Cuticular Hydrocarbon Composition Reflects Genetic Relationship Among Colonies of the Introduced Termite Reticulitermes santonensis Feytaud

Nestmate recognition plays a key role in kin selection to maintain colony integrity in social insects. Previous studies have demonstrated that nestmate recognition is dependent on detection of cuticular hydrocarbons. However, the absence of intraspecific aggression between some colonies of Isoptera and social Hymenoptera questions whether kin recognition must occur in social insects. The purpose of this study was to determine if cuticular hydrocarbon similarity and high genetic relatedness could explain the lack of intraspecific aggression among and within colonies of the introduced subterranean termite Reticulitermes santonensis. We performed both GC analysis of cuticular hydrocarbons and genotyping by using 10 DNA microsatellite loci on the same 10 workers from each of 14 parisian colonies. Multivariate analyses demonstrated correspondence between cuticular hydrocarbon patterns and genetic variation. By using a redundancy analysis combining chemical and genetic data, we found that a few hydrocarbons (mainly short vs. long chains; saturated vs. unsaturated alkanes) were associated with most genetic variation. We also found a strong positive correlation between chemical and genetic distances between colonies, thus providing evidence of a genetic basis for cuticular hydrocarbon variation. However, genetic distance did not account for all chemical variation, thus suggesting that some hydrocarbon variation was environmentally derived. Investigation at the intracolony level indicated that cuticular hydrocarbons did not depend on colony social structure. Based on our findings, we speculate that the absence of intraspecific aggression in R. santonensis may result from a loss of diversity in genetically derived recognition compounds in this species that presumably descended from R. flavipes populations imported from North America.     

DIET-MEDIATED INTER-COLONIAL AGGRESSION IN THE FORMOSAN SUBTERRANEAN TERMITE <Emphasis Type="Italic">Coptotermes formosanus</Emphasis>

In most social insects, intercolonial and interspecific aggression are expressions of territoriality. In termites, cuticular hydrocarbons (CHCs) have been extensively studied for their role in nestmate recognition and aggressive discrimination of nonnest-mates. More recently, molecular genetic techniques have made it possible to determine relatedness between colonies and to investigate the influence of genetics on aggression. In the Formosan subterranean termite, Coptotermes formosanus, however, the role of CHCs and genetic relatedness in inter-colony aggression has been ambiguous, suggesting the involvement of additional factors in nest-mate recognition. In this study we assess the range of aggression in this termite species and characterize the influence of genetic relatedness, CHC profiles and diet on aggression levels. We collected four colonies of C. formosanus, feeding either on bald cypress or birch, from three locations in Louisiana. Inter-colony aggression ranged from low to high. Differences in CHC profiles, as well as genetic distances between colonies determined by using microsatellite DNA markers, showed no significant correlation with aggression. However, termite diet (host tree) played a significant role in determining the level of aggression. Thus, two distantly related colonies, each feeding on different diets, showed high aggression that significantly diminished if they were fed on the same wood in the laboratory (spruce). Using headspace solid phase microextraction, we found three compounds from workers fed on birch that were absent in workers fed on spruce. Such diet-derived chemicals may be involved in the complex determination of nest-mate recognition in C. formosanus.     

Cuticular Hydrocarbon Phenotypes Do not Indicate Cryptic Species in Fungus-Growing Termites (Isoptera: Macrotermitinae)

In several termite species, distinct differences in the composition of cuticular hydrocarbons among colonies correspond to high genetic divergence of mitochondrial DNA sequences. These observations suggest that hydrocarbon phenotypes represent cryptic species. Different cuticular hydrocarbon phenotypes also are found among colonies of fungus-growing termites of the genus Macrotermes. To determine if these hydrocarbon differences in Macrotermes also indicate cryptic species, we sequenced the mitochondrial CO I gene from species in West and East Africa. Among individuals of a supposed species but belonging to different cuticular hydrocarbon phenotypes, the genetic distances are much smaller than distances between species. Unlike what has been observed in other termites, Macrotermes hydrocarbon phenotypes do not represent cryptic species. Our findings suggest fundamental differences in the evolution and/or function of cuticular hydrocarbons among different termite lineages. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Induced mimicry of colony odors in ants

