Prolonged faunal turnover in earliest ants revealed by North American Cretaceous amber.

All ∼14,000 extant ant species descended from the same common ancestor, which lived ∼140-120 million years ago (Ma).1,2 While modern ants began to diversify in the Cretaceous, recent fossil evidence has demonstrated that older lineages concomitantly occupied the same ancient ecosystems.3 These early-diverging ant lineages, or stem ants, left no modern descendants; however, they dominated the fossil record throughout the Cretaceous until their ultimate extinction sometime around the K-Pg boundary. Even as stem ant lineages appear to be diverse and abundant throughout the Cretaceous, the extent of their longevity in the fossil record and circumstances contributing to their extinction remain unknown.3 Here we report the youngest stem ants, preserved in ∼77 Ma Cretaceous amber from North Carolina, which illustrate unexpected morphological stability and lineage persistence in this enigmatic group, rivaling the longevity of contemporary ants. Through phylogenetic reconstruction and morphometric analyses, we find evidence that total taxic turnover in ants was not accompanied by a fundamental morphological shift, in contrast to other analogous stem extinctions such as theropod dinosaurs. While stem taxa showed broad morphological variation, high-density ant morphospace remained relatively constant through the last 100 million years, detailing a parallel, but temporally staggered, evolutionary history of modern and stem ants.

Morphology of the Mandibular and Intramandibular Glands of Army Ant Workers of Labidus praedator (Smith 1858) and Labidus coecus (Latreille 1802) (Formicidae: Dorylinae).

Social insects are characterized by having a wide diversity of exocrine glands, with highlights for ants with about 85 glands spreading throughout the body. The mandibular and intramandibular glands are associated with the production of pheromones. The army ants (Dorylinae) play an important role in the structure of the invertebrate community because they are efficient predators and provide suitable conditions for various animals following their invasions in the food search. Labidus coecus (Latreille) is an underground-ameliorating ant and Labidus praedator (Smith) is a generalist surface predator which can deplete invertebrate biomass by up to 75%. This work investigated the morphology of the mandibular and intramandibular glands of L. praedator and L. coecus workers. The glands were analyzed by light microscopy, histochemistry, and scanning electron microscopy. The mandibular and intramandibular glands of the two species were classified as class III glands. The data on the morphology of the mandibular glands has revealed that they have characteristics in common with other subfamilies. The intramandibular glands of the two species of Labidus have similar morphology and chemical composition, which indicates that the components of these glands can have the same function despite their different habits.

Morphology and ultrastructure of the mandibular gland in Camponotus japonicus.

The mandibular gland is an important exocrine gland connected to the mandibles. In ants, it plays a crucial role in alarm communication and reproduction. While the overall morphology of the mandibular gland does not vary much between ant species, significant differences exist in the ultrastructural characteristics and gland size in some reported cases. However, there have been few systematic comparative studies on this subject. In this paper, we conducted microscopic observations to analyze the morphological and ultrastructural similarities and differences of the mandibular gland in different castes of Camponotus japonicus. Our findings revealed that the mandibular glands of males were significantly larger than those of females. In terms of relative size, within the female castes the mandibular glands of minor workers were the largest. Additionally, our observations of the ultrastructure showed that secretory substances were most abundant in queens. All female castes and males showed the presence of crystalline structures in the secretory cells. The content of these biocrystals decreased from minor workers to major workers, queens, and males. In males, however, crystalline structures were rare and the most abundant substances are droplet-like secretions. The differences in morphology and ultrastructure between different castes suggest that the function of the mandibular gland varies among castes. In conclusion, our study provides insights into the morphological and ultrastructural characteristics of the mandibular gland in different castes of C. japonicus. Further research is needed to fully understand the specific functions and mechanisms of this gland in ants.

Variation in Pheidole nodus (Hymenoptera: Formicidae) functional morphology across urban parks.

