Evolution of cuticular hydrocarbon diversity in ants

The cuticular hydrocarbons (CHCs) of ants provide important cues for nest-mate and caste recognition. There is enormous diversity in the composition of these CHCs, but the manner in which this diversity has evolved is poorly understood. We gathered data on CHC profiles for 56 ant species, relating this information to their phylogeny. We deduced the mode of evolution of CHC profiles by reconstructing character evolution and then relating the number of changes in CHC components along each branch of the phylogeny to the length of the branch. There was a strong correlation between branch length and number of component changes, with fewer changes occurring on short branches. Our analysis thereby indicated a gradual mode of evolution. Different ant species tend to use specific CHC structural types that are exclusive of other structural types, indicating that species differences may be generated in part by switching particular biosynthetic pathways on or off in different lineages. We found limited, and contradictory, evidence for abiotic factors (temperature and rainfall) driving change in CHC profiles.     

The evolution of a complex trait: cuticular hydrocarbons in ants evolve independent from phylogenetic constraints

Cuticular hydrocarbons (CHCs) are ubiquitous and highly diverse in insects, serving as communication signal and waterproofing agent. Despite their vital function, the causes, mechanisms and constraints on CHC diversification are still poorly understood. Here, we investigated phylogenetic constraints on the evolution of CHC profiles, using a global data set of the species‐rich and chemically diverse ant genus Crematogaster. We decomposed CHC profiles into quantitative (relative abundances, chain length) and qualitative traits (presence/absence of CHC classes). A species‐level phylogeny was estimated using newly generated and previously published sequences from five nuclear markers. Moreover, we reconstructed a phylogeny for the chemically diverse Crematogaster levior species group using cytochrome oxidase I. Phylogenetic signal was measured for these traits on genus and clade level and within the chemically diverse C. levior group. For most quantitative CHC traits, phylogenetic signal was low and did not differ from random expectation. This was true on the level of genus, clade and species group, indicating that CHC traits are evolutionary labile. In contrast, the presence or absence of alkenes and alkadienes was highly conserved within the C. levior group. Hence, the presence or absence of biosynthetic pathways may be phylogenetically constrained, especially at lower taxonomic levels. Our study shows that CHC composition can evolve rapidly, allowing insects to quickly adapt their chemical profiles to external selection pressures, whereas the presence of biosynthetic pathways appears more constrained. However, our results stress the importance to consider the taxonomic level when investigating phylogenetic constraints.     

Release from prey preservation behavior via prey switch allowed diversification of cuticular hydrocarbon profiles in digger wasps

The cuticle of insects is covered by a layer of hydrocarbons (CHC), whose original function is the protection from desiccation and pathogens. However, in most insects CHC profiles are species specific. While this variability among species was largely linked to communication and recognition functions, additional selective forces may shape insect CHC profiles. Here, we show that in Philanthinae digger wasps (Crabronidae) the CHC profile coevolved with a peculiar brood‐care strategy. In particular, we found that the behavior to embalm prey stored in the nest with hydrocarbons is adaptive to protect larval food from fungi in those species hunting for Hymenoptera. The prey embalming secretion is identical in composition to the alkene‐dominated CHC profile in these species, suggesting that their profile is adaptively conserved for this purpose. In contrast, prey embalming is not required in those species that switched to Coleoptera as prey. Released from this chemical brood‐care strategy, Coleoptera‐hunting species considerably diversified their CHC profiles. Differential needs to successfully protect prey types used as larval food have thus driven the diversification of CHCs profiles of female Philanthinae wasps. To the best of our knowledge, this is the first evidence of a direct link between selection pressure for food preservation and CHC diversity.     

