Chemical species recognition in an adaptive radiation of Hawaiian Tetragnatha spiders (Araneae: Tetragnathidae).

Studies of adaptive radiations have played a central role in our understanding of reproductive isolation. Yet the focus has been on human-biased visual and auditory signals, leaving gaps in our knowledge of other modalities. To date, studies on chemical signals in adaptive radiations have focused on systems with multimodal signalling, making it difficult to isolate the role chemicals play in reproductive isolation. In this study we examine the use of chemical signals in the species recognition and adaptive radiation of Hawaiian Tetragnatha spiders by focusing on entire communities of co-occurring species, and conducting behavioural assays in conjunction with chemical analysis of their silks using gas chromatography-mass spectrometry. Male spiders significantly preferred the silk extracts of conspecific mates over those of sympatric heterospecifics. The compounds found in the silk extracts, long chain alkyl methyl ethers, were remarkably species-specific in the combination and quantity. The differences in the profile were greatest between co-occurring species and between closely related sibling species. Lastly, there were significant differences in the chemical profile between two populations of a particular species. These findings provide key insights into the role chemical signals play in the attainment and maintenance of reproductive barriers between closely related co-occurring species.

Reference genome of the bicolored carpenter ant, Camponotus vicinus.

Carpenter ants in the genus Camponotus are large, conspicuous ants that are abundant and ecologically influential in many terrestrial ecosystems. The bicolored carpenter ant, Camponotus vicinus Mayr, is distributed across a wide range of elevations and latitudes in western North America, where it is a prominent scavenger and predator. Here, we present a high-quality genome assembly of C. vicinus from a sample collected in Sonoma County, California, near the type locality of the species. This genome assembly consists of 38 scaffolds spanning 302.74 Mb, with contig N50 of 15.9 Mb, scaffold N50 of 19.9 Mb, and BUSCO completeness of 99.2%. This genome sequence will be a valuable resource for exploring the evolutionary ecology of C. vicinus and carpenter ants generally. It also provides an important tool for clarifying cryptic diversity within the C. vicinus species complex, a genetically diverse set of populations, some of which are quite localized and of conservation interest.

The role of body size and cuticular hydrocarbons in the desiccation resistance of invasive Argentine ants (Linepithema humile).

An insect's cuticle is typically covered in a layer of wax prominently featuring various hydrocarbons involved in desiccation resistance and chemical communication. In Argentine ants (Linepithema humile), cuticular hydrocarbons (CHCs) communicate colony identity, but also provide waterproofing necessary to survive dry conditions. Theory suggests different CHC compound classes have functional trade-offs, such that selection for compounds used in communication would compromise waterproofing, and vice versa. We sampled sites of invasive L. humile populations from across California to test whether CHC differences between them can explain differences in their desiccation survival. We hypothesized that CHCs whose abundance was correlated with environmental factors would determine survival during desiccation, but our regression analysis did not support this hypothesis. Interestingly, we found the abundance of most CHCs had a negative correlation with survival, regardless of compound class. We suggest that the CHC differences between L. humile nests in California are insufficient to explain their differential survival against desiccation, and that body mass is a better predictor of desiccation survival at this scale of comparison.

Phylogeography and population genetics of a widespread cold-adapted ant, Prenolepis imparis.

As species arise, evolve and diverge, they are shaped by forces that unfold across short and long timescales and at both local and vast geographical scales. It is rare, however, to be able document this history across broad sweeps of time and space in a single species. Here, we report the results of a continental-scale phylogenomic analysis across the entire range of a widespread species. We analysed sequences of 1402 orthologous ultraconserved element (UCE) loci from 75 individuals to identify population genetic structure and historical demographic patterns across the continent-wide range of a cold-adapted ant, the winter ant, Prenolepis imparis. We recovered five well-supported, genetically isolated clades representing lineages that diverged from 8.2-2.2 million years ago. These include: (i) an early diverging lineage located in Florida, (ii) a lineage that spans the southern United States, (iii) populations that extend across the midwestern and northeastern United States, (iv) populations from the western United States and (v) populations in southwestern Arizona and Mexico. Population genetic analyses revealed little or no gene flow among these lineages, but patterns consistent with more recent gene flow among populations within lineages, and localized structure with migration in the western United States. High support for five major geographical lineages and lack of evidence of contemporary gene flow indicate in situ diversification across the species' range, producing relatively ancient lineages that persisted through subsequent climate change and glaciation during the Quaternary.