Background: Habitat fragmentation and consequent population isolation in urban areas can impose significant selection pressures on individuals and species confined to urban islands, such as parks. Despite many comparative studies on the diversity and structure of ant community living in urban areas, studies on ants' responses to these highly variable ecosystems are often based on assemblage composition and interspecific mean trait values, which ignore the potential for high intraspecific functional trait variation among individuals. Methods: Here, we examined differences in functional traits among populations of the generalist ant Pheidole nodus fragmented between urban parks. We used pitfall trapping, which is more random and objective than sampling colonies directly, despite a trade-off against sample size. We then tested whether trait-filtering could explain phenotypic differences among urban park ant populations, and whether ant populations in different parks exhibited different phenotypic optima, leading to positional shifts in anatomical morphospace through the regional ant meta-population. Results: Intraspecific morphological differentiation was evident across this urban region. Populations had different convex hull volumes, positioned differently over the morphospace. Conclusions: Fragmentation and habitat degradation reduced phenotypic diversity and, ultimately, changed the morphological optima of populations in this urban landscape. Considering ants' broad taxonomic and functional diversity and their important role in ecosystems, further work over a variety of ant taxa is necessary to ascertain those varied morphological response pathways operating in response to population segregation in urban environments.

Morphology of the metapleural gland and its associated novel atrial cone gland in Strumigenys ants.

The metapleural gland is a unique feature of the ant phenotype, but diversity in its anatomy and function across the ants is not well documented or understood. We studied the morphology of the metapleural gland in 20 mainly Oriental Strumigenys species using histology, scanning and transmission electron microscopy, and micro-CT. The gland is formed by a cluster of class-3 cells, their secretion is transported through a bundle of ducts into the sclerotized atrium and is guided along a series of parallel cuticular ridges towards the atrial opening. Among the examined species, queens have more gland cells than conspecific workers, while the examined males do not have the gland. The social parasite S. mutica has the most developed metapleural gland. In addition, we describe a novel class-1 atrial cone gland which is associated with the metapleural gland. The epithelium of this cone gland forms an invagination into the ventral atrium of the metapleural gland. The cuticular cone may be lacking in some Afrotropical and Neotropical species, although these may still contain the epithelial gland. The functional and evolutionary drivers of morphological variation in the exocrine system across species form interesting questions for future work.

A caste differentiation mutant elucidates the evolution of socially parasitic ants.

Most ant species have two distinct female castes-queens and workers-yet the developmental and genetic mechanisms that produce these alternative phenotypes remain poorly understood. Working with a clonal ant, we discovered a variant strain that expresses queen-like traits in individuals that would normally become workers. The variants show changes in morphology, behavior, and fitness that cause them to rely on workers in wild-type (WT) colonies for survival. Overall, they resemble the queens of many obligately parasitic ants that have evolutionarily lost the worker caste and live inside colonies of closely related hosts. The prevailing theory for the evolution of these workerless social parasites is that they evolve from reproductively isolated populations of facultative intermediates that acquire parasitic phenotypes in a stepwise fashion. However, empirical evidence for such facultative ancestors remains weak, and it is unclear how reproductive isolation could gradually arise in sympatry. In contrast, we isolated these variants just a few generations after they arose within their WT parent colony, implying that the complex phenotype reported here was induced in a single genetic step. This suggests that a single genetic module can decouple the coordinated mechanisms of caste development, allowing an obligately parasitic variant to arise directly from a free-living ancestor. Consistent with this hypothesis, the variants have lost one of the two alleles of a putative supergene that is heterozygous in WTs. These findings provide a plausible explanation for the evolution of ant social parasites and implicate new candidate molecular mechanisms for ant caste differentiation.

Transcriptomic analysis of mosaic brain differentiation underlying complex division of labor in a social insect.

