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.

Candidate genes involved in cuticular hydrocarbon differentiation between cryptic, parabiotic ant species.

Insect cuticular hydrocarbons (CHCs) are highly diverse and have multiple functions, including communication and waterproofing. CHC profiles form species-specific, complex blends of up to 150 compounds. Especially in ants, even closely related species can have largely different profiles, raising the question how CHC differences are mirrored in the regulation of biosynthetic pathways. The neotropical ants Crematogaster levior and Camponotus femoratus both consist of two cryptic species each that are morphologically similar, but express strongly different CHC profiles. This is ideal to study the molecular basis of CHC differences. We thus investigated gene expression differences in fat-body transcriptomes of these ants. Despite common garden conditions, we found several thousand differentially expressed transcripts within each cryptic species pair. Many of these were related to metabolic processes, probably accounting for physiological differences. Moreover, we identified candidate genes from five gene families involved in CHC biosynthesis. By assigning candidate transcripts to orthologs in Drosophila, we inferred which CHCs might be influenced by differential gene expression. Expression of these candidate genes was often mirrored in the CHC profiles. For example, Cr. levior A, which has longer CHCs than its cryptic sister species, had a higher expression of elongases and a lower expression of fatty acyl- CoA reductases. This study is one of the first to identify CHC candidate genes in ants and will provide a basis for further research on the genetic basis of CHC biosynthesis.

Little parallelism in genomic signatures of local adaptation in two sympatric, cryptic sister species.

Species living in sympatry and sharing a similar niche often express parallel phenotypes as a response to similar selection pressures. The degree of parallelism within underlying genomic levels is often unexplored, but can give insight into the mechanisms of natural selection and adaptation. Here, we use multi-dimensional genomic associations to assess the basis of local and climate adaptation in two sympatric, cryptic Crematogaster levior ant species along a climate gradient. Additionally, we investigate the genomic basis of chemical communication in both species. Communication in insects is mainly mediated by cuticular hydrocarbons (CHCs), which also protect against water loss and, hence, are subject to changes via environmental acclimation or adaptation. The combination of environmental and chemical association analyses based on genome-wide Pool-Seq data allowed us to identify single nucleotide polymorphisms (SNPs) associated with climate and with chemical differences. Within species, CHC changes as a response to climate seem to be driven by phenotypic plasticity, since there is no overlap between climate- and CHC-associated SNPs. The only exception is the odorant receptor OR22c, which may be a candidate for population-specific CHC recognition in one of the species. Within both species, climate is significantly correlated with CHC differences, as well as to allele frequency differences. However, associated candidate SNPs, genes and functions are largely species-specific and we find evidence for minimal parallel evolution only on the level of genomic regions (J = 0.04). This highlights that even closely related species may follow divergent evolutionary trajectories when expressing similar adaptive phenotypes.

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.

Influence of Mutualistic Lifestyle, Mutualistic Partner, and Climate on Cuticular Hydrocarbon Profiles in Parabiotic Ants.

A vital trait in insects is their cuticular hydrocarbon (CHC) profile, which protects the insect against desiccation and serves in chemical communication. Due to these functions, CHC profiles are shaped by both climatic conditions and biotic interactions. Here, we investigated CHC differentiation in the neotropical parabiotic ant species Crematogaster levior and Camponotus femoratus, which mutualistically share a nest. Both consist of two cryptic species each (Cr. levior A and B and Ca. femoratus PAT and PS) that differ genetically and possess strongly different CHC profiles. We characterized and compared CHC profiles of the four cryptic species in detail. Our results suggest that Cr. levior A, Ca. femoratus PAT and Ca. femoratus PS adapted their CHC profiles to the parabiotic lifestyle by producing longer-chain CHCs. At the same time, they changed their major CHC classes, and produce more alkadienes and methyl-branched alkenes compared to Cr. levior B or non-parabiotic species. The CHC profiles of Cr. levior B were more similar to related, non-parabiotic species of the Orthocrema clade than Cr. levior A, and the chain lengths of B were similar to the reconstructed ancestral state. Signals of both the parabiotic partner (biotic conditions) and climate (abiotic conditions) were found in the CHC profiles of all four cryptic species. Our data suggest that mutualisms shaped the CHC profiles of the studied species, in particular chain length and CHC class composition. Beside this, signals of the parabiotic partners indicate potential impacts of biotic interactions, via chemical mimicry or chemical camouflage.

Hybrid Genome Assembly of a Neotropical Mutualistic Ant.

The success of social insects is largely intertwined with their highly advanced chemical communication system that facilitates recognition and discrimination of species and nest-mates, recruitment, and division of labor. Hydrocarbons, which cover the cuticle of insects, not only serve as waterproofing agents but also constitute a major component of this communication system. Two cryptic Crematogaster species, which share their nest with Camponotus ants, show striking diversity in their cuticular hydrocarbon (CHC) profile. This mutualistic system therefore offers a great opportunity to study the genetic basis of CHC divergence between sister species. As a basis for further genome-wide studies high-quality genomes are needed. Here, we present the annotated draft genome for Crematogaster levior A. By combining the three most commonly used sequencing techniques-Illumina, PacBio, and Oxford Nanopore-we constructed a high-quality de novo ant genome. We show that even low coverage of long reads can add significantly to overall genome contiguity. Annotation of desaturase and elongase genes, which play a role in CHC biosynthesis revealed one of the largest repertoires in ants and a higher number of desaturases in general than in other Hymenoptera. This may provide a mechanistic explanation for the high diversity observed in C. levior CHC profiles.

Coping with the climate: cuticular hydrocarbon acclimation of ants under constant and fluctuating conditions.

Terrestrial arthropods achieve waterproofing by a layer of cuticular hydrocarbons (CHCs). At the same time, CHCs also serve as communication signals. To maintain waterproofing under different climate conditions, insects adjust the chemical composition of their CHC layer, but this may affect the communication via CHCs. The detailed acclimatory changes of CHCs and how these influence their physical properties are still unknown. Here, we studied acclimation in two closely related ant species with distinct CHC profiles, Myrmica rubra and Myrmica ruginodis, in response to constant or fluctuating temperature and humidity regimes. We measured how acclimation affected CHC composition and viscosity, and the ants' drought survival. In both species, CHC composition showed strong, predictable responses to temperature regimes. Warm-acclimated individuals had higher proportions of linear alkanes, and less methyl-branched or unsaturated CHCs. These changes coincided with higher solid content and viscosity of CHCs in warm-acclimated ants. Temperature fluctuation caused effects similar to those observed under constant-cool conditions in Mrubra, but led to entirely different profiles in Mruginodis, suggesting that fluctuating and constant conditions pose very different challenges. Acclimation to dry conditions led to higher absolute amounts of CHCs, which increased the ants' drought survival, whereas temperature acclimation did not. Hence, the temperature-induced CHC changes cannot be explained by the need for waterproofing alone. Although these changes could be non-adaptive, we propose that they serve to maintain a constant CHC viscosity, which may be essential for communication and other functions.