The cuticular hydrocarbons ofFormica selysi (Formicinae) andMonica rubida (Myrmicinae) reared in single species and in mixed species colonies were determined using gas chromatography (GC) and GC-mass spectrometry. In colonies containing both species, each species modified its species-specific recognition odor. This odor is composed, at least partly, of cuticular hydrocarbons. The cuticular hydrocarbons ofM. rubida consist only of saturated alkanes (n-alkanes and branched alkanes). InF. selysi the mixture also contains unsaturated compounds (monoenes and dienes). In hetero-specific colonies, a new chemical signature developed. This signature resulted from qualitative and quantitative changes in the spectrum of hydrocarbons produced by each species and permitted the two species to inhabit the same nest without displaying interspecific aggression. The readjustment seemed to be more an active synthesis or an active transfer than simply a passive transfer from one species to the other. This may imply that the ants synthesized some components of the hydrocarbon signature of the other species. These synthesizing processes may be activated under particular social environmental conditions.     

Cuticular hydrocarbons and defensive compounds ofReticulitermes flavipes (Kollar) andR. santonensis (feytaud): Polymorphism and chemotaxonomy

Colonies ofReticulitermes flavipes andR. santonensis were collected from the southeastern United States (Georgia) and the southwest of France (Charente-maritime). Defensive compounds and cuticular hydrocarbons were identified by gas chromatography-mass spectrometry and quantified by gas chromatography using an internal standard for each caste and all colonies. These analyses show that although the cuticular hydrocarbons ofR. santonensis in Europe andR. flavipes in Georgia are identical, their relative proportions are different. However, the defensive compounds synthesized by their soldiers are different. A strong chemical polymorphism between sympatric colonies ofR. flavipes in the SW United States was detected in terms of both the hydrocarbons of the workers and soldiers and in the defensive secretions of the soldiers. The six defensive secretion phenotypes are based on the presence or absence of terpenes whereas the cuticular hydrocarbon phenotypes are based on significant differences in the proportions of the various components. A multivariate analysis (analysis of principal components) clearly permitted discrimination of four phenotypes (three inR. flavipes and one inR. santonensis) without intermediates. The hydrocarbons responsible for these variations were identified, and it was shown that the variations are neither seasonal nor geographic. The phenotypes of the cuticular hydrocarbons (workers and soldiers) and defensive compounds are linked in each colony, forming in three groups inR. flavipes Georgia, one subdivided into four subgroups according to the defensive secretion phenotypes. The role of these polymorphisms is discussed and ethological tests indicate that the chemical polymorphism do not determine aggressive behavior. The taxonomic significance of these results is considered and two hypothesis are formulated: (1) We only detected a strong genetic polymorphism in one unique species, and we believe thatR. santonensis was introduced into Europe in the last century from oneR. flavipes colony. (2) Chemical variability characterizes the sibling species that can be grouped into the same subspeciesR. flavipes. Unknown mechanisms of reproductive isolation separate them.     

Segregation of colony odor in the desert ant Cataglyphis niger

There are two separate, and presumably opposing, processes that affect colony odor in the desert ant Cataglyphis niger: (1) biosynthesis and turnover of these chemicals by individual ants, and (2) homogenization of colony odor through exchange of cues. The first increases signal variability; the latter decreases it. The impact of these factors was tested by splitting colonies and monitoring the profile changes occurring in the postpharyngeal glands (PPG) and cuticular hydrocarbons.From each of two polygynous nests four daughter colonies were formed, three monogynous and one queenless. Thereafter, 10 ants from each were randomly selected each month, for three successive months, for analyses of their PPG and cuticular hydrocarbons. From two colonies we also obtained ants from a known matriline. Over time, there was a shift in hydrocarbon profiles of both the PPG and cuticular washes in each of the tested colonies. Moreover, by subjecting selected hydrocarbon constituents to a discriminant analyses based on their relative proportions, all of the daughter colonies (queenright and queenless) were distinguishable from each other and from their respective mother colonies. In each of the queenright daughter colonies, the queen profile was indiscriminable from that of the workers and often was in the center of the group. Full sisters were clearly distinguishable from their nestmates, emphasizing the genetic versus environmental processes that govern colony odor. The effect of time was always superior to the separation effect in contributing to odor segregation. Comparison of the Mahalanobis distances indicated that the shift in hydrocarbon seems to proceed along parallel lines rather than in divergence. However, there was no overt aggression between ants that originated from the different subgroups in dyadic encounters. It appears that in this species a three-month separation period is not sufficient to change the hydrocarbon profile beyond the recognition threshold.     