Concerted developmental programming may constrain changes in component structures of the brain, thus limiting the ability of selection to form an adaptive mosaic of size-variable brain compartments independent of total brain size or body size. Measuring patterns of gene expression underpinning brain scaling in conjunction with anatomical brain atlases can aid in identifying influences of concerted and/or mosaic evolution. Species exhibiting exceptional size and behavioral polyphenisms provide excellent systems to test predictions of brain evolution models by quantifying brain gene expression. We examined patterns of brain gene expression in a remarkably polymorphic and behaviorally complex social insect, the leafcutter ant Atta cephalotes. The majority of significant differential gene expression observed among three morphologically, behaviorally, and neuroanatomically differentiated worker size groups was attributable to body size. However, we also found evidence of differential brain gene expression unexplained by worker morphological variation and transcriptomic analysis identified patterns not linearly correlated with worker size but sometimes mirroring neuropil scaling. Additionally, we identified enriched gene ontology terms associated with nucleic acid regulation, metabolism, neurotransmission, and sensory perception, further supporting a relationship between brain gene expression, brain mosaicism, and worker labor role. These findings demonstrate that differential brain gene expression among polymorphic workers underpins behavioral and neuroanatomical differentiation associated with complex agrarian division of labor in A. cephalotes.

Comparative morphology and ultrastructure of the labial gland among castes of Camponotus japonicus (Hymenoptera: Formicidae).

The labial gland is a major exocrine gland in the thoracic cavity of ants, which is connected to the mouth area and opens at the base of the labium. The labial gland in the various castes and males of Camponotus japonicus were examined using dissection, light microscopy, scanning and transmission electron microscopy. Our findings reveal clear caste differences in the appearance of the labial gland, and suggest a relationship between gland structure and its behavioral role in queens, males and workers: queens possess the largest reservoir sac; males have the smallest; workers have abundant secretion droplets in the gland cells of the tubules; epithelial thickness of gland cells varies considerably between workers and reproductive ants. The apical cell region has a high density of microvilli and polymorphic mitochondria, whereas the central cell region is rich in rough endoplasmic reticulum (RER) which means its main secretions are proteinaceous compounds. We believe that the labial gland has a different function in the castes of C. japonicus: the gland of workers and queens may be used to feed the larvae; workers participate in trophallaxis in the nest, while the males are not involved in these activities. Calculations of the relative size of the labial gland for each caste and males indicated that minor workers have the relative highest developed labial gland, supporting a role in trophallaxis. In addition, we found a phenomenon of enlarged labial gland in minor workers, which accounted for almost 22%, but the reason for this is unknown.

Unparalleled details of soft tissues in a Cretaceous ant.

For social insects such as ants, the internal organs are likely important in understanding their eusocial behavior and evolution. Such organs, however, are rarely preserved on fossils. In each of the few cases reporting exceptionally fossilized soft tissues in arthropods, the nervous, muscular and cardiovascular systems have been described individually, but never in combination. Here, we report a female specimen (gyne) of the extinct ant group-†Zigrasimecia-included in a Cretaceous amber piece from Kachin, Myanmar, with an almost complete system formed by various internal organs. These include the brain, the main exocrine system, part of the digestive tract, and several muscle clusters. This research expands our knowledge of internal anatomy in stem group ants. As the gyne bears a morphologically unique labrum, our specimen's internal and external features support the notion that the early ant may have special ecological habits during the Cretaceous period.

Microstructures at the distal tip of ant chemosensory sensilla.

Ants and other eusocial insects emit and receive chemical signals to communicate important information within the colony. In ants, nestmate recognition, task allocation, and reproductive distribution of labor are largely mediated through the detection of cuticular hydrocarbons (CHCs) that cover the exoskeleton. With their large size and limited volatility, these CHCs are believed to be primarily detected through direct contact with the antennae during behavioral interactions. Here we first use scanning electron microscopy to investigate the unique morphological features of CHC-sensitive basiconic sensilla of two ant species, the black carpenter ant Camponotus pennsylvanicus and the Indian jumping ant Harpegnathos saltator. These basiconic sensilla possess an abundance of small pores typical of most insect olfactory sensilla, but also have a large concave depression at the terminal end. Basiconic sensilla are enriched at the distal segments of the antennae in both species, which aligns with their proposed role in contact chemosensation of CHCs. A survey of these sensilla across additional ant species shows varied microstructures at their tips, but each possess surface textures that would also increase sensory surface area. These unique ant chemosensory sensilla represent yet another example of how specialized structures have evolved to serve the functional requirements of eusocial communication.

Morphology and ultrastructure of the prepharyngeal and pharyngeal glands in the ant Camponotus japonicus.

