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Chagas disease is a lethal, neglected tropical disease. Unfortunately, aggressive insecticide-spraying campaigns have not been able to eliminate domestic infestation of Triatoma dimidiata, the native vector in Guatemala. To target interventions toward houses most at risk of infestation, comprehensive socioeconomic and entomologic surveys were conducted in two towns in Jutiapa, Guatemala. Given the exhaustively large search space associated with combinations of risk factors, traditional statistics are limited in their ability to discover risk factor interactions. Two recently developed statistical evolutionary algorithms, specifically designed to accommodate risk factor interactions and heterogeneity, were applied to this large combinatorial search space and used in tandem to identify sets of risk factor combinations associated with infestation. The optimal model includes 10 risk factors in what is known as a third-order disjunctive normal form (i.e., infested households have chicken coops AND deteriorated bedroom walls OR an accumulation of objects AND dirt floors AND total number of occupants ≥ 5 AND years of electricity ≥ 5 OR poor hygienic condition ratings AND adobe walls AND deteriorated walls AND dogs). Houses with dirt floors and deteriorated walls have been reported previously as risk factors and align well with factors currently targeted by Ecohealth interventions to minimize infestation. However, the tandem evolutionary algorithms also identified two new socioeconomic risk factors (i.e., households having many occupants and years of electricity ≥ 5). Identifying key risk factors may help with the development of new Ecohealth interventions and/or reduce the survey time needed to identify houses most at risk.

Reproductive division of labor in the social insects is typically determined by environmental cues; however, genetic effects on caste have been discovered in a growing set of ant taxa. An extreme form of genetic caste determination is "social hybridogenesis," in which co-occurring genetic lineages obligately interbreed to produce workers, whereas daughter queens are of pure-lineage ancestry. In this study, we tested the hypothesis that social hybridogenesis in the genus Pogonomyrmex resulted from one or more interspecific hybridization events, and if so, whether individual lineages were of hybrid ancestry. We reconstructed evolutionary relationships of four lineage pairs to populations of two closely related non-hybridogenetic species, Pogonomyrmex barbatus and Pogonomyrmex rugosus, using nuclear SNP loci and mitochondrial sequencing. The nuclear phylogeny supported a hybridization hypothesis, with one member of each pair nested within P. rugosus, whereas the other was nested within P. barbatus. The source populations corresponded with two distinct geographic areas at the eastern and western edges of a zone of contact. Relatively little gene flow was detected between interbreeding lineages, either historically or currently. This suggests that shifts in reproductive caste determination may reinforce reproductive incompatibility, in a manner similar to the evolution of hybridogenesis in nonsocial systems.

Geographic variation in low temperatures at poleward range margins of terrestrial species often mirrors population variation in cold resistance, suggesting that range boundaries may be set by evolutionary constraints on cold physiology. The northeastern woodland ant Aphaenogaster picea occurs up to approximately 45°N in central Maine. We combined presence/absence surveys with classification tree analysis to characterize its northern range limit and assayed two measures of cold resistance operating on different timescales to determine whether and how marginal populations adapt to environmental extremes. The range boundary of A. picea was predicted primarily by temperature, but low winter temperatures did not emerge as the primary correlate of species occurrence. Low summer temperatures and high seasonal variability predicted absence above the boundary, whereas high mean annual temperature (MAT) predicted presence in southern Maine. In contrast, assays of cold resistance across multiple sites were consistent with the hypothesis of local cold adaptation at the range edge: among populations, there was a 4-min reduction in chill coma recovery time across a 2° reduction in MAT. Baseline resistance and capacity for additional plastic cold hardening shifted in opposite directions, with hardening capacity approaching zero at the coldest sites. This trade-off between baseline resistance and cold-hardening capacity suggests that populations at range edges may adapt to colder temperatures through genetic assimilation of plastic responses, potentially constraining further adaptation and range expansion.

Assays for parasite detection in insect vectors provide important information for disease control. American Trypanosomiasis (Chagas disease) is the most devastating vector-borne illness and the fourth most common in Central America behind HIV/AIDS and acute respiratory and diarrheal infections (Peterson et al., 2019). Under-detection of parasites is a general problem which may be influenced by parasite genetic variation; however, little is known about the genetic variation of the Chagas parasite, especially in this region. In this study we compared six assays for detecting the Chagas parasite, Trypanosoma cruzi: genomic reduced representation sequencing (here referred to as genotype-by-sequencing or GBS), two with conventional PCR (i.e., agarose gel detection), two with qPCR, and microscopy. Our results show that, compared to GBS genomic analysis, microscopy and PCR under-detected T. cruzi in vectors from Central America. Of 94 samples, 44% (50/94) were positive based on genomic analysis. The lowest detection, 9% (3/32) was in a subset assayed with microscopy. Four PCR assays, two with conventional PCR and two with qPCR showed intermediate levels of detection. Both qPCR tests and one conventional PCR test targeted the 195 bp repeat of satellite DNA while the fourth test targeted the 18S gene. Statistical analyses of the genomic and PCR results indicate that the PCR assays significantly under detect infections of Central American T. cruzi genotypes.