Social closure,aggressive behavior,and cuticular hydrocarbon profiles in the polydomous antCataglyphis iberica (hymenoptera,Formicidae)

Nestmate recognition was studied in the polydomous antCataglyphis iberica (Formicinae) in the laboratory. The study examined six colonies collected from two different populations 600 km apart in the Iberian peninsula (Barcelona and Murcia). Introduction of an alien worker into an allocolonial arena always ended in death to the intruder, demonstrating that in this species societies are extremely closed. Dyadic encounters composed of individuals from different colonies in a neutral arena confirmed the existence of high aggression between allocolonial individuals. We also investigated variability in the composition of the major cuticular hydrocarbons between the colonies used in the behaviorial tests. There were marked quantitative differences between the profiles of ants from the two populations, suggesting that the populations are completely segregated. Cuticular profiles within a population tended to be more similar, but were nevertheless colony specific. The degree of colony closure inC. iberica seemed to be independent of geographic distance since aggression between the colonies was always at its maximum, irrespective of their population origin.     

Harvester Ants Utilize Cuticular Hydrocarbons in Nestmate Recognition

Cuticular hydrocarbons appear to play a role in ant nestmate recognition, but few studies have tested this hypothesis experimentally with purified hydrocarbon extracts. We exposed captive colonies of the harvester ant Pogonomyrmex barbatus to small glass blocks coated with whole cuticular lipid extracts and the purified hydrocarbon portion of extracts from nestmate and nonnestmate workers. As an estimate of agonistic behavior, we measured the proportion of ants in contact with blocks that flared their mandibles. Blocks coated with cuticular extracts from nonnestmates were contacted by more workers in one of two experiments and elicited higher levels of aggression in both experiments than blocks bearing extracts from nestmates. The cuticular hydrocarbon fraction of extracts alone was sufficient to elicit agonistic behavior toward nonnestmates. The results demonstrate that harvester ants can perceive differences in cuticular hydrocarbon composition, and can use those differences in nestmate recognition.     

Role of cuticular hydrocarbons of aphid parasitoids in their relationship to aphid-attending ants

Lysiphlebus cardui, the dominant aphidiid parasitoid of the black bean aphid,Aphis fabae cirsiiacanthoidis (Afc), on creeping thistle, is able to forage in ant-attended aphid colonies without being attacked by ants. Several behavioral observations and experimental studies led to the hypothesis thatL. cardui mimics the cuticular hydrocarbon profile of its host aphid. Chemical analysis of the cuticular extracts revealed that bothL. cardui and Afc exclusively possess saturated hydrocarbons:n-alkanes, monomethyl (MMA), dimethyl (DMA), and trimethyl alkanes (TMA). Comparison of the hydrocarbon profiles of parasitoid and aphid showed great qualitative resemblance between parasitoid and host:L. cardui possesses almost all host-specific compounds in addition to species-specific hydrocarbons of mainly higher molecular weight (>C₃₀). However, there is a lesser quantitative correspondence between parasitoid and host aphid. Furthermore, we analyzed the cuticular hydrocarbon profile of another parasitoid of Afc,Trioxys angelicae. This aphidiid species is vigorously attacked and finally killed by honeydewcollecting ants when encountered in aphid colonies. Its cuticular hydrocarbon profile is characterized by the presence of large amounts of (Z)-11-alkenes of chain lenghts C₂₇, C₂₉, C₃₁, and C₃₃, in addition to alkanes and presumably trienes. The role of the unsaturated hydrocarbons onT. angelicae as recognition cues for aphid-attending ants is discussed.     