The prepharyngeal gland (prePG) and pharyngeal gland (PG) make up the largest exocrine structures in the head of the ant Camponotus japonicus. We used microscopy to study the histological and ultrastructural features of both glands in different castes. The number of secretory units in the prePG is considerably higher than in other ant species and shows a complex duct system which is made up by duct cells, secondary ducts and a main duct. These lead the secretions of hundreds to thousands of secretory cells into the prepharynx through a modified sieve plate at each side. The glove-shaped PG shows clear caste differences in tubule number. The ultrastructure of both the prePG and PG shows abundant mitochondria and secretion vesicles. Moreover, the prePG is loaded with rough endoplasmic reticulum (RER) which means its main secretions are proteinaceous compounds, while the PG is dominated by smooth endoplasmic reticulum (SER) which means the main secretions are lipids. The morphological differences like cell number of the prePG and tubule number of the PG indicate different secretory abilities of each caste. We for the first time introduce histology-based relative size to indicate secretory activity. The proportionally high development of the prePG in minor workers supports a role in trophallaxis.

Shifts in morphological covariation and evolutionary rates across multiple acquisitions of the trap-jaw mechanism in Strumigenys.

A long-standing question in comparative biology is how the evolution of biomechanical systems influences morphological evolution. The need for functional fidelity implies that the evolution of such systems should be associated with tighter morphological covariation, which may promote or dampen rates of morphological evolution. I examine this question across multiple evolutionary origins of the trap-jaw mechanism in the genus Strumigenys. Trap-jaw ants have latch-mediated, spring-actuated systems that amplify the power output of their mandibles. I use Bayesian estimates of covariation and evolutionary rates to test the hypotheses that the evolution of this high-performance system is associated with tighter morphological covariation in the head and mandibles relative to nontrap-jaw forms and that this leads to shifts in rates of morphological evolution. Contrary to these hypotheses, there is no evidence of a large-scale shift to higher covariation in trap-jaw forms, while different traits show both increased and decreased evolutionary rates between forms. These patterns may be indicative of many-to-one mapping and/or mechanical sensitivity in the trap-jaw LaMSA system. Overall, it appears that the evolution of trap-jaw forms in Strumigenys did not require a correlated increase in morphological covariation, partly explaining the proclivity with which the system has evolved.

Do the different life history strategies of ants and honeybees determine fat body morphology?

The separation of two sister groups such as ants and bees in the Cretaceous involved the development of distinctive characteristics to occupy separate ecological niches. From the point of view of biology and ecology, it is important to see how different life history strategies affect the physiology of these insects. The fat body is the most metabolically important tissue in the organism of each insect. Therefore, we conducted a comparative analysis of the morphological image of the subcuticular fat body in different localisation/segments in Formica (Seviformica) cinerea and Apis mellifera mellifera foragers, because of the similarity of their functions in colonies. We observed that the fat bodies of ants and bees were composed of the same cell types: trophocytes and oenocytes. However, in each of the segments, the fat body cells in ants were bigger and there were fewer of them in comparison with bees. The dorsal part of the fat body of ants had a bilayer structure, where the outer layer was formed by binucleated oenocytes. Binucleated oenocytes were also found in the inner layer near the heart and tracheole. In bees, the fat body was unilayered and the trophocytes and oenocytes were present side by side. The similarities and, in particular, the differences in the structure of the fat body are the adaptation of these sister groups to life in a diverse environment.

Morphology and ultrastructure of the infrabuccal pocket in Strumigenys ants.

The morphology of the infrabuccal pocket has been studied with light and electron microscopy (SEM, TEM) in 19 species of Strumigenys ants. The structural organization is similar in workers, queens and males, and supports the involvement of the pocket in the filtration of food particles before they can enter the digestive tract. A carpet of posteriorly oriented bristle hairs on the hypopharynx first guide ingested food into the pocket, where large solid particles are compacted into a pellet that will be regurgitated. The remaining products enter the digestive tract through a filtering wall of parallel hair combs lining the pharynx interior that are directed against the food flow. This mechanical filtering allows only liquids and sufficiently small food particles to enter the digestive system. The wall of the infrabuccal pocket is differentiated into a conspicuous glandular epithelium, of which the ultrastructural characteristics can be understood as an adaptation against the frequent shape changes of the pocket. The gland elaborates a non-proteinaceous secretion, although its functional significance still remains unknown.

