with winged/wingless polyphenism thus allow for the production of highly variable mosaic intercastes, and colonies incubate the advantageous mosaics.

Minute workers and large soldiers in the subterranean ant Carebara perpusilla: Musculoskeletal consequences of Haller's rule in the thorax.

Many organismal traits vary with body size, often reflecting trade-offs in the face of size-dependent constraints. For example, Haller's rule, the allometric pattern whereby smaller organisms have proportionally larger brains, can have carry-on effects on head design as the brain competes for space with other structures. Ant species with polymorphic worker castes are interesting cases for helping us understand these allometric effects. Here, we examine the effects of miniaturization on the ant power core, the mesosoma (thorax), with particular attention to how the scaling of nervous system structures affects the skeletomuscular elements involved with load bearing and locomotion. Using X-ray computed microtomography (microCT), we studied the thorax of Carebara perpusilla, an African ant species that has minute workers (1.5 mm-long) and larger soldiers (3.0 mm-long), allowing strong intraspecific comparisons. We find that the thoracic nervous system is relatively larger in minute workers, similar to Haller's rule, with consequences on the skeletomuscular organisation. Minute workers have relatively smaller petiole muscles and indirect head muscles, but relatively larger external trochanter muscles and direct head muscles. We link these allometric trade-offs to miniaturization and division of labor, and discuss how thorax design underlies the success of minute ants.

Glandular innovations for a tunnelling life: Silk and associated leg glands in Melissotarsus and Rhopalomastix queen and worker ants.

As in other Hymenoptera, adult ants cannot secrete silk, unlike the larvae that spin a cocoon prior to metamorphosis. Fisher and Robertson (1999) first showed the existence of a silk gland in the head of adult Melissotarsus beccarii workers, and we confirm this with detailed histology and ultrastructural comparisons of both queens and workers. This African genus exhibits extreme morphological adaptations (legs, head shape and mandibular muscles) for tunnelling behaviour inside living trees, that underlie an obligate mutualism with scale insects. Rhopalomastix is its sister genus distributed across Asia, and we show that queens and workers also have a silk gland. This lineage of minute workers relies on silk to secure their network of tunnels against other arboreal ants. We show striking differences between these genera in the anatomy and ultrastructure of the cells that secrete silk, especially numerous vacuoles and an unexpectedly branched end apparatus in Melissotarsus. Moreover, the legs of Melissotarsus are much more specialized for tunnelling, and this includes highly expanded basitarsi. The latter house the novel 'Delage-Darchen gland', and we document its anatomy and ultrastructure, suggesting a proteinaceous secretion to harden roofs made of silk combined with wood fragments. The restriction of the Delage-Darchen gland to Melissotarsus, combined with a modified silk gland (an almost three-fold increase in the number of secretory cells, and ultrastructural differences suggestive of higher secretory activity), are evidence of an outstanding evolutionary divergence relative to Rhopalomastix. Synthesis of silk by adults is a significant innovation among ants, but its augmented production in Melissotarsus makes them better adapted for the hazards of arboreal life.

Shift in colonial reproductive strategy associated with a tropical-temperate gradient in Rhytidoponera ants.

Offspring quality and quantity are subject to a trade-off. Depending on species, ant colonies can produce either numerous winged queens that start new colonies alone (independent colony foundation [ICF]) or one propagule consisting of reproductives helped by workers (fission). Some species do both. In the Rhytidoponera impressa group, colonies with winged queens use ICF, whereas colonies with mated workers use fission. We assessed the effect of latitude and environment on colonial reproduction by collecting 79 colonies along the Australian east coast. A population dynamical model predicted that from tropical north to temperate south, seasonal fluctuations and harsher conditions should lead to a decrease in ICF success (-48%), making fission a suitable alternative. Yet, our empirical data showed that ICF persists at a higher rate than expected, presumably because it allows aerial dispersal, unlike fission. Investigation of colony demography and measurement of lean and fat weights in virgin winged queens and workers showed that, from north to south, colonies increase quality of queens relative to workers (+50%) at the cost of quantity (-86%). This modification limits the decrease in ICF success (-34%). A tremendous range of offspring phenotypes associated with quality-quantity trade-offs make ants competitive in diverse habitats.

A mutualism without honeydew: what benefits for Melissotarsus emeryi ants and armored scale insects (Diaspididae)?