CLINES IN CUTICULAR HYDROCARBONS IN TWO DROSOPHILA SPECIES WITH INDEPENDENT POPULATION HISTORIES

We took a comparative approach utilizing clines to investigate the extent to which natural selection may have shaped population divergence in cuticular hydrocarbons (CHCs) that are also under sexual selection in Drosophila. We detected the presence of CHC clines along a latitudinal gradient on the east coast of Australia in two fly species with independent phylogenetic and population histories, suggesting adaptation to shared abiotic factors. For both species, significant associations were detected between clinal variation in CHCs and temperature variation along the gradient, suggesting temperature maxima as a candidate abiotic factor shaping CHC variation among populations. However, rainfall and humidity correlated with CHC variation to differing extents in the two species, suggesting that response to these abiotic factors may vary in a species‐specific manner. Our results suggest that natural selection, in addition to sexual selection, plays a significant role in structuring among‐population variation in sexually selected traits in Drosophila.     

Absence of cuticular alkenes allows lycaenid larvae to avoid predation by Formica japonica ants

Chemical mimicry and camouflage based on cuticular hydrocarbons (CHCs) are adaptive strategies that are frequently observed in myrmecophilous insects. The larvae of several lycaenid butterfly species that exhibit obligate associations with specific ant species have been reported to use chemical mimicry. However, little is known about the strategies used by the larvae of species that have facultative associations with multiple ant species. We attempted to reveal the effects of larval CHC profiles on interactions with Formica japonica workers, using three lycaenid species, two facultative ant‐associated (Lycaeides argyrognomon and Zizeeria maha) and one non‐ant‐associated (Lycaena phlaeas), which commonly possess n‐alkanes as the major CHCs. In field bioassays, the lycaenid larvae were attacked by ant workers less often than larvae of Papilio polytes (Papilionidae), the CHCs of which were rich in 7‐alkenes. Treating the lycaenid larvae with 7‐heptacosene and 9‐heptacosene significantly activated ant aggression (biting), whereas treating them with n‐heptacosane, n‐octacosane and 13‐methylheptacosane had little effect. Furthermore, larvae of Pieris rapae (Pieridae), possessing n‐alkanes as the dominant CHCs, suffered an intermediate level of ant biting between the lycaenid and Pa. polytes larvae. However, treatments of the P. rapae larvae with 7‐heptacosene and 9‐heptacosene significantly affected the frequency of ant biting. These findings suggest that the absence of alkenes in larval CHC profiles is an effective means of circumventing predation by ants and allows lycaenid larvae to inhabit the foraging territory of predaceous ants, at least to some extent.     

A geographical mosaic of coevolution in a slave-making host-parasite system

Three different isolated populations of the slave‐making ant Rossomyrmex minuchae, sympatric with its obligate host Proformica longiseta, are known from the high mountains of southern Spain. To test the prediction that the slave‐maker and its host represent a coevolutionary geographical mosaic, we studied the variation in the cuticular hydrocarbons (CHCs) as the trait most likely to show the selection mosaic, plus trait remixing by the gene flow in the populations of each species by means of microsatellites. We found within populations, host and parasite had more similar CHC profiles than between the populations or between parasites and allopatric hosts. The differences between the CHC profiles of the host and parasite, which may be responsible for the level of tolerance towards the parasite, varied between the populations suggesting the existence of a selection mosaic of coevolution. Furthermore, P. longiseta showed higher gene flow than R. minuchae, which would allow local variation in the coevolution of the host and parasite while allowing some trait remixing.     

The genetics of cuticular hydrocarbon profiles in the Fruit Fly Drosophila simulans

Female mate choice is one mechanism of sexual selection and, provided there is adequate genetic variation in the male traits that are the target of this selection, they will evolve via female choice. Cuticular hydrocarbons (CHCs) are important in Drosophila mate choice, but relatively little is known about the underlying genetic architecture of CHC profiles in Drosophila simulans. Here, we used gas chromatography-mass spectrometry to investigate patterns of genetic variation in the CHC profiles of male and female D. simulans using isofemale lines. We found substantial genetic variation for CHC profiles and individual CHC components, and individual CHCs were frequently strongly genetically correlated, with a tendency for negative covariance between long- and short-chain CHCs in males. Intersexual genetic covariances were often weak and frequently differed in sign. These findings are novel and significant, highlighting the previously unexplored genetic architecture of CHCs in D. simulans and suggest that this architecture may facilitate sex-specific CHC evolution.     