Thermal tolerance of an organism depends on both the ability to dynamically adjust to a thermal stress and preparatory developmental processes that enhance thermal resistance. However, the extent to which standing genetic variation in thermal tolerance alleles influence dynamic stress responses vs. preparatory processes is unknown. Here, using the model species Drosophila melanogaster, we used a combination of Genome Wide Association mapping (GWAS) and transcriptomic profiling to characterize whether genes associated with thermal tolerance are primarily involved in dynamic stress responses or preparatory processes that influence physiological condition at the time of thermal stress. To test our hypotheses, we measured the critical thermal minimum (CTmin) and critical thermal maximum (CTmax) of 100 lines of the Drosophila Genetic Reference Panel (DGRP) and used GWAS to identify loci that explain variation in thermal limits. We observed greater variation in lower thermal limits, with CTmin ranging from 1.81 to 8.60°C, while CTmax ranged from 38.74 to 40.64°C. We identified 151 and 99 distinct genes associated with CTmin and CTmax, respectively, and there was strong support that these genes are involved in both dynamic responses to thermal stress and preparatory processes that increase thermal resistance. Many of the genes identified by GWAS were involved in the direct transcriptional response to thermal stress (72/151 for cold; 59/99 for heat), and overall GWAS candidates were more likely to be differentially expressed than other genes. Further, several GWAS candidates were regulatory genes that may participate in the regulation of stress responses, and gene ontologies related to development and morphogenesis were enriched, suggesting many of these genes influence thermal tolerance through effects on development and physiological status. Overall, our results suggest that thermal tolerance alleles can influence both dynamic plastic responses to thermal stress and preparatory processes that improve thermal resistance. These results also have utility for directly comparing GWAS and transcriptomic approaches for identifying candidate genes associated with thermal tolerance.

To date, the phylogeny of Triatoma dimidiata sensu lato (s. l.) (Hemiptera: Reduviidae: Triatominae), the epidemiologically most important Chagas disease vector in Central America and a secondary vector in Mexico and northern South America, has only been investigated by one multi-copy nuclear gene (Internal Transcribed Spacer – 2) and a few mitochondrial genes. We examined 450 specimens sampled across most of its native range from Mexico to Ecuador using reduced representation next-generation sequencing encompassing over 16,000 single nucleotide polymorphisms (SNPs). Using a combined phylogenetic and species delimitation approach we uncovered two distinct species, as well as a well-defined third group that may contain multiple species. The findings are discussed with respect to possible drivers of diversification and the epidemiological importance of the distinct species and groups.

Few studies have quantified the relative importance of direct effects of climate change on communities versus indirect effects that are mediated thorough species interactions, and the limited evidence is conflicting. Trait-based approaches have been popular in studies of climate change, but can they be used to estimate direct versus indirect effects? At the species level, thermal tolerance is a trait that is often used to predict winners and losers under scenarios of climate change. But thermal tolerance might also inform when species interactions are likely to be important because only subsets of species will be able to exploit the available warmer climatic niche space, and competition may intensify in the remaining, compressed cooler climatic niche space. Here, we explore the relative roles of the direct effects of temperature change and indirect effects of species interactions on forest ant communities that were heated as part of a large-scale climate manipulation at high- and low-latitude sites in eastern North America. Overall, we found mixed support for the importance of negative species interactions (competition), but found that the magnitude of these interaction effects was predictable based on the heat tolerance of the focal species. Forager abundance and nest site occupancy of heat-intolerant species were more often influenced by negative interactions with other species than by direct effects of temperature. Our findings suggest that measures of species-specific heat tolerance may roughly predict when species interactions will influence responses to global climate change.

Geographic variation in low temperatures at poleward range margins of terrestrial species often mirrors population variation in cold resistance, suggesting that range boundaries may be set by evolutionary constraints on cold physiology. The northeastern woodland ant