Venom Alkaloid and Cuticular Hydrocarbon Profiles Are Associated with Social Organization,Queen Fertility Status,and Queen Genotype in the Fire Ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Queens in social insect colonies advertise their presence in the colony to: a) attract workers’ attention and care; b) gain acceptance by workers as replacement or supplemental reproductives; c) prevent reproductive development in nestmates. We analyzed the chemical content of whole body surface extracts of adult queens of different developmental and reproductive stages, and of adult workers from monogyne (single colony queen) and polygyne (multiple colony queens) forms of the fire ant Solenopsis invicta. We found that the composition of the most abundant components, venom alkaloids, differed between queens and workers, as well as between reproductive and non-reproductive queens. Additionally, workers of the two forms could be distinguished by alkaloid composition. Finally, sexually mature, non-reproductive queens from polygyne colonies differed in their proportions of cis-piperidine alkaloids, depending on their Gp-9 genotype, although the difference disappeared once they became functional reproductives. Among the unsaturated cuticular hydrocarbons characteristic of queens, there were differences in amounts of alkenes/alkadienes between non-reproductive polygyne queens of different Gp-9 genotypes, between non-reproductive and reproductive queens, and between polygyne and monogyne reproductive queens, with the amounts increasing at a relatively higher rate through reproductive ontogeny in queens bearing the Gp-9 b allele. Given that the genotype-specific piperidine differences reflect differences in rates of reproductive maturation between queens, we speculate that these abundant and unique compounds have been co-opted to serve in fertility signaling, while the cuticular hydrocarbons now play a complementary role in regulation of social organization by signaling queen Gp-9 genotype.     

Cuticular hydrocarbons of dampwood termites,Zootermopsis: Intra- and intercolony variation and potential as taxonomic characters

Colonies ofZootermopsis were collected from the central Sierra Nevada and the Monterey Penninsula in California, and from southern Arizona. Cuticular hydrocarbons were identified by gas chromatography-mass spectrometry (GC-MS) and quantified by gas-liquid chromatography (GLC) for each caste of all colonies. Four consistent and distinct cuticular hydrocarbon patterns, or chemical phenotypes, were identified. Unique and abundant monomethyl- and dimethylalkanes, and ann-alkene provided easy separation of the various phenotypes. Significant differences in the proportions of the various components were found among castes within a colony and colonies within phenotypes from California. Differences in the hydrocarbon proportions for castes were not consistent between colonies. The current taxonomy of the genusZootermopsis recognizes three species. Our identification of four consistent, unique cuticular hydrocarbon phenotypes from the three described species should alert systematists and others to a major concern. If there are truly only three extant species, then the hypothesis that cuticular hydrocarbon profiles in this genus are species specific is not acceptable. Conversely, if cuticular hydrocarbon profiles are truly species specific, then there is at least one new, undescribed species ofZootermopsis.Isoptera: Termopsidae.     

Cuticular Chemistry of Males and Females in the Ant Formica fusca

Communication between organisms involves visual, auditory, and olfactory pathways. In solitary insects, chemical recognition cues are influenced mainly by selection regimes related to species recognition and sexual selection. In social insects, chemical recognition cues have an additional role in mediating recognition of society members and, thereby, allowing kin selection to operate. Here, we examined whether cuticular hydrocarbon profiles are sex-specific and whether males and young queens of the ant Formica fusca have colony-specific profiles. We also investigated whether there is a relationship between genetic relatedness and chemical diversity within colonies. We demonstrated that female and male sexuals do not have unique sex-specific compounds, but that there are quantitative chemical differences between the sexes. Out of the 51 cuticular hydrocarbon compounds identified, 10 showed a significant quantitative difference between males and females. We also showed that both males and females have a significant colony-specific component in their profiles. Finally, we found a negative correlation between within-colony relatedness and within-colony chemical diversity of branched, but not linear compounds. This suggests that colonies with multiple matri- or patrilines also have a significantly greater chemical diversity.     

Colony-Specific Cuticular Hydrocarbon Profile in Formica argentea Ants

The cuticular hydrocarbons of the ant Formica argentea were identified by gas chromatography/mass spectrometry. Behavioral bioassays tested the role of each class of cuticular hydrocarbon in nestmate recognition, and statistical analyses looked for potential colony-specific signatures. The cuticular hydrocarbons of F. argentea consist of n-alkanes, alkenes, and methyl-branched alkanes. Behavioral bioassays demonstrated that changes in the alkene and methyl-branched alkane signature of F. argentea increased aggression, but changes in alkanes did not. Statistical analyses demonstrated that F. argentea workers present a colony-specific hydrocarbon profile based on their methyl-branched C₂₉ alkane signature. Using this signature alone, it is possible to group worker ants statistically by nest, suggesting that methyl-branched C₂₉ alkanes may be important in nestmate recognition for this species. These results support the idea that variation in positional isomers of cuticular hydrocarbons of the same carbon chain length may provide enough information for nestmate recognition. Although the addition of alkenes increased aggression in F. argentea, alkenes did not provide a colony-specific signature. This study reinforces the idea that investigators studying nestmate recognition should not examine cuticular hydrocarbon profiles as a whole but rather, should look for colony-specific signatures embedded in parts of the profile.     