Mutualisms between ants and sap-sucking insects generally involve clear benefits for both partners: the ants provide protection in exchange for honeydew. However, a single ant genus associates with armoured scale insects (Diaspididae) that do not excrete honeydew. We studied three colonies of Melissotarsus emeryi ants from two localities in Mozambique. Vast numbers of the diaspidid Morganella conspicua occupied galleries dug by the ants under the bark of living trees. Unlike free-living M. conspicua and other diaspidids, M. conspicua living with ants are known to lack shields, likely because they gain protection against enemies and desiccation. Nevertheless, we documented the occurrence of rare individuals with shields inside ant galleries, indicating that their glands continue to secrete wax and proteins as building material. This is likely to constitute a significant portion of the ants' diet, in addition to diaspidid exuviae and excretions from the Malpighian tubules. Indeed, Melissotarsus workers cannot walk outside the galleries due to modified middle legs, forcing them to obtain all nourishment within the tree. Melissotarsus founding queens, however, must locate a suitable host tree while flying, and acquire diaspidid crawlers. This mutualism involves ants that are highly specialised to chew through living wood, and diaspidids that can also live freely outside the bark. It is extremely widespread in Africa and Madagascar, recorded from 20 tree families, and harmful effects on plant hosts require rapid study.

Regulation of reproduction in a queenless ant: aggression, pheromones and reduction in conflict.

In the monogynous queenless ant Diacamma ceylonense, the future reproductive (future gamergate) is very aggressive towards infertile workers during the first days of her adult life. Overt aggression disappears at about three weeks, when the future gamergate begins to lay male-destined eggs and is ready to mate. Over the same period, her cuticular hydrocarbon profile alters, changing from a chemical signature similar to that of a sterile worker towards that of a gamergate. In nature, these behavioural and chemical changes will coincide with a reduction in conflict within the nest: faced with a virgin future gamergate, infertile workers have an interest in producing male-destined eggs; however, once the gamergate produces female eggs, they have an interest in rearing her offspring. This demonstration of a shift from physical inhibition to chemical signalling is interpreted in terms of sociogenetic theory, the role of cuticular hydrocarbons as an indicator of fertility in insects and the fact that the regulation of reproduction in Diacamma involves mechanisms redolent of both queenless and queenright ant species.

Bigger helpers in the ant Cataglyphis bombycina: increased worker polymorphism or novel soldier caste?

INTRODUCTION: The mechanisms by which development favors or constrains the evolution of new phenotypes are incompletely understood. Polyphenic species may benefit from developmental plasticity not only regarding ecological advantages, but also potential for evolutionary diversification. For instance, the repeated evolution of novel castes in ants may have been facilitated by the existence of alternative queen and worker castes and their respective developmental programs. MATERIAL AND METHODS: Cataglyphis bombycina is exceptional in its genus because winged queens and size-polymorphic workers occur together with bigger individuals having saber-shaped mandibles. We measured seven body parts in more than 150 individuals to perform a morphometric analysis and assess the developmental origin of this novel phenotype. RESULTS: Adults with saber-shaped mandibles differ from both workers and queens regarding the size of most body parts. Their relative growth rates are identical to workers for some pairs of body parts, and identical to queens for other pairs of body parts; critical sizes differ in all cases. CONCLUSIONS: Big individuals are a third caste, i.e. soldiers, not major workers. Novel traits such as elongated mandibles are combined with a mix of queen and worker growth rates. We also reveal the existence of a dimorphism in the queen caste (microgynes and macrogynes). We discuss how novel phenotypes can evolve more readily in the context of an existing polyphenism. Both morphological traits and growth rules from existing queen and worker castes can be recombined, hence mosaic phenotypes are more likely to be viable. In C. bombycina, such a mosaic phenotype appears to function both for defense (saber-shaped mandibles) and fat storage (big abdomen). Recycling of developmental programs may have contributed to the morphological diversity and ecological success of ants.

Evolution of thorax architecture in ant castes highlights trade-off between flight and ground behaviors.