Given the abundance, broad distribution, and diversity of roles that ants play in many ecosystems, they are an ideal group to serve as ecosystem indicators of climatic change. At present, only a few whole-genome sequences of ants are available (19 of >16,000 species), mostly from tropical and sub-tropical species. To address this limited sampling, we sequenced genomes of temperate-latitude species from the genus Aphaenogaster, a genus with important seed dispersers. In total, we sampled seven colonies of six species: Aphaenogaster ashmeadi, Aphaenogaster floridana, Aphaenogaster fulva, Aphaenogaster miamiana, Aphaenogaster picea, and Aphaenogaster rudis. The geographic ranges of these species collectively span eastern North America from southern Florida to southern Canada, which encompasses a latitudinal gradient in which many climatic variables are changing rapidly. For the six genomes, we assembled an average of 271,039 contigs into 47,337 scaffolds. The Aphaenogaster genomes displayed high levels of completeness with 96.1% to 97.6% of Hymenoptera BUSCOs completely represented, relative to currently sequenced ant genomes which ranged from 88.2% to 98.5%. Additionally, the mean genome size was 370.5 Mb, ranging from 310.3 to 429.7, which is comparable to that of other sequenced ant genomes (212.8–396.0 Mb) and flow cytometry estimates (210.7–690.4 Mb). In an analysis of currently sequenced ant genomes and the new Aphaenogaster sequences, we found that after controlling for both spatial autocorrelation and phylogenetics ant genome size was marginally correlated with sample site climate similarity. Of all examined climate variables, minimum temperature, and annual precipitation had the strongest correlations with genome size, with ants from locations with colder minimum temperatures and higher levels of precipitation having larger genomes. These results suggest that climate extremes could be a selective force acting on ant genomes and point to the need for more extensive sequencing of ant genomes.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and transmitted by triatomine insect vectors. In Guatemala, insecticide spraying is an integral part of management of the main vector, Triatoma dimidiata. Spraying typically has low efficacy, which may be due to incomplete elimination from infested houses, within-village dispersal, or influx from other villages or sylvan environments. To evaluate how these mechanisms contribute to reinfestation, we conducted a time-course analysis of T. dimidiata infestation, abundance and household genetic structure in two nearby villages in Jutiapa, Guatemala; houses in the first village were surveyed, treated with insecticide if infested and then re-surveyed at eight and 22 months following spraying, while the second village served as an untreated control to quantify changes associated with seasonal dispersal. Insects were genotyped at 2–3000 SNP loci for kinship and population genetic analyses. Insecticide application reduced overall infestation and abundance, while the untreated village was stable over time. Nevertheless, within two years 35.5% of treated houses were reinfested and genetic diversity had largely recovered. Insects collected from reinfested houses post-spraying were most closely related to pre-spray collections from the same house, suggesting that infestations had not been fully eliminated. Immigration by unrelated insects was also detected within a year of spraying; when it occurred, dispersal was primarily local from neighboring houses. Similar dispersal patterns were observed following the annual dispersal season in the untreated village, with high-infestation houses serving as sources for neighboring homes. Our findings suggest that the efficacy of pyrethroid application is rapidly diminished by both within-house breeding by survivors and annual cycles of among-house movement. Given these patterns, we conclude that house structural improvements, an integral part of the Ecohealth approach that makes houses refractory to vector colonization and persistence, are critical for long-term reduction of T. dimidiata infestation.

Given the abundance, broad distribution, and diversity of roles that ants play in many ecosystems, they are an ideal group to serve as ecosystem indicators of climatic change. At present, only a few whole-genome sequences of ants are available (19 of >16,000 species), mostly from tropical and sub-tropical species. To address this limited sampling, we sequenced genomes of temperate-latitude species from the genus Aphaenogaster, a genus with important seed dispersers. In total, we sampled seven colonies of six species: Aphaenogaster ashmeadi, Aphaenogaster floridana, Aphaenogaster fulva, Aphaenogaster miamiana, Aphaenogaster picea, and Aphaenogaster rudis. The geographic ranges of these species collectively span eastern North America from southern Florida to southern Canada, which encompasses a latitudinal gradient in which many climatic variables are changing rapidly. For the six genomes, we assembled an average of 271,039 contigs into 47,337 scaffolds. The Aphaenogaster genomes displayed high levels of completeness with 96.1% to 97.6% of Hymenoptera BUSCOs completely represented, relative to currently sequenced ant genomes which ranged from 88.2% to 98.5%. Additionally, the mean genome size was 370.5 Mb, ranging from 310.3 to 429.7, which is comparable to that of other sequenced ant genomes (212.8–396.0 Mb) and flow cytometry estimates (210.7–690.4 Mb). In an analysis of currently sequenced ant genomes and the new Aphaenogaster sequences, we found that after controlling for both spatial autocorrelation and phylogenetics ant genome size was marginally correlated with sample site climate similarity. Of all examined climate variables, minimum temperature, and annual precipitation had the strongest correlations with genome size, with ants from locations with colder minimum temperatures and higher levels of precipitation having larger genomes. These results suggest that climate extremes could be a selective force acting on ant genomes and point to the need for more extensive sequencing of ant genomes.