Behavioural variation and plasticity along an invasive ant introduction pathway.

Once established in new areas, introduced species may exhibit changes in their biology due to phenotypic plasticity, novel selection pressures and genetic drift. Moreover, the introduction process itself has been hypothesised to act as a selective filter for traits that promote invasiveness. We tested the hypothesis that behaviours thought to promote invasiveness-such as increased foraging activity and aggression-are selected for during invasion by comparing traits among native and introduced populations of the widespread Argentine ant (Linepithema humile). We studied Argentine ant populations in the native range in Argentina and in three invaded regions along an introduction pathway: California, Australia and New Zealand. In each region, we set up 32 experimental colonies to measure foraging activity and interspecific aggression in a subset of the study regions. These colonies were subject to experimental manipulation of carbohydrate availability and octopamine, a biogenic amine known to modulate behaviour in insects, to measure variation in behavioural plasticity. We found variation in foraging activity among populations, but this variation was not consistent with selection on behaviour in relation to the invasion process. We found that colonies with limited access to carbohydrates exhibited unchanged exploratory behaviour, but higher exploitation activity and lower aggression. Colonies given octopamine consistently increased foraging behaviour (both exploration and exploitation), as well as aggression when also sugar-deprived. There was no difference in the degree of behavioural response to our experimental treatments along the introduction pathway. We did not find support for selection of behavioural traits associated with invasiveness along the Argentine ant's introduction pathway or clear evidence for an association between the introduction process and variation in behavioural plasticity. These results indicate that mechanisms promote behavioural variation in a similar fashion both in native and introduced ranges. Our results challenge the assumption that introduced populations always perform better in key behavioural traits hypothesised to be associated with invasion success.

Desiccation Resistance and Micro-Climate Adaptation: Cuticular Hydrocarbon Signatures of Different Argentine Ant Supercolonies Across California.

Cuticular hydrocarbons (CHCs), the dominant fraction of the insects' epicuticle and the primary barrier to desiccation, form the basis for a wide range of chemical signaling systems. In eusocial insects, CHCs are key mediators of nestmate recognition, and colony identity appears to be maintained through a uniform CHC profile. In the unicolonial Argentine ant Linepithema humile, an unparalleled invasive expansion has led to vast supercolonies whose nestmates can still recognize each other across thousands of miles. CHC profiles are expected to display considerable variation as they adapt to fundamentally differing environmental conditions across the Argentine ant's expanded range, yet this variation would largely conflict with the vastly extended nestmate recognition based on CHC uniformity. To shed light on these seemingly contradictory selective pressures, we attempt to decipher which CHC classes enable adaptation to such a wide array of environmental conditions and contrast them with the overall CHC profile uniformity postulated to maintain nestmate recognition. n-Alkanes and n-alkenes showed the largest adaptability to environmental conditions most closely associated with desiccation, pointing at their function for water-proofing. Trimethyl alkanes, on the other hand, were reduced in environments associated with higher desiccation stress. However, CHC patterns correlated with environmental conditions were largely overriden when taking overall CHC variation across the expanded range of L. humile into account, resulting in conserved colony-specific CHC signatures. This delivers intriguing insights into the hierarchy of CHC functionality integrating both adaptation to a wide array of different climatic conditions and the maintenance of a universally accepted chemical profile.

Social insect genomes exhibit dramatic evolution in gene composition and regulation while preserving regulatory features linked to sociality.