Temporal changes in colony cuticular hydrocarbon patterns ofSolenopsis invicta

Heritable cuticular hydrocarbon patterns ofSolenopsis invicta workers are consistent within colonies for a given sampling time but vary sufficiently from colony to colony to distinguish the colonies from each other. In addition, cuticular hydrocarbon patterns change within colonies over time. Nestmate recognition cues found on the individual's cuticle, can be from heritable or environmental sources, and are a subset of colony odor. The cuticular hydrocarbons can be used as a model for heritable nestmate recognition cues. We propose that because potential nestmate recognition cues, both environmental and genetic, are dynamic in nature rather than static, during its lifetime a worker must continually update its perception (template) of colony odor and nestmate recognition cues.     

Wax Lipids Signal Nest Identity in Bumblebee Colonies

The signalling functions of cuticular lipids, particularly cuticular hydrocarbons, have gained considerable attention in social insect communication. Information transfer between individuals by means of these substances has been examined extensively. However, communication with cuticular lipids is not limited to inter-individual recognition. Cuticular compounds can also have a signalling function in the nest environment. Workers of the bumblebee Bombus terrestris leave cuticular lipid traces, so-called footprints, that mark their nest entrance. In addition, there is evidence that bumblebees sense nesting material to identify their colony. In this study, we examined the signalling potential of bumblebee wax, and tested if bumblebee workers are able to identify their colony with the help of wax scent. Chemical analyses of wax extracts using coupled gas chromatography–mass spectrometry showed that wax from colonies of the bumblebee B. terrestris contained a complex blend of cuticular lipids, dominated by hydrocarbons and wax esters. Comparing the relative compound amounts of wax samples from different colonies, we found that wax scent patterns varied with nest identity. Olfactometer bioassays showed that bumblebees were able to discriminate between wax scents from their own and a foreign colony. Our findings suggest that wax emits characteristic olfactory profiles that are used by workers to recognize their colony.     

Intercaste,intercolony, and temporal variation in cuticular hydrocarbons ofCopotermes formosanus shiraki (Isoptera: Rhinotermitidae)

We characterized the variation in cuticular hydrocarbon mixtures between seven colonies of the Formosan subterranean termite,Coptotermes formosanus Shiraki, from the same population. We report differences between castes, between colonies, and within the population over time to assess seasonality. Colonies ofC. formosanus from Oahu, Hawaii, were sampled for 25 months. Each month, one sample each of 200 workers, 50 soldiers, nymphs, or alates from each colony was subjected to gas chromatography-mass spectrometry (GC-MS) analysis of the cuticular hydrocarbons. We resolved 39 individual peaks and identified 52 individual or isomeric mixtures of hydrocarbons. Onlyn-alkanes and methyl-branched alkanes occur; no olefins were found. Internally branched monomethylalkanes were the most abundant class of hydrocarbons, representing 45% to 50% of the total 9-;11-;13-Methyl-heptacosane accounted for over 30% of the total hydrocarbon for all castes. 2-Methyl- and 3-methylalkanes comprise approximately 30% of the total. Internally branched dimethylalkanes constitute 15% to 20% of the total cuticular hydrocarbon. Only one trimethylalkane, 13,15,17-trimethylnonacosane, was found in small amounts. The hydrocarbon mixtures of all four castes were similar. Quantitative differences in hydrocarbon mixtures among the castes were easily displayed using canonical discriminant analysis. Soldiers and workers are significantly different from one another and from nymphs and alates. Nineteen peaks are statistically significant between workers and soldiers. Nymphs and alates were not statistically different. We detected statistically significant quantitative differences between colonies in 18 peaks for workers and 12 peaks for soldiers. Each of the colonies ofC. formosanus can be separated from the others by the proportions of their hydrocarbon components. We detected statistically significant differences between months of the year for 12 peaks for workers and four peaks for soldiers; two peaks each for workers and soldiers showed distinct, seasonal trends. This seasonal shift in proportions of hydrocarbons correlates with the production of alates.     