The ant abdomen: The skeletomuscular and soft tissue anatomy of Amblyopone australis workers (Hymenoptera: Formicidae).

Recent studies of insect anatomy evince a trend towards a comprehensive and integrative investigation of individual traits and their evolutionary relationships. The abdomen of ants, however, remains critically understudied. To address this shortcoming, we describe the abdominal anatomy of Amblyopone australis Erichson, using a multimodal approach combining manual dissection, histology, and microcomputed tomography. We focus on skeletomusculature, but additionally describe the metapleural and metasomal exocrine glands, and the morphology of the circulatory, digestive, reproductive, and nervous systems. We describe the muscles of the dorsal vessel and the ducts of the venom and Dufour's gland, and characterize the visceral anal musculature. Through comparison with other major ant lineages, apoid wasps, and other hymenopteran outgroups, we provide a first approximation of the complete abdominal skeletomuscular groundplan in Formicidae, with a nomenclatural schema generally applicable to the hexapod abdomen. All skeletal muscles were identifiable with their homologs, while we observe potential apomorphies in the pregenital skeleton and the sting musculature. Specifically, we propose the eighth coxocoxal muscle as an ant synapomorphy; we consider possible transformation series contributing to the distribution of states of the sternal apodemes in ants, Hymenoptera, and Hexapoda; and we address the possibly synapomorphic loss of the seventh sternal-eighth gonapophyseal muscles in the vespiform Aculeata. We homologize the ovipositor muscles across Hymenoptera, and summarize demonstrated and hypothetical muscle functions across the abdomen. We also give a new interpretation of the proximal processes of gonapophyses VIII and the ventromedial processes of gonocoxites IX, and make nomenclatural suggestions in the context of evolutionary anatomy and ontology. Finally, we discuss the utility of techniques applied and emphasize the value of primary anatomical research.

A new ant-butterfly symbiosis in the forest canopy fills an evolutionary gap.

Myrmecophilous butterflies can establish complex symbiotic relationships with ants. A caterpillar wandering among the brood of the aggressive ponerine ant Neoponera villosa was found inside the core of a nest built in the myrmecophytic bromeliad Aechmea bracteata. This is the first caterpillar found living inside a ponerine ant nest. Its DNA barcode was sequenced, and an integrative approach was used to identify it as Pseudonymphidia agave, a poorly known member of the subtribe Pachythonina in the riodinid tribe Nymphidiini. The cuticle of the tank-like caterpillar lacks projections or tubercles and is covered dorsally by specialized flat setae that form an armor of small plates. Ant-organs potentially related to caterpillar-ant signaling, such as perforated cupola organs and tentacle nectary organs, are present. These morphological traits, together with evidence of social integration (direct contact with host brood, protective morphology, slow movement, no host aggressiveness), suggest that P. agave is a symbiotic, social parasite of N. villosa, preying on its host brood. However, several knowledge gaps remain, including oviposition site, dependence on bromeliad association, steps to colony integration, and larval diet through development. Carnivory has been reported in all known members of the subtribe Pachythonina (caterpillars prey on honeydew-producing hemipterans) suggesting a shift to myrmecophagy inside the ant nests as a possible evolutionary transition.

Nano-CT imaging of larvae in the ant Pheidole hyatti reveals coordinated growth of a rudimentary organ necessary for soldier development.