Genomes of eusocial insects code for dramatic examples of phenotypic plasticity and social organization. We compared the genomes of seven ants, the honeybee, and various solitary insects to examine whether eusocial lineages share distinct features of genomic organization. Each ant lineage contains ∼4000 novel genes, but only 64 of these genes are conserved among all seven ants. Many gene families have been expanded in ants, notably those involved in chemical communication (e.g., desaturases and odorant receptors). Alignment of the ant genomes revealed reduced purifying selection compared with Drosophila without significantly reduced synteny. Correspondingly, ant genomes exhibit dramatic divergence of noncoding regulatory elements; however, extant conserved regions are enriched for novel noncoding RNAs and transcription factor-binding sites. Comparison of orthologous gene promoters between eusocial and solitary species revealed significant regulatory evolution in both cis (e.g., Creb) and trans (e.g., fork head) for nearly 2000 genes, many of which exhibit phenotypic plasticity. Our results emphasize that genomic changes can occur remarkably fast in ants, because two recently diverged leaf-cutter ant species exhibit faster accumulation of species-specific genes and greater divergence in regulatory elements compared with other ants or Drosophila. Thus, while the "socio-genomes" of ants and the honeybee are broadly characterized by a pervasive pattern of divergence in gene composition and regulation, they preserve lineage-specific regulatory features linked to eusociality. We propose that changes in gene regulation played a key role in the origins of insect eusociality, whereas changes in gene composition were more relevant for lineage-specific eusocial adaptations.

Dead ant walking: a myrmecophilous beetle predator uses parasitoid host location cues to selectively prey on parasitized ants.

Myrmecophiles (i.e. organisms that associate with ants) use a variety of ecological niches and employ different strategies to survive encounters with ants. Because ants are typically excellent defenders, myrmecophiles may choose moments of weakness to take advantage of their ant associates. This hypothesis was studied in the rove beetle, Myrmedonota xipe, which associates with Azteca sericeasur ants in the presence of parasitoid flies. A combination of laboratory and field experiments show that M. xipe beetles selectively locate and prey upon parasitized ants. These parasitized ants are less aggressive towards beetles than healthy ants, allowing beetles to eat the parasitized ants alive without interruption. Moreover, behavioural assays and chemical analysis reveal that M. xipe are attracted to the ant's alarm pheromone, the same secretion used by the phorid fly parasitoids in host location. This strategy allows beetles access to an abundant but otherwise inaccessible resource, as A. sericeasur ants are typically highly aggressive. These results are the first, to our knowledge, to demonstrate a predator sharing cues with a parasitoid to gain access to an otherwise unavailable prey item. Furthermore, this work highlights the importance of studying ant-myrmecophile interactions beyond just their pairwise context.

Differential Sharing of Chemical Cues by Social Parasites Versus Social Mutualists in a Three-Species Symbiosis.

Chemical recognition systems are crucial for maintaining the unity of social insect colonies. It has been proposed that colonies form a common chemical signature, called the gestalt odor, which is used to distinguish colony members and non-members. This chemical integration is achieved actively through social interactions such as trophallaxis and allogrooming, or passively such as through exposure to common nest material. When colonies are infiltrated by social parasites, the intruders often use some form of chemical mimicry. However, it is not always clear how this chemical mimicry is accomplished. Here, we used a three-species nesting symbiosis to test the differences in chemical integration of mutualistic (parabiotic) and parasitic ant species. We found that the parasite (Solenopsis picea) obtains chemical cues from both of the two parabiotic host ant species. However, the two parabiotic species (Crematogaster levior and Camponotus femoratus) maintain species-specific cues, and do not acquire compounds from the other species. Our findings suggest that there is a fundamental difference in how social mutualists and social parasites use chemicals to integrate themselves into colonies.

Cuticular Hydrocarbon Cues Are Used for Host Acceptance by Pseudacteon spp. Phorid Flies that Attack Azteca sericeasur Ants.