Genetic Distance and Age Affect the Cuticular Chemical Profiles of the Clonal Ant Cerapachys biroi

Although cuticular hydrocarbons (CHCs) have received much attention from biologists because of their important role in insect communication, few studies have addressed the chemical ecology of clonal species of eusocial insects. In this study we investigated whether and how differences in CHCs relate to the genetics and reproductive dynamics of the parthenogenetic ant Cerapachys biroi. We collected individuals of different ages and subcastes from several colonies belonging to four clonal lineages, and analyzed their cuticular chemical signature. CHCs varied according to colonies and clonal lineages in two independent data sets, and correlations were found between genetic and chemical distances between colonies. This supports the results of previous research showing that C. biroi workers discriminate between nestmates and non-nestmates, especially when they belong to different clonal lineages. In C. biroi, the production of individuals of a morphological subcaste specialized in reproduction is inversely proportional to colony-level fertility. As chemical signatures usually correlate with fertility and reproductive activity in social Hymenoptera, we asked whether CHCs could function as fertility-signaling primer pheromones determining larval subcaste fate in C. biroi. Interestingly, and contrary to findings for several other ant species, fertility and reproductive activity showed no correlation with chemical signatures, suggesting the absence of fertility related CHCs. This implies that other cues are responsible for subcaste differentiation in this species.     

Interfamily variation in comb wax hydrocarbons produced by honey bees

The hydrocarbons of honeybee comb wax vary significantly between colonies. This variation is explained in part by genetic (familial) differences among colonies. Even though significant differences in wax hydrocarbons exist among families, there is a high level of consistency within and among families in a correlation analysis, indicating structural constancy in comb wax. The significance of these results in interpreting the potential role of comb wax in the nestmate recognition system of the honeybee is discussed.     

Diet-Related Modification of Cuticular Hydrocarbon Profiles of the Argentine Ant, <Emphasis Type="Italic">Linepithema humile</Emphasis>, Diminishes Intercolony Aggression

Territorial boundaries between conspecific social insect colonies are maintained through a highly developed nestmate recognition system modulated by heritable and, in some instances, nonheritable cues. Argentine ants, Linepithema humile, use both genetic and environmentally derived cues to discriminate nestmates from nonnestmates. We explored the possibility that intraspecific aggression in the Argentine ant might diminish when colonies shared a common diet. After segregating recently field-collected colony pairs into high or moderate aggression categories, we examined the effect of one of three diets: two hydrocarbon-rich insect prey, Blattella germanica and Supella longipalpa, and an artificial (insect-free) diet, on the magnitude of aggression loss. Aggression diminished between colony pairs that were initially moderately aggressive. However, initially highly aggressive colony pairs maintained high levels of injurious aggression throughout the study, independent of diet type. Each diet altered the cuticular hydrocarbon profile by contributing unique, diet-specific cues. We suggest that acquisition of common exogenous nestmate recognition cues from shared food sources may diminish aggression and promote fusion in neighboring colonies of the Argentine ant.     

Genetic Relatedness and Chemical Profiles in an Unusually Peaceful Eusocial Bee

Colonies of the stingless bee Tetragonilla collina frequently occur in unusually high densities and in direct neighborhood (nest aggregations), in rainforests of Southeast Asia. To investigate whether close relatedness and/or similar chemical profiles facilitate the co-occurrence of multiple T. collina colonies, we investigated aggressive behavior, genetic relatedness and cuticular hydrocarbon (CHC) profiles within and between colonies and nest aggregations. Although 17 out of 19 colonies within aggregations were largely unrelated, intraspecific aggression between different colonies was basically absent both within and among aggregations. This lack of aggression should favor social parasitism and hence the occurrence of unrelated individuals within a colony. However, low within-colony relatedness was found in only five out of 19 colonies where it may be explained by queen turnover or the occurrence of foreign workers. CHC profiles of colonies within and among aggregations were statistically different. However, many workers could chemically not be assigned to their maternal colony, indicating considerable overlap among colonies in odor profiles of workers. Moreover, odor profiles tended to be more similar within than among aggregations, although most colonies were unrelated. Thus, CHC profiles were a poor indicator of relatedness in T. collina. The lack of correlation between relatedness and chemical similarity in T. collina may be explained by the incorporation of resin-derived terpenes in their CHC profiles. The composition of these terpenes was highly similar among colonies, particularly within aggregations, hence potentially decreasing chemical distinctiveness and increasing behavioral tolerance.