The growth of imaginal discs in holometabolous insects is coordinated with larval growth to ensure the symmetrical and proportional development of the adult appendages. In ants, the differential growth of these discs generates distinct castes-the winged male and queen castes and the wingless worker caste. In the hyperdiverse ant genus Pheidole, the worker caste is composed of two morphologically distinct subcastes: small-headed minor workers and larger, big-headed, soldiers. Although these worker subcastes are completely wingless, soldier larvae develop rudimentary forewing discs that function in generating the disproportionate head-to-body scaling and size of soldiers. It remains unclear, however, how rudimentary forewing discs in soldier larvae are coordinated with other imaginal discs. Here we show, using quantitative nano-CT imaging and three-dimensional analyses, that the increase in the volume of the soldier rudimentary forewing discs is coordinated with larval size as well as with the increase in the volume of the leg and eye-antennal (head) discs. However, relative to larval size, we found that when the rudimentary forewing discs appear during the last larval instar, they are relatively smaller but increase in volume faster than that of the head (eye-antennal) and leg discs. These findings show that the rudimentary wing disc in soldier larvae has evolved novel patterns of inter-organ coordination as compared with other insects to generate the big-headed soldier caste in Pheidole. More generally, our study raises the possibility that novel patterns of inter-organ coordination are a general feature of rudimentary organs that acquire novel regulatory functions during development and evolution.

Inhibition of HSP90 causes morphological variation in the invasive ant Cardiocondyla obscurior.

Canalization underlies the expression of steady phenotypes in the face of unsteady environmental conditions or varying genetic backgrounds. The chaperone HSP90 has been identified as a key component of the molecular machinery regulating canalization and a growing body of research suggests that HSP90 could act as a general capacitator in evolution. However, empirical data about HSP90-dependent phenotypic variation and its evolutionary impact is still scarce, particularly for non-model species. Here we report how pharmacological suppression of HSP90 increases morphological variation up to 87% in the invasive ant Cardiocondyla obscurior. We show that workers treated with the HSP90 inhibitor 17-DMAG are significantly more diverse compared to untreated workers in two of four measured traits: maximal eye distance and maximal propodeal spine distance. We further find morphological differentiation between natural populations of C. obscurior in the same traits that responded to our pharmacological treatment. These findings add support for the putative impact of HSP90 on canalization, the modularity of phenotypic traits, and its potential role in morphological evolution of ants.

The origin of wing polyphenism in ants: An eco-evo-devo perspective.

The evolution of eusociality, where solitary individuals integrate into a single colony, is a major transition in individuality. In ants, the origin of eusociality coincided with the origin of a wing polyphenism approximately 160 million years ago, giving rise to colonies with winged queens and wingless workers. As a consequence, both eusociality and wing polyphenism are nearly universal features of all ants. Here, we synthesize fossil, ecological, developmental, and evolutionary data in an attempt to understand the factors that contributed to the origin of wing polyphenism in ants. We propose multiple models and hypotheses to explain how wing polyphenism is orchestrated at multiple levels, from environmental cues to gene networks. Furthermore, we argue that the origin of wing polyphenism enabled the subsequent evolution of morphological diversity across the ants. We finally conclude by outlining several outstanding questions for future work.

The Pronotum of Worker of Camponotus borellii Emery (Hymenoptera: Formicidae): How Can It Affect Performance of the Head, Work Division, and Development of the Worker Caste?

In polymorphic ants, whose workers display continuous size distribution, each subcaste occupies a phenotypic space, usually with diffuse morphological boundaries. These morphological differences are closely associated to size by allometry although the environment also plays a key role that affects the fitness of the species. In Camponotus borellii Emery, the species selected as a study model, workers exhibit a continuous increase in size; geometric morphometric (GM) was used over four morphological traits: head capsule, clypeus, pronotum, and mesosoma, in order to assess (1) changes in shape, among the worker caste; (2) the influence of allometry on such changes; and (3) pronotum shape in respect to the head so as to infer which factors may influence the polymorphic development of the worker caste. The results indicated that the pronotum is organized into two highly integrated functional modules (neck and shield), corresponding to one developmental module. GM shows a similar pattern to that obtained for linear morphometry, though the worker ratio was different along continuous size distribution due to shape changes in two traits, with are also useful for delimiting modular units: (1) rounded shape of the posterior region of the head in minor workers; (2) shape of the pronotum, especially its anterior region, henceforth, neck, which widens as a consequence of the higher development of its central region, henceforth, shield, in major workers. The relevance of these results is discussed regarding functional morphology (pronotum in relation to the head), work division, and development of the worker caste.