Parasitoids often use complex cues to identify suitable hosts in their environment. Phorid fly parasitoids that develop on one or a few host species often use multiple cues, ranging from general to highly specific, to home in on an appropriate host. Here, we describe the hierarchy of cues that Pseudacteon phorid flies use to identify Azteca ant hosts. We show, through behavioral observations in the field, that phorid flies are attracted to two cryptic Azteca species, but only attack Azteca sericeasur (Hymenoptera: Formicidae: Dolichoderinae). To test whether the phorid flies use cuticular hydrocarbons (CHCs) to distinguish between the two Azteca taxa, we first documented and compared cuticular hydrocarbons of the two Azteca taxa using gas chromatography/mass spectrometry. Then, using cuticular hydrocarbon-transfer experiments with live ants, we characterized the cuticular hydrocarbons of A. sericeasur as a short-range, host location cue used by P. lasciniosus (Diptera: Phoridae) to locate the ants.

The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle.

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host-microbe symbioses.

Draft genome of the globally widespread and invasive Argentine ant (Linepithema humile).

Ants are some of the most abundant and familiar animals on Earth, and they play vital roles in most terrestrial ecosystems. Although all ants are eusocial, and display a variety of complex and fascinating behaviors, few genomic resources exist for them. Here, we report the draft genome sequence of a particularly widespread and well-studied species, the invasive Argentine ant (Linepithema humile), which was accomplished using a combination of 454 (Roche) and Illumina sequencing and community-based funding rather than federal grant support. Manual annotation of >1,000 genes from a variety of different gene families and functional classes reveals unique features of the Argentine ant's biology, as well as similarities to Apis mellifera and Nasonia vitripennis. Distinctive features of the Argentine ant genome include remarkable expansions of gustatory (116 genes) and odorant receptors (367 genes), an abundance of cytochrome P450 genes (>110), lineage-specific expansions of yellow/major royal jelly proteins and desaturases, and complete CpG DNA methylation and RNAi toolkits. The Argentine ant genome contains fewer immune genes than Drosophila and Tribolium, which may reflect the prominent role played by behavioral and chemical suppression of pathogens. Analysis of the ratio of observed to expected CpG nucleotides for genes in the reproductive development and apoptosis pathways suggests higher levels of methylation than in the genome overall. The resources provided by this genome sequence will offer an abundance of tools for researchers seeking to illuminate the fascinating biology of this emerging model organism.

Draft genome of the red harvester ant Pogonomyrmex barbatus.

We report the draft genome sequence of the red harvester ant, Pogonomyrmex barbatus. The genome was sequenced using 454 pyrosequencing, and the current assembly and annotation were completed in less than 1 y. Analyses of conserved gene groups (more than 1,200 manually annotated genes to date) suggest a high-quality assembly and annotation comparable to recently sequenced insect genomes using Sanger sequencing. The red harvester ant is a model for studying reproductive division of labor, phenotypic plasticity, and sociogenomics. Although the genome of P. barbatus is similar to other sequenced hymenopterans (Apis mellifera and Nasonia vitripennis) in GC content and compositional organization, and possesses a complete CpG methylation toolkit, its predicted genomic CpG content differs markedly from the other hymenopterans. Gene networks involved in generating key differences between the queen and worker castes (e.g., wings and ovaries) show signatures of increased methylation and suggest that ants and bees may have independently co-opted the same gene regulatory mechanisms for reproductive division of labor. Gene family expansions (e.g., 344 functional odorant receptors) and pseudogene accumulation in chemoreception and P450 genes compared with A. mellifera and N. vitripennis are consistent with major life-history changes during the adaptive radiation of Pogonomyrmex spp., perhaps in parallel with the development of the North American deserts.

An assemblage-level comparison of genetic diversity and population genetic structure between island and mainland ant populations.

Island biotas provide unparalleled opportunities to examine evolutionary processes. Founder effects and bottlenecks, for example, typically decrease genetic diversity in island populations, while selection for reduced dispersal can increase population structure. Given that support for these generalities mostly comes from single-species analyses, assemblage-level comparisons are needed to clarify how (i) colonization affects the gene pools of interacting insular organisms, and (ii) patterns of genetic differentiation vary within assemblages of organisms. Here, we use genome-wide sequence data from ultraconserved elements (UCEs) to compare genetic diversity and population structure of mainland and island populations of nine ant species in coastal southern California. As expected, island populations (from Santa Cruz Island) had lower expected heterozygosity and Watterson's theta compared to mainland populations (from the Lompoc Valley). Island populations, however, exhibited smaller genetic distances among samples, indicating less population subdivision. Within the focal assemblage, pairwise Fst values revealed pronounced interspecific variation in mainland-island differentiation, which increases with gyne body size. Our results reveal population differences across an assemblage of interacting species, and illuminate general patterns of insularization in ants. Compared to single-species studies, our analysis of nine conspecific population pairs from the same island-mainland system offers a powerful approach to studying fundamental evolutionary processes.