Chagas disease, considered a neglected disease by the World Health Organization, is caused by the protozoan parasite Trypanosoma cruzi, and transmitted by >140 triatomine species across the Americas. In Central America, the main vector is Triatoma dimidiata, an opportunistic blood meal feeder inhabiting both domestic and sylvatic ecotopes. Given the diversity of interacting biological agents involved in the epidemiology of Chagas disease, having simultaneous information on the dynamics of the parasite, vector, the gut microbiome of the vector, and the blood meal source would facilitate identifying key biotic factors associated with the risk of T. cruzi transmission. In this study, we developed a RADseq-based analysis pipeline to study mixed-species DNA extracted from T. dimidiata abdomens. To evaluate the efficacy of the method across spatial scales, we used a nested spatial sampling design that spanned from individual villages within Guatemala to major biogeographic regions of Central America. Information from each biotic source was distinguished with bioinformatics tools and used to evaluate the prevalence of T. cruzi infection and predominant Discrete Typing Units (DTUs) in the region, the population genetic structure of T. dimidiata, gut microbial diversity, and the blood meal history. An average of 3.25 million reads per specimen were obtained, with approximately 1% assigned to the parasite, 20% to the vector, 11% to bacteria, and 4% to putative blood meals. Using a total of 6,405 T. cruzi SNPs, we detected nine infected vectors harboring two distinct DTUs: TcI and a second unidentified strain, possibly TcIV. Vector specimens were sufficiently variable for population genomic analyses, with a total of 25,710 T. dimidiata SNPs across all samples that were sufficient to detect geographic genetic structure at both local and regional scales. We observed a diverse microbiotic community, with significantly higher bacterial species richness in infected T. dimidiata abdomens than those that were not infected. Unifrac analysis suggests a common assemblage of bacteria associated with infection, which co-occurs with the typical gut microbial community derived from the local environment. We identified vertebrate blood meals from five T. dimidiata abdomens, including chicken, dog, duck and human; however, additional detection methods would be necessary to confidently identify blood meal sources from most specimens. Overall, our study shows this method is effective for simultaneously generating genetic data on vectors and their associated parasites, along with ecological information on feeding patterns and microbial interactions that may be followed up with complementary approaches such as PCR-based parasite detection, 18S eukaryotic and 16S bacterial barcoding., Author summary Chagas disease is caused by the parasite Trypanosoma cruzi, which is spread by triatomine kissing bugs. There are many biotic factors that influence the risk of disease transmission, including the strain of the parasite, the vector movement patterns, the community of microbes interacting with the parasite inside the vector's gut, and the availability of suitable vertebrate hosts. DNA from all of these species can be found in the gut of an infected bug, providing an opportunity to investigate all of them simultaneously by genetically analyzing this single tissue. In this study, we developed a DNA-based method to retrieve, separate, and analyze genetic information from the abdomens of 32 T. dimidiata kissing bug vectors collected across Central America. We found two distinct strains of T. cruzi, and four T. dimidiata genetic clusters associated with environmental and geographical characteristics. These populations harbored different bacterial gut communities that were augmented by specifically infection-associated bacteria when the vector was infected by the parasite. In some cases, we could identify what the insect had recently fed on, including chicken, duck, dog and human. Having simultaneous information on all of these organisms may help to fine-tune control strategies that influence the risk of T. cruzi transmission.

Although harvester ant queens only lay eggs for most of their lives, they interact socially when they start colonies, behaving aggressively or cooperatively depending on the population they come from. We investigated whether brain neurotransmitters known to control behaviors from insects to humans were linked to queen behaviors. We found that neurotransmitter levels changed and one, serotonin, was elevated specifically when queens became aggressive, even though this switch occurred at different times in different populations.

A central goal of biology is to uncover the genetic basis for the origin of new phenotypes. A particularly effective approach is to examine the genomic architecture of species that have secondarily lost a phenotype with respect to their close relatives. In the eusocial Hymenoptera, queens and workers have divergent phenotypes that may be produced via either expression of alternative sets of caste-specific genes and pathways or differences in expression patterns of a shared set of multifunctional genes. To distinguish between these two hypotheses, we investigated how secondary loss of the worker phenotype in workerless ant social parasites impacted genome evolution across two independent origins of social parasitism in the ant generaPogonomyrmex and Vollenhovia. We sequenced the genomes of three social parasites and their most-closely related eusocial host species and compared gene losses in social parasites with gene expression differences between host queens and workers. Virtually all annotated genes were expressed to some degree in both castes of the host, with most shifting in queen-worker bias across developmental stages. As a result, despite 4 1M y of divergence from the last common ancestor that had workers, the social parasite ss howed strikingly little evidence of gene loss, damaging mutations, or shifts in selection regime resulting from loss of the worker caste. This suggests that regulatory changes within a multifunctional genome, rather than sequence differences, have played a predominant role in the evolution of social parasitism, and perhaps also in the many gains and losses of phenotypes in the social insects.

Biological invasions are largely thought to be contemporary, having recently increased sharply in the wake of globalization. However, human commerce had already become global by the mid-16th century when the Spanish connected the New World with Europe and Asia via their Manila galleon and West Indies trade routes. We use genetic data to trace the global invasion of one of the world's most widespread and invasive pest ants, the tropical fire ant, Solenopsis geminata. Our results reveal a pattern of introduction of Old World populations that is highly consistent with historical trading routes suggesting that Spanish trade introduced the tropical fire ant to Asia in the 16th century. We identify southwestern Mexico as the most likely source for the invasive populations, which is consistent with the use of Acapulco as the major Spanish port on the Pacific Ocean. From there, the Spanish galleons brought silver to Manila, which served as a hub for trade with China. The genetic data document a corresponding spread of S. geminata from Mexico via Manila to Taiwan and from there, throughout the Old World. Our descriptions of the worldwide spread of S. geminata represent a rare documented case of a biological invasion of a highly invasive and globally distributed pest species due to the earliest stages of global commerce.

Tradeoffs in functional traits facilitate species coexistence within communities and are important contributors to community assembly. Two aspects of a life-history tradeoff known to structure ant communities include foraging efficiency (i.e. resource discovery and retrieval) and competitive dominance (territoriality and survivorship following competition). This work examined these aspects in workers of two fire ant species (Solenopsis xyloni and Solenopsis geminata) and their F1 hybrids using laboratory experiments. Parental foraging strategies were consistent with a tradeoff between discovery efficiency and competitive territorial dominance. Desert-adapted S. xyloni was the most efficient forager but the least effective territory defender; mesic-adapted S. geminata was a less efficient forager, but fared best in competition trials. Hybrid workers were generally intermediate, leading to increased survival by hybrids in competitive encounters with S. geminata compared with the performance of S. xyloni. These findings suggest that traits associated with foraging and competitive ability in the parental species are subject to environmental filtering; across habitats, species with a similar dominance rank in their respective communities differ in their placement along the discovery–dominance axis. Interspecific hybridisation may play a role in extending eastern range limits of S. xyloni via shifting colony allocation towards the strategy of its competitor. In the overlap zone, S. xyloni with hybrid workers may be sufficiently competitive to persist with S. geminata, while colonies with S. xyloni workers cannot, providing a substantial selective advantage to hybridisation for S. xyloni queens that may explain the unusual hybridogenetic social structure of this population.