The concerted evolution of morphological and behavioral specializations has compelling examples in ant castes. Unique to ants is a marked divergence between winged queens and wingless workers, but morphological specializations for behaviors on the ground have been overlooked. We analyzed thorax morphology of queens and workers in species from 21 of the 25 ant subfamilies. We uncovered unique skeletomuscular modifications in workers that presumably increase power and flexibility of head-thorax articulation, emphasizing that workers are not simply wingless versions of queens. We also identified two distinct types of queens and showed repeated evolutionary associations with strategies of colony foundation. Solitary founding queens that hunt have a more worker-like thorax. Our results reveal that ants invest in the relative size of thorax segments according to their tasks. Versatility of head movements allows for better manipulation of food and objects, which arguably contributed to the ants' ecological and evolutionary success. DOI: http://dx.doi.org/10.7554/eLife.01539.001.

Evolution of a soldier caste specialized to lay unfertilized eggs in the ant genus Crematogaster (subgenus Orthocrema).

Among social Hymenoptera, only some ant genera have more than one morphological kind of non-reproductive adults. Individuals that are bigger than ordinary workers can function for defence and/or food storage. In Crematogaster (Orthocrema) smithi from Arizona, a third caste exists in addition to winged queens and workers; it is intermediate in size, weight and morphology, and individuals lay many unfertilized eggs that are mostly eaten by larvae (Heinze et al., 1995, 1999). We studied another three species belonging to the subgenus Orthocrema: Crematogaster pygmaea from Brazil, Crematogaster biroi and Crematogaster schimmeri from Taiwan. Using scanning electron microscopy and ovarian dissections, we show that 'intermediates' are a patchwork of queen-like and worker-like traits, just as in C. smithi; importantly the combinations differ across species. 'Intermediates' are numerically few in the colonies, and in C. pygmaea they are produced seasonally. Using histology we confirmed the lack of a spermatheca, thus they are not ergatoid queens. Based on the similarity of their mosaic phenotypes with those in other ant lineages, we suggest that Orthocrema 'intermediates' are a soldier caste with a specialized trophic function. This soldier caste has been reported in other Orthocrema species from Madagascar, Guinea and Costa Rica, suggesting that it is widespread in this subgenus.

Selection against aerial dispersal in ants: two non-flying queen phenotypes in Pogonomyrmex laticeps.

The South American seed-harvester ant Pogonomyrmex laticeps has dimorphic queens: ergatoid (permanently wingless) and brachypterous (short, non-functional wings). Surveys in western Argentina indicated that colonies near Chilecito, La Rioja Province, produced only ergatoid queens, while those near Punta Balasto, Catamarca Province (263 km away), produced only brachypterous queens. Brachypterous queens were significantly larger than ergatoid queens for 10 of 11 external characters, but both phenotypes had comparable reproductive potential, i.e., a spermatheca and a similar number of ovarioles. Using normal winged queens of the closely related P. uruguayensis for comparison, we determined that both queen phenotypes in P. laticeps had a full set of dorsal thoracic sclerites, albeit each sclerite was much reduced, whereas workers had a thorax without distinct dorsal sclerites. Sclerites were fused and immobile in ergatoid queens, while they were separable and fully articulated in brachypterous queens. Both phenotypes lacked the big indirect flight muscles, but brachypterous queens retained the tiny direct flight muscles. Overall, this dimorphism across populations indicates that there are alternative solutions to selective pressures against flying queens. We lack field data about colony founding strategy (independent or dependent) for either queen phenotype, but colonies at both sites produced numerous gynes, and we infer that all foundresses initiate colonies independently and are obligate foragers.

How many gamergates is an ant queen worth?

Ant reproductives exhibit different morphological adaptations linked to dispersal and fertility. By reviewing the literature on taxa where workers can reproduce sexually (i.e. become gamergates) we show that (1) species with a single gamergate generally have lost the winged queen caste, whereas only half of the species with several gamergates have, and (2) single-gamergate species have smaller colonies than multiple-gamergate species. Comparison with "classical" ants without gamergates, where having one vs having several winged queens are two distinct syndromes, suggests that having one vs having several gamergates are not. Gamergate number does not affect the success of colony fission, but retention of the queen caste permits the option of independent foundation.

Degeneration of sperm reservoir and the loss of mating ability in worker ants.