The role of cuticular hydrocarbons as chemical cues for nestmate recognition in the invasive Argentine ant (<Emphasis Type="Italic">Linepithema humile</Emphasis>)

Argentine ants (Linepithema humile) in their native South American range, like most other ant species, form spatially restricted colonies that display high levels of aggression toward other such colonies. In their introduced range, Argentine ants are unicolonial and form massive supercolonies composed of numerous nests among which territorial boundaries are absent. Here we examine the role of cuticular hydrocarbons (CHCs) in nestmate recognition of this highly damaging invasive ant using three supercolonies from its introduced range. We conducted behavioral assays to test the response of Argentine ants to workers treated with colonymate or non-colonymate CHCs. Additionally, we quantified the amount of hydrocarbons transferred to individual ants and performed gas chromatography-mass spectrometry (GC/MS) to qualitatively characterize our manipulation of CHC profiles. The GC/MS data revealed marked differences in the hydrocarbon profiles across supercolonies and indicated that our treatment effectively masked the original chemical profile of the treated ants with the profile belonging to the foreign individuals. We found that individual workers treated with foreign CHCs were aggressively rejected by their colonymates and this behavior appears to be concentration-dependent: larger quantities of foreign CHCs triggered higher levels of aggression. Moreover, this response was not simply due to an increase in the amount of CHCs applied to the cuticle since treatment with high concentrations of nestmate CHCs did not trigger aggression.The results of this study bolster the findings of previous studies on social insects that have implicated CHCs as nestmate recognition cues and provide insight into the mechanisms of nestmate recognition in the invasive Argentine ant. Received 6 February 2007; revised 31 May and 27 July 2007; accepted 16 August 2007.     

Genetic and environmental influences on the cuticular hydrocarbon profiles of Goniozus wasps

The cuticular hydrocarbon (CHC) profiles of insects are well known to be variable. This variation may be due to genetic influences, environmental influences, or both. Most prior studies have focused on social insects, mainly those in the Hymenoptera, and have shown that hydrocarbons play an important role mediating social behaviour, particularly via kin recognition. Here, we assess the CHC profiles of three species of parasitoid wasps in the genus Goniozus (Hymenoptera: Bethylidae), some of which are known to attune their behaviour according to both environmentally based and genetically based recognition of kin. We find that CHC profiles vary according to both the genetic background (wasp species) and the developmental environment (host species) of individual parasitoids. This indicates that kin recognition could be based on CHC profiles in these parasitoids, as it is in social Hymenoptera. Because the CHC profiles of species within the genus Goniozus are dissimilar, we also conclude that chemical analysis could be used as a taxonomic tool alongside morphological and molecular genetic identification for Goniozus and other species.     

Crowd control: sex ratio affects sexually selected cuticular hydrocarbons in male Drosophila serrata

Although it is advantageous for males to express costly sexually selected signals when females are present, they may also benefit from suppressing these signals to avoid costly interactions with rival males. Cuticular chemical profiles frequently function as insect sexual signals; however, few studies have asked whether males alter these signals in response to their social environment. In Drosophila serrata, an Australian fly, there is sexual selection for a multivariate combination of male cuticular hydrocarbons (CHCs). Here, we show that the ratio of females to males that an adult male experiences has a strong effect on his CHC expression, with female‐biased adult sex ratios eliciting greater expression of CHC profiles associated with higher male mating success. Classical models predict that male reproductive investment should be highest when there is a small but nonzero number of rivals, but we found that males expressed the most attractive combination of CHCs when there were no rivals. We found that male CHCs were highly sensitive to adult sex ratio, with males expressing higher values of CHC profiles associated with greater mating success as the ratio of females to males increased. Moreover, sex ratio has a stronger effect on male CHC expression than adult density. Finally, we explore whether sex ratio affects the variance among a group of males in their CHC expression, as might be expected if individuals respond differently to a given social environment, but find little effect. Our results reveal that subtle differences in social environment can induce plasticity in male chemical signal expression.     