The fate of queen foundress associations in ants varies across taxa: in some, lethal fighting results in survival of a single queen, while in others, queens coexist long term. One hypothesis for this difference is that selection favors fighting when group sizes are small and tolerance when groups are large. In an experiment with the ant Messor pergandei, we formed small, medium, and large groups with newly mated queens from field populations that have different mean group sizes and differ in whether multiple queens occur in older established colonies. We found that whether queens are eliminated by fighting depends upon region of origin and not group size: regardless of co-foundress number, queens from sites with single-queen adult field colonies displayed agonistic behaviors and their colonies reduced to a single queen, while queens from sites with multiple-queen colonies did not fight and co-foundresses coexisted long term. Worker aggression towards and elimination of queens were also correlated with region of origin. Where fighting occurred, queens were as likely to be killed by workers as by other queens. An aggressive display was the most common form of agonistic interaction among queens, while fighting was relatively rare. We hypothesize that queen displays evolved in response to worker attacks because they increase the probability that workers will eliminate competitor queens. Our results suggest that the evolutionary interests of workers, as well as queens, could be important in determining the evolution and maintenance of queen elimination in foundress associations.

Ecological diversification into thermally divergent habitats can push species toward their physiological limits, requiring them to accommodate temperature extremes through plastic or evolutionary changes that increase persistence under the local thermal regime. One way to withstand thermal stress is to increase production of heat shock proteins, either by maintaining higher baseline abundance within cells or by increasing the magnitude of induction in response to heat stress. We evaluated whether environmental variation was associated with expression of three heat shock protein genes in two closely-related species of woodland ant, Aphaenogaster picea and A. rudis. We compared adult workers from colonies collected from 25 sites across their geographic ranges. Colonies were maintained at two different laboratory temperatures, and tested for the independent effects of environment, phylogeny, and acclimation temperature on baseline and heat-induced gene expression. The annual maximum temperature at each collection site (Tmax) was not a significant predictor of either baseline expression or magnitude of induction of any of the heat shock protein genes tested. A phylogenetic effect was detected only for basal expression of Hsp40, which was lower in the most southern populations of A. rudis and higher in a mid-range population of possible hybrid ancestry. In contrast, a higher acclimation temperature significantly increased baseline expression of Hsc70-4, and increased induction of Hsp40 and Hsp83. Thus, physiological acclimation to temperature variation appears to involve modulation of the heat shock response, whereas other mechanisms are likely to be responsible for evolutionary shifts in thermal performance associated with large-scale climate gradients.

Background The distributions of species and their responses to climate change are in part determined by their thermal tolerances. However, little is known about how thermal tolerance evolves. To test whether evolutionary extension of thermal limits is accomplished through enhanced cellular stress response (enhanced response), constitutively elevated expression of protective genes (genetic assimilation) or a shift from damage resistance to passive mechanisms of thermal stability (tolerance), we conducted an analysis of the reactionome: the reaction norm for all genes in an organism’s transcriptome measured across an experimental gradient. We characterized thermal reactionomes of two common ant species in the eastern U.S, the northern cool-climate Aphaenogaster picea and the southern warm-climate Aphaenogaster carolinensis, across 12 temperatures that spanned their entire thermal breadth. Results We found that at least 2 % of all genes changed expression with temperature. The majority of upregulation was specific to exposure to low temperatures. The cool-adapted A. picea induced expression of more genes in response to extreme temperatures than did A. carolinensis, consistent with the enhanced response hypothesis. In contrast, under high temperatures the warm-adapted A. carolinensis downregulated many of the genes upregulated in A. picea, and required more extreme temperatures to induce down-regulation in gene expression, consistent with the tolerance hypothesis. We found no evidence for a trade-off between constitutive and inducible gene expression as predicted by the genetic assimilation hypothesis. Conclusions These results suggest that increases in upper thermal limits may require an evolutionary shift in response mechanism away from damage repair toward tolerance and prevention. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2466-z) contains supplementary material, which is available to authorized users.

Summary 1. Caste determination in eusocial insects is the process by which individuals differentiate into reproducer or helper phenotypes. 2. Environmental caste determination (ECD) is predicted to be more efficient than genetic caste determination (GCD), yet GCD occurs in several populations of Pogonomyrmex harvester ants. 3. We tested whether GCD reduces efficiency by comparing colony growth rates of two GCD lineage pairs (H and J) with two closely related ECD congeners, P. rugosus and P. barbatus, under laboratory conditions over a range of temperatures. In addition, we directly compared metabolic rates of GCD and ECD larvae using flow-through respirometry. 4. Unexpectedly, colonies from GCD lineages grew faster than colonies of P. rugosus across all temperatures, and grew at the same rate as colonies of P. barbatus. Slower colony growth rates of P. rugosus were caused by lower queen fecundity and slower larval development. Variation in developmental rate was not due to differences in larval metabolic rates, which did not differ among taxa. 5. These results suggest that GCD in Pogonomyrmex does not impose significant productivity costs during colony growth. Instead, efficiency costs are compensated by other physiological mechanisms which may or may not be directly related to the mode of caste determination. 6. Persistence of GCD populations in contact with ECD competitors likely stems from a life-history trade-off favouring different taxa across the geographic range of the complex: the slow growing but starvation-resistant P. rugosus dominates in resource-poor regions, while faster growing and competitive GCD populations predominate in more mesic habitats.