Workers never mate in the large majority of ants, and they have usually lost the spermatheca, an organ specialized for long-term storage of sperm. Such 'non-sexual' workers are restricted to laying unfertilized eggs that give rise to males, and they cannot compete with the queens for the production of female offspring. In sharp contrast, workers in 200-300 species from phylogenetically basal subfamilies can reproduce sexually ('gamergates') because they retain a functional spermatheca like the queens. Importantly, 'non-sexual' workers in closely related species have a vestigial spermatheca. In this study, we compared the reservoir epithelium of 'sexual' workers to that of congeneric queens and 'non-sexual' workers using 21 species of Amblyoponinae, Ponerinae and Ectatomminae. We show that a pronounced thickening of the epithelium near the opening of the sperm duct is strictly associated with sexual reproduction in both castes. This is unlike 'non-sexual' workers in which this epithelium is always very thin, with few organelles; but all other structures remain intact. We discuss this evolutionary degeneration of the spermatheca and how it relates to behavioural or physiological modifications linked to mating. Our results help understand the loss of sexual reproduction by ant workers, a critical step in the extreme specialization of their phenotype.

Winged queens replaced by reproductives smaller than workers in Mystrium ants.

In ants, winged queens that are specialized for independent colony foundation can be replaced by wingless reproductives better adapted for colony fission. We studied this shift in reproductive strategy by comparing two Mystrium species from Madagascar using morphometry, allometry and dissections. Mystrium rogeri has a single dealate queen in each colony with a larger thorax than workers and similar mandibles that allow these queens to hunt during non-claustral foundation. In contrast, Mystrium 'red' lacks winged queens and half of the female adults belong to a wingless 'intermorph' caste smaller and allometrically distinct from the workers. Intermorphs have functional ovaries and spermatheca while those of workers are degenerate. Intermorphs care for brood and a few mate and reproduce making them an all-purpose caste that takes charge of both work and reproduction. However, their mandibles are reduced and inappropriate for hunting centipedes, unlike the workers' mandibles. This together with their small thorax disallow them to perform independent colony foundation, and colonies reproduce by fission. M. rogeri workers have mandibles polymorphic in size and shape, which allow for all tasks from brood care to hunting. In M. 'red', colonial investment in reproduction has shifted from producing expensive winged queens to more numerous helpers. M. 'red' intermorphs are the first case of reproductives smaller than workers in ants and illustrate their potential to diversify their caste system for better colonial economy.

Testing homology with morphology, development and gene expression: sex-specific thoracic appendages of the ant Diacamma.

Females of the ants belonging to the queenless genus Diacamma have a pair of unique tiny thoracic appendages, called "gemmae," located on the mesothoracic segment. They are covered with sensory hairs, filled with exocrine glands and are involved in the behavioral regulation of reproduction. We report here a morphological, developmental, and genetic study of the development of the gemmae. Both male and female larvae have dorsal mesothoracic discs, although differing in shape and fate. In Diacamma ceylonense, we show that, contrary to butterflies, these discs specify parts of the adult thorax in addition to wing tissues, as in Drosophila. We have cloned and studied the expression of wingless (wg) and scalloped (sd), two genes known to play a critical role in wing morphogenesis in Drosophila. In the fly's mesothoracic dorsal disc, sd is specifically expressed in the wing pouch. In Diacamma, we show that sd is also expressed in male dorsal thoracic discs, whereas its expression was undetectable in females. From this result and observations of shape and growth of cultured isolated discs, we suggest that gemmae originate from a more ventral part of the dorsal disc than the wing pouch and discuss the pro and cons of gemma/wing homology.

Queen-worker differences in spermatheca reservoir of phylogenetically basal ants.

Ant queens mate when young and store sperm in their spermatheca to fertilize eggs for several years until their death. In contrast, workers in most species never mate. We have compared the histological organization of spermathecae in 25 poneromorph species exhibiting various degrees of queen-worker dimorphism. The spermathecae of both castes in all species are similar in having a reservoir connected by a sperm duct to the ovary, and a paired gland opening into this duct. The reservoir of queens typically has a columnar epithelium in the hilar region (near the opening of the sperm duct), whereas the epithelium in the distal region is cuboidal. Abundant mitochondria together with apical microvilli and basal invaginations indicate an osmoregulatory function. In contrast, the reservoir epithelium of workers is flattened throughout and lacks these transport characteristics. This single difference shows the importance of a columnar epithelium in the reservoir for sperm storage. However, our data have not revealed inter-specific variations in the development of the hilar region linked with higher fecundity. We have found no consistent differences in associated structures, such as the spermatheca gland or sperm ducts, or in the musculature between queens and workers.