Cuticular hydrocarbons as potential mediators of cryptic species divergence in a mutualistic ant association

Upon advances in sequencing techniques, more and more morphologically identical organisms are identified as cryptic species. Often, mutualistic interactions are proposed as drivers of diversification. Species of the neotropical parabiotic ant association between Crematogaster levior and Camponotus femoratus are known for highly diverse cuticular hydrocarbon (CHC) profiles, which in insects serve as desiccation barrier but also as communication cues. In the present study, we investigated the association of the ants’ CHC profiles with genotypes and morphological traits, and discovered cryptic species pairs in both genera. To assess putative niche differentiation between the cryptic species, we conducted an environmental association study that included various climate variables, canopy cover, and mutualistic plant species. Although mostly sympatric, the two Camponotus species seem to prefer different climate niches. However in the two Crematogaster species, we could not detect any differences in niche preference. The strong differentiation in the CHC profiles may thus suggest a possible role during speciation itself either by inducing assortative mating or by reinforcing sexual selection after the speciation event. We did not detect any further niche differences in the environmental parameters tested. Thus, it remains open how the cryptic species avoid competitive exclusion, with scope for further investigations.     

Replicated evolutionary divergence in the cuticular hydrocarbon profile of male crickets associated with the loss of song in the Hawaiian archipelago

Female choice based on male secondary sexual traits is well documented, although the extent to which this selection can drive an evolutionary divergence in male traits among populations is less clear. Male field crickets Teleogryllus oceanicus attract females using a calling song and once contacted switch to courtship song to persuade them to mate. These crickets also secrete onto their cuticle a cocktail of long‐chained fatty acids or cuticular hydrocarbons (CHCs). Females choose among potential mates based on the structure of male acoustic signals and on the composition of male CHC profiles. Here, we utilize two naturally occurring mutations that have arisen independently on two Hawaiian islands and render the male silent to ask whether the evolutionary loss of acoustic signalling can drive an evolutionary divergence in the alternative signalling modality, male CHC profiles. Q_ST‐F_ST comparisons revealed strong patterns of CHC divergence among three populations of crickets on the islands of Hawaii, Oahu and Kauai. Contrasts between wild‐type and flatwing males on the islands of Oahu and Kauai indicated that variation in male CHC profiles within populations is associated with the loss of acoustic signalling; flatwing males had a relatively low abundance of long‐chained CHCs relative to the short‐chained CHCs that females find attractive. Given their dual functions in desiccation resistance and sexual signalling, insect CHCs may be particularly important traits for reproductive isolation and ultimately speciation.     

The evolution of queen pheromones in the ant genus Lasius

Queen pheromones are among the most important chemical messages regulating insect societies yet they remain largely undiscovered, hindering research into interesting proximate and ultimate questions. Identifying queen pheromones in multiple species would give new insight into the selective pressures and evolutionary constraints acting on these ubiquitous signals. Here, we present experimental and comparative evidence that 3‐methylalkanes, hydrocarbons present on the queen's cuticle, are a queen pheromone throughout the ant genus Lasius. Interspecific variation in the chemical profile is consistent with 3‐methylalkanes evolving more slowly than other types of hydrocarbons, perhaps due to differential selection or evolutionary constraints. We argue that the sensory ecology of the worker response imposes strong stabilizing selection on queen pheromones relative to other hydrocarbons. 3‐Methylalkanes are also strongly physiologically and genetically coupled with fecundity in at least one Lasius species, which may translate into evolutionary constraints. Our results highlight how honest signalling could minimize evolutionary conflict over reproduction, promoting the evolution and maintenance of eusociality.     