The long-term persistence of hybridogenetic systems, in which one genetic lineage is dependent on another as a sperm host, is paradoxical because they require continual coexistence between ecological competitors. Several species of ants display a social form of hybridogenesis, in which two distinct genetic lineages obligately interbreed to produce sterile workers, while intra-lineage progeny become the reproductives. A recent model suggests that persistence of such a system depends on the relative strengths of negative frequency-dependent selection acting during colony growth and positive frequency-dependent selection during reproduction. We used path analysis to investigate the effect of lineage frequency on reproductive output and colony sex ratio over a single reproductive season in a natural population of the H1/H2 lineage pair in the genus Pogonomyrmex. Results suggest that lineage frequency does impact reproduction via two opposing routes: the more common lineage procures more same-lineage mates, resulting in a higher proportion of same-lineage mates increases reproduction through higher queen-egg availability, but the rarer lineage procures more alternate-lineage mates, yielding a more genetically diverse worker caste that increases productivity. As a result, lineage frequency has only a weakly positive overall effect on colony reproduction, and overall frequency-dependence in this system across the entire life cycle is likely to be negative as predicted by the persistence model.

The establishment of new species by hybridization is difficult because it requires the development of reproductive isolation (RI) in sympatry to escape the homogenizing effects of gene flow from the parental species. Here we investigated the role of two pre- and two postzygotic mechanisms of RI in a system comprising two interdependent Pogonomyrmex harvester ant lineages (the H1 and H2 lineages) of hybrid origin and one of their parental species (P. rugosus). Similar to most other ants, P. rugosus is characterized by an environmental system of caste determination with female brood developing either into queens or workers depending on nongenetic factors. By contrast, there is a strong genetic component to caste determination in the H1 and H2 lineages because the developmental fate of female brood depends on the genetic origin of the parents, with interlineage eggs developing into workers and intralineage eggs developing into queens. The study of a mixed mating aggregation revealed strong differences in mating flight timing between P. rugosus and the two lineages as a first mechanism of RI. A second important prezygotic mechanism was assortative mating. Laboratory experiments also provided support for one of the two investigated mechanisms of postzygotic isolation. The majority of offspring produced from the few matings between P. rugosus and the lineages aborted at the egg stage. This hybrid inviability was under maternal influence, with hybrids produced by P. rugosus queens being always inviable whereas a small proportion of H2 lineage queens produced large numbers of adult hybrid offspring. Finally, we found no evidence that genetic caste determination acted as a second postzygotic mechanism reducing gene flow between P. rugosus and the H lineages. The few viable P. rugosus-H hybrids were not preferentially shunted into functionally sterile workers but developed into both workers and queens. Overall, these results reveal that the nearly complete (99.5%) RI between P. rugosus and the two hybrid lineages stems from the combination of two typical prezygotic mechanisms (mating time divergence and assortative mating) and one postzygotic mechanism (hybrid inviability).

Some populations of Pogonomyrmex harvester ants comprise pairs of highly differentiated lineages with queens mating at random with several males of their own and of the alternate lineage. These queens produce two types of diploid offspring, those fertilized by males of the queens' lineage which develop into new queens and those fertilized by males of the other lineage which mostly develop into functionally sterile workers. This unusual mode of genetic caste determination has been found in 26 populations and a total of four lineage pairs (F(1)-F(2), G(1)-G(2), H(1)-H(2) and J(1)-J(2)) have been described in these populations. Despite the fact that a few interlineage queens are produced, previous studies revealed that there is a complete lack of genetic introgression between lineages. Here we quantify the proportion of interlineage queens produced in each of the four lineage pairs and determine the fate of these queens. In the F(1)-F(2), G(1)-G(2) and H(1)-H(2) lineage pairs, interlineage queens were produced by a minority of colonies. These colonies exclusively produced interlineage queens and workers, suggesting that interlineage eggs can develop into queens in these three pairs of lineages in the absence of competition with pure-lineage brood. An analysis of three key stages of the colony life cycle revealed that colonies headed by interlineage queens failed to grow sufficiently to produce reproductive individuals. In laboratory comparisons, interlineage queens produced fewer viable eggs, with the effect that they raised fewer workers and lost more weight per worker produced than pure-lineage queens. In the J(1)-J(2) lineage pair, we did not find a single interlineage queen, raising the possibility that interlineage eggs have completely lost the ability to develop into queens in this lineage pair. Hence, two distinct mechanisms seem to account for the complete lack of between-lineage gene flow in the F(1)-F(2), G(1)-G(2), H(1)-H(2) and J(1)-J(2) lineage pairs.