Dissecting ant recognition systems in the age of genomics.

Hamilton is probably best known for his seminal work demonstrating the role of kin selection in social evolution. His work made it clear that, for individuals to direct their altruistic behaviours towards appropriate recipients (kin), mechanisms must exist for kin recognition. In the social insects, colonies are typically comprised of kin, and colony recognition cues are used as proxies for kinship cues. Recent years have brought rapid advances in our understanding of the genetic and molecular mechanisms that are used for this process. Here, I review some of the most notable advances, particularly the contributions from recent ant genome sequences and molecular biology.

Learning and discrimination of cuticular hydrocarbons in a social insect.

Social insect cuticular hydrocarbon (CHC) mixtures are among the most complex chemical cues known and are important in nest-mate, caste and species recognition. Despite our growing knowledge of the nature of these cues, we have very little insight into how social insects actually perceive and discriminate among these chemicals. In this study, we use the newly developed technique of differential olfactory conditioning to pure, custom-designed synthetic colony odours to analyse signal discrimination in Argentine ants, Linepithema humile. Our results show that tri-methyl alkanes are more easily learned than single-methyl or straight-chain alkanes. In addition, we reveal that Argentine ants can discriminate between hydrocarbons with different branching patterns and the same chain length, but not always between hydrocarbons with the same branching patterns but different chain length. Our data thus show that biochemical characteristics influence those compounds that ants can discriminate between, and which thus potentially play a role in chemical signalling and nest-mate recognition.

A silica gel based method for extracting insect surface hydrocarbons.

Here, we describe a novel method for the extraction of insect cuticular hydrocarbons using silica gel, herein referred to as "silica-rubbing". This method permits the selective sampling of external hydrocarbons from insect cuticle surfaces for subsequent analysis using gas chromatography-mass spectrometry (GC-MS). The cuticular hydrocarbons are first adsorbed to silica gel particles by rubbing the cuticle of insect specimens with the materials, and then are subsequently eluted using organic solvents. We compared the cuticular hydrocarbon profiles that resulted from extractions using silica-rubbing and solvent-soaking methods in four ant and one bee species: Linepithema humile, Azteca instabilis, Camponotus floridanus, Pogonomyrmex barbatus (Hymenoptera: Formicidae), and Euglossa dilemma (Hymenoptera: Apidae). We also compared the hydrocarbon profiles of Euglossa dilemma obtained via silica-rubbing and solid phase microextraction (SPME). Comparison of hydrocarbon profiles obtained by different extraction methods indicates that silica rubbing selectively extracts the hydrocarbons that are present on the surface of the cuticular wax layer, without extracting hydrocarbons from internal glands and tissues. Due to its surface specificity, efficiency, and low cost, this new method may be useful for studying the biology of insect cuticular hydrocarbons.

The Effect of Diet on Colony Recognition and Cuticular Hydrocarbon Profiles of the Invasive Argentine Ant, Linepithema humile.