Examining the Role of Cuticular Hydrocarbons in Firefly Species Recognition

During animal courtship, multiple signals transmitted in different sensory modalities may be used to recognize potential mates. In fireflies (Coleoptera: Lampyridae), nocturnally active species rely on long-range bioluminescent signals for species, sex, and mate recognition, while several diurnally active species rely on pheromonal signals. Although in many insects non-volatile cuticular hydrocarbons (CHC) also function in species and sex discrimination, little is known about the potential role of CHC in fireflies. Here, we used gas chromatography to characterize species and sex differences in the CHC profiles of several North American fireflies, including three nocturnal and two diurnal species. Additionally, we conducted behavioral bioassays to determine whether firefly males (the searching sex) were differentially attracted to extracts from conspecific vs. heterospecific females. Gas chromatography revealed that nocturnal Photinus fireflies had low or undetectable CHC levels in both sexes, while diurnal fireflies showed higher CHC levels. No major sex differences in CHC profiles were observed for any firefly species. Behavioral bioassays demonstrated that males of the diurnal firefly Ellychnia corrusca were preferentially attracted to chemical extracts from conspecific vs. heterospecific females, while males of the remaining species showed no discrimination. These results suggest that while CHC may function as species recognition signals for some diurnal fireflies, these compounds are unlikely to be important contact signals in nocturnal Photinus fireflies.     

Female choice for male cuticular hydrocarbon profile in decorated crickets is not based on similarity to their own profile

Indirect genetic benefits derived from female mate choice comprise additive (good genes) and nonadditive genetic benefits (genetic compatibility). Although good genes can be revealed by condition‐dependent display traits, the mechanism by which compatibility alleles are detected is unclear because evaluation of the genetic similarity of a prospective mate requires the female to assess the genotype of the male and compare it to her own. Cuticular hydrocarbons (CHCs), lipids coating the exoskeleton of most insects, influence female mate choice in a number of species and offer a way for females to assess genetic similarity of prospective mates. Here, we determine whether female mate choice in decorated crickets is based on male CHCs and whether it is influenced by females' own CHC profiles. We used multivariate selection analysis to estimate the strength and form of selection acting on male CHCs through female mate choice, and employed different measures of multivariate dissimilarity to determine whether a female's preference for male CHCs is based on similarity to her own CHC profile. Female mating preferences were significantly influenced by CHC profiles of males. Male CHC attractiveness was not, however, contingent on the CHC profile of the choosing female, as certain male CHC phenotypes were equally attractive to most females, evidenced by significant linear and stabilizing selection gradients. These results suggest that additive genetic benefits, rather than nonadditive genetic benefits, accrue to female mate choice, in support of earlier work showing that CHC expression of males, but not females, is condition dependent.     

Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. IX. Host plant and population specific epicuticular hydrocarbon expression influences mate choice and sexual selection

Sexual signals in cactophilic Drosophila mojavensis include cuticular hydrocarbons (CHCs), contact pheromones that mediate female discrimination of males during courtship. CHCs, along with male courtship songs, cause premating isolation between diverged populations, and are influenced by genotype × environment interactions caused by different host cacti. CHC profiles of mated and unmated adult flies from a Baja California and a mainland Mexico population of D. mojavensis reared on two host cacti were assayed to test the hypothesis that male CHCs mediate within‐population female discrimination of males. In multiple choice courtship trials, mated and unmated males differed in CHC profiles, indicating that females prefer males with particular blends of CHCs. Mated and unmated females significantly differed in CHC profiles as well. Adults in the choice trials had CHC profiles that were significantly different from those in pair‐mated adults from no‐choice trials revealing an influence of sexual selection. Females preferred different male CHC blends in each population, but the influence of host cactus on CHC variation was significant only in the mainland population indicating population‐specific plasticity in CHCs. Different groups of CHCs mediated female choice‐based sexual selection in each population suggesting that geographical and ecological divergence has the potential to promote divergence in mate communication systems.     