Background The eusocial Hymenoptera have radiated across a wide range of thermal environments, exposing them to significant physiological stressors. We reconstructed the evolutionary history of three families of Heat Shock Proteins (Hsp90, Hsp70, Hsp40), the primary molecular chaperones protecting against thermal damage, across 12 Hymenopteran species and four other insect orders. We also predicted and tested for thermal inducibility of eight Hsps from the presence of cis-regulatory heat shock elements (HSEs). We tested whether Hsp induction patterns in ants were associated with different thermal environments. Results We found evidence for duplications, losses, and cis-regulatory changes in two of the three gene families. One member of the Hsp90 gene family, hsp83, duplicated basally in the Hymenoptera, with shifts in HSE motifs in the novel copy. Both copies were retained in bees, but ants retained only the novel HSE copy. For Hsp70, Hymenoptera lack the primary heat-inducible orthologue from Drosophila melanogaster and instead induce the cognate form, hsc70-4, which also underwent an early duplication. Episodic diversifying selection was detected along the branch predating the duplication of hsc70-4 and continued along one of the paralogue branches after duplication. Four out of eight Hsp genes were heat-inducible and matched the predictions based on presence of conserved HSEs. For the inducible homologues, the more thermally tolerant species, Pogonomyrmex barbatus, had greater Hsp basal expression and induction in response to heat stress than did the less thermally tolerant species, Aphaenogaster picea. Furthermore, there was no trade-off between basal expression and induction. Conclusions Our results highlight the unique evolutionary history of Hsps in eusocial Hymenoptera, which has been shaped by gains, losses, and changes in cis-regulation. Ants, and most likely other Hymenoptera, utilize lineage-specific heat inducible Hsps, whose expression patterns are associated with adaptive variation in thermal tolerance between two ant species. Collectively, our analyses suggest that Hsp sequence and expression patterns may reflect the forces of selection acting on thermal tolerance in ants and other social Hymenoptera. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0573-0) contains supplementary material, which is available to authorized users.

Genetic caste determination has been described in two populations of Pogonomyrmex harvester ants, each comprising a pair of interbreeding lineages. Queens mate with males of their own and of the alternate lineage and produce two types of diploid offspring, those fertilized by males of the queens' lineage which develop into queens and those fertilized by males of the other lineage which develop into workers. Each of the lineages has been shown to be itself of hybrid origin between the species Pogonomyrmex barbatus and Pogonomyrmex rugosus, which both have typical, environmentally determined caste differentiation. In a large scale genetic survey across 35 sites in Arizona, New Mexico and Texas, we found that genetic caste determination associated with pairs of interbreeding lineages occurred frequently (in 26 out of the 35 sites). Overall, we identified eight lineages with genetic caste determination that always co-occurred in the same complementary lineage pairs. Three of the four lineage pairs appear to have a common origin while their relationship with the fourth remains unclear. The level of genetic differentiation among these eight lineages was significantly higher than the differentiation between P. rugosus and P. barbatus, which questions the appropriate taxonomic status of these genetic lineages. In addition to being genetically isolated from one another, all lineages with genetic caste determination were genetically distinct from P. rugosus and P. barbatus, even when colonies of interbreeding lineages co-occurred with colonies of either putative parent at the same site. Such nearly complete reproductive isolation between the lineages and the species with environmental caste determination might prevent the genetic caste determination system to be swept away by gene flow.

Hybrid speciation occurs when combination of two interspecific genomes results in individuals that are of high fitness but reproductively incompatible with the parental species. Although hybrid speciation is a relatively common source of new species in plants, it appears to be a much rarer occurrence in animal taxa. Here we report on reproductive isolation and range overlap between the rough harvester ant Pogonomyrmex rugosus and two lineages with hybrid genotypes (H 1 and H2). Both lineages obligately interbreed and produce genetically distinct queen and worker offspring, a phenomenon referred to as genetic caste determination (GCD). Diploid offspring produced by gametes of the same lineage develop only into queens, whereas diploid offspring derived from gametes of distinct lineages develop into workers. We investigated small-scale patterns of gene flow between the parent and the two H lineages by sampling along an 80-km transect between a pure P. rugosus population and a two-lineage population. Microsatellite and mitochondrial markers both indicated virtually no gene flow between the parent species and either lineage even at sites where parental and H-lineage colonies co-occurred. The geographic ranges of the parental species and the two-lineage population were essentially parapatric, with a surprisingly narrow band of overlap and evidence of spatial structuring even at microgeographic scales within the transition zone. This suggests that ecological competition with the parent species plays a significant role in determining the evolutionary persistence and current distribution of the hybrid lineages and the genetic caste system.

Caste differentiation and reproductive division of labor are the hallmarks of insect societies [1]. In ants and other social Hymenoptera, development of female larvae into queens or workers generally results from environmentally induced differences in gene expression [2–4]. However, several cases in which certain gene combinations may determine reproductive status have been described in bees [5] and ants [6–9]. We investigated experimentally whether genotype directly influences caste determination in two populations of Pogonomyrmex harvester ants in which genotype-caste associations have been observed. Each population contains two genetic lineages [10]. Queens are polyandrous [11, 12] and mate with males of both lineages [6, 7], but in mature colonies, over 95% of daughter queens have a pure-lineage genome, whereas all workers are of F1 interlineage ancestry [6–8]. We found that this pattern is maintained throughout the colony life cycle, even when only a single caste is being produced. Through controlled crosses, we demonstrate that pure-lineage eggs fail to develop into workers even when interlineage brood are not present. Thus, environmental caste determination in these individuals appears to have been lost in favor of a hardwired genetic mechanism. Our results reveal that genetic control of reproductive fate can persist without loss of the eusocial caste structure.