Ants are some of the most abundant and ecologically successful terrestrial organisms, and invasive ants rank among the most damaging invasive species. The Argentine ant is a particularly well-studied invader, in part, because of the extreme social structure, known as unicoloniality, that occurs in introduced populations. Unicoloniality is characterized by the formation of geographically vast supercolonies, within which territorial behavior and intraspecific aggression are absent. Although there is considerable evidence supporting a genetic basis for the odor cues involved in colony recognition, some studies have suggested that diet may also influence colony recognition cues and, thus, colony structure. Here, we test the role for insect-derived recognition cues by performing a diet supplementation experiment in a natural field setting, and a more extreme dietary manipulation experiment in the lab. After one month, in both the field and the lab, we found that aggressive supercolonies remained aggressive toward each other and non-aggressive nests (from the same supercolony) remained non-aggressive, regardless of dietary treatment. In one lab treatment, we did observe a significant decrease in the level of aggression between different supercolonies that were fed the same diet, but aggression was still frequent. We did not see any evidence for cuticular hydrocarbon odor cues being transferred from prey to ants in any of the field treatments. In the more extreme lab treatment, however, several cuticular hydrocarbons were acquired from both roach and cricket insect prey (but not Drosophila). Based on these data, we conclude that dietary changes are unlikely to underlie changes in behavior or colony structure in Argentine ants in real-world settings. However, these results indicate that caution is warranted when interpreting the behaviors of animals that have been reared on diets that are substantially different from natural populations.

Taxonomically restricted genes are associated with the evolution of sociality in the honey bee.

BACKGROUND: Studies have shown that taxonomically restricted genes are significant in number and important for the evolution of lineage specific traits. Social insects have gained many novel morphological and behavioral traits relative to their solitary ancestors. The task repertoire of an advanced social insect, for example, can be 40-50 tasks, about twice that of a solitary wasp or bee. The genetic basis of this expansion in behavioral repertoire is still poorly understood, and a role for taxonomically restricted genes has not been explored at the whole genome level. RESULTS: Here we present comparative genomics results suggesting that taxonomically restricted genes may have played an important role in generating the expansion of behavioral repertoire associated with the evolution of eusociality. First, we show that the current honey bee official gene set contains about 700 taxonomically restricted genes. These are split between orphans, genes found only in the Hymenoptera, and genes found only in insects. Few of the orphans or genes restricted to the Hymenoptera have been the focus of experimental work, but several of those that have are associated with novel eusocial traits or traits thought to have changed radically as a consequence of eusociality. Second, we predicted that if taxonomically restricted genes are important for generating novel eusocial traits, then they should be expressed with greater frequency in workers relative to the queen, as the workers exhibit most of the novel behavior of the honey bee relative to their solitary ancestors. We found support for this prediction. Twice as many taxonomically restricted genes were found amongst the genes with higher expression in workers compared to those with higher expression in queens. Finally, we compiled an extensive list of candidate taxonomically restricted genes involved in eusocial evolution by analyzing several caste specific gene expression data sets. CONCLUSIONS: This work identifies a large number of candidate taxonomically restricted genes that may have played a role in eusocial evolution. This work thus lays the foundation for future functional genomics work on the evolution of novelty in the context of social behavior. We also present preliminary evidence, based on biased patterns of gene expression, that taxonomically restricted genes may have played a role in the evolution of caste systems, a characteristic lineage specific social trait.

Experience influences aggressive behaviour in the Argentine ant.

All animals interact with conspecifics during their life, and nearly all also display some form of aggression. An enduring challenge, however, is to understand how the experiences of an individual animal influence its later behaviours. Several studies have shown that prior winning experience increases the probability of initiating fights in later encounters. Using behavioural assays in the laboratory, we provide evidence that, in Argentine ants (Linepithema humile), the mere exposure to an opponent, without the encounter escalating to a fight, also increases the probability that it will display aggression in later encounters. Argentine ant workers differ in their propensity to attack non-colonymates, with some ants repeatedly aggressive and others consistently more docile. Although 78 per cent of the workers were consistent in their behaviour from one encounter to the next, workers that did change their behaviour after an encounter with a non-colonymate more often changed from non-aggressive to aggressive, rather than the reverse. Surprisingly, a single encounter with a non-colonymate increased a worker's propensity to fight in encounters up to a week later. An encounter with a non-colonymate also increased the probability that a worker would attack ants from a colony that it had not previously encountered. Thus, these interactions lowered the overall aggression threshold, rather than stimulating a specific aggressive response to a particular foreign colony. Finally, our data suggest that aggression towards non-colonymates increases with age.