Interaction between temperature and male pheromone in sexual isolation in Drosophila melanogaster

In Drosophila, female hydrocarbons are known to be involved in premating isolation between different species and pheromonal races. The role of male‐specific hydrocarbon polymorphism is not as well documented. The dominant cuticular hydrocarbon (CHC) in male D. melanogaster is usually 7‐tricosene (7‐T), with the exception of African populations, in which 7‐pentacosene (7‐P) is dominant. Here, we took advantage of a population from the Comoro Islands (Com), in which males fell on a continuum of low to high levels of 7‐T, to perform temperature selection and selection on CHCs' profiles. We conducted several experiments on the selected Com males to study the plasticity of their CHCs in response to temperature shift, their role in resistance to desiccation and in sexual selection. We then compared the results obtained for selected lines to those from three common laboratory strains with different and homogenous hydrocarbon profiles: CS, Cot and Tai. Temperature selection modified the CHC profiles of the Com males in few generations of selection. We showed that the 7‐P/7‐T ratio depends on temperature with generally more 7‐P at higher temperatures and observed a relationship between chain length and resistance to desiccation in both temperature‐ and phenotypically selected Com lines. There was partial sexual isolation between the flies with clear‐cut phenotypes within the phenotypically selected lines and the laboratory strains. These results indicate that the dominant male pheromones are under environmental selection and may have played a role in reproductive isolation.     

Cuticular hydrocarbon profiles differ between ant body parts: implications for communication and our understanding of CHC diffusion

Insect cuticular hydrocarbons (CHCs) serve as communication signals and protect against desiccation. They form complex blends of up to 150 different compounds. Due to differences in molecular packing, CHC classes differ in melting point. Communication is especially important in social insects like ants, which use CHCs to communicate within the colony and to recognize nestmates. Nestmate recognition models often assume a homogenous colony odor, where CHCs are collected, mixed, and redistributed in the postpharyngeal gland (PPG). Via diffusion, recognition cues should evenly spread over the body surface. Hence, CHC composition should be similar across body parts and in the PPG. To test this, we compared CHC composition among whole-body extracts, PPG, legs, thorax, and gaster, across 17 ant species from 3 genera. Quantitative CHC composition differed between body parts, with consistent patterns across species and CHC classes. Early-melting CHC classes were most abundant in the PPG. In contrast, whole body, gaster, thorax, and legs had increasing proportions of CHC classes with higher melting points. Intraindividual CHC variation was highest for rather solid, late-melting CHC classes, suggesting that CHCs differ in their diffusion rates across the body surface. Our results show that body parts strongly differ in CHC composition, either being rich in rather solid, late-melting, or rather liquid, early-melting CHCs. This implies that recognition cues are not homogenously present across the insect body. However, the unequal diffusion of different CHCs represents a biophysical mechanism that enables caste differences despite continuous CHC exchange among colony members.     

Adaptive dynamics of cuticular hydrocarbons in Drosophila

Cuticular hydrocarbons (CHCs) are hydrophobic compounds deposited on the arthropod cuticle that are of functional significance with respect to stress tolerance, social interactions and mating dynamics. We characterized CHC profiles in natural populations of Drosophila melanogaster at five levels: across a latitudinal transect in the eastern United States, as a function of developmental temperature during culture, across seasonal time in replicate years, and as a function of rapid evolution in experimental mesocosms in the field. Furthermore, we also characterized spatial and temporal changes in allele frequencies for SNPs in genes that are associated with the production and chemical profile of CHCs. Our data demonstrate a striking degree of parallelism for clinal and seasonal variation in CHCs in this taxon; CHC profiles also demonstrate significant plasticity in response to rearing temperature, and the observed patterns of plasticity parallel the spatiotemporal patterns observed in nature. We find that these congruent shifts in CHC profiles across time and space are also mirrored by predictable shifts in allele frequencies at SNPs associated with CHC chain length. Finally, we observed rapid and predictable evolution of CHC profiles in experimental mesocosms in the field. Together, these data strongly suggest that CHC profiles respond rapidly and adaptively to environmental parameters that covary with latitude and season, and that this response reflects the process of local adaptation in natural populations of D. melanogaster.