Division of labor is a key factor in the ecological success of social groups. Recent work suggests that division of labor can emerge even without specific adaptations for task specialization and that it can appear in incipient social groups as a self-organizational property. We investigated experimentally how selection and self-organization may interact during the evolution of division of labor by examining task performance in groups of normally solitary versus normally social ant queens. We created social pairs of colony-founding queens from two populations of the ant Pogonomyrmex californicus, one in which queens are normally solitary and one in which queens form foundress groups, and observed their behavior during nest excavation. In both populations, one of the two queens usually performed most of the excavation, becoming the excavation specialist. We could predict which queen would become the specialist based on their relative propensities to perform the task in other contexts, consistent with a variance-based model of task specialization. The occurrence of specialization even when group members were not adapted to social life suggests that division of labor may well have been present in incipient queen groups. However, division of labor can result in cost skew among group members, and thus, paradoxically, within-group selection may constrain or even reduce specialization. Consistent with this effect, pairs of normally solitary queens were significantly more asymmetrical in their task performance than normally social pairs, in which both queens nearly always performed the behavior to some degree.

This hypothesis-driven laboratory exercise teaches how DNA evidence can be used to investigate an organism's evolutionary history while providing practical modeling of the fundamental processes of gene transcription and translation. We used an inquiry-based approach to construct a laboratory around a nontrivial, open-ended evolutionary question about the relationship of five species of Drosophila. In the course of answering this question, students at the early college biology level learn how the information in DNA can be extracted and used by both the cell and scientists. This dual proximate-ultimate approach introduces students to the techniques of PCR, DNA sequencing, and phylogenetic sequence analysis while simultaneously providing a concrete pen-and-paper model of the cellular processes of transcription and translation. The laboratory has been successfully employed over 3 years with first-year college students and has proven its versatility by being easily adapted to a "dry lab" form with advanced high school students.

The process of reproductive caste determination in eusocial insect colonies is generally understood to be mediated by environmental, rather than genetic factors. We present data demonstrating unexpected genetic differences between reproductive castes in a variant of the rough harvester ant, Pogonomyrmex rugosus var. fuscatus. Across multiple loci, queens were consistently more homozygous than expected, while workers were more heterozygous. Adult colony queens were divided into two highly divergent genetic groups, indicating the presence of two cryptic species, rather than a single population. The observed genetic differences between castes reflect differential representation of heterospecific and conspecific patrilines in these offspring groups. All workers were hybrids; by contrast, winged queens were nearly all pure-species. The complete lack of pure-species workers indicates a loss of worker potential in pure-species female offspring. Hybrids appear to be bipotential, but do not normally develop into reproductives because they are displaced by pure-species females in the reproductive pool. Genetic differences between reproductive castes are expected to be rare in non-hybridizing populations, but within hybrid zones they may be evolutionarily stable and thus much more likely to occur.

A number of hypotheses have been proposed to explain the evolution of group colony foundation (pleometrosis) in ants, but evaluating their importance under natural conditions is difficult. The desert seed-harvester ant, Messor pergandei, shows regional variation in the incidence of pleometrosis. I investigated whether there are ecological differences between the two regions that may explain the observed behavioral divergence. Pleometrosis was significantly associated with reduced precipitation, lower vegetation biomass, and lower adult colony density. These differences are consistent with the hypothesis that additional worker production in group colonies increases foraging success rather than competitive ability. The data also suggest an additional hypothesis, desiccation resistance, that should be explored. Annual variability within regions was high and far exceeded the magnitude of the differences seen between regions. Future laboratory and field experiments should take such variability into account.

Sociality can promote both cooperation and competition. Although we assume social organisms possess adaptations that allow them to form groups and deal with within-group conflicts, these are often subtle and difficult to detect. Using a simple form of sociality displayed by young ant foundresses of the desert seed-harvester ant, Messor pergandei, as a model system, I exploited intraspecific geographic variation in social behaviour to investigate how the social context has altered individual behavioural strategies. I created social groups composed of two normally nonsocial foundresses, normally social foundresses and mixed groups, and compared foundresses' aggression and willingness to invest resources towards colony productivity. Nonsocial foundresses, presumably not adapted to social life, invested significantly more resources into colony growth than normally social foundresses, and experienced reduced survivorship in mixed groups. In contrast, social foundresses displayed two novel competitive behavioural strategies when confronted with nonsocial partners: retaliatory aggression and egg consumption. These results document that the evolution of social behaviour, rather than promoting cooperation, is driven by the evolution of competitive strategies designed to exploit group members and reduce the risk of being exploited by others.

The evolution of cooperative colony foundation (pleometrosis) in ants has been attributed to conversion of extra resources into increased competitive ability. Most cooperative founding species provide these additional resources from internal fat stores; however, in those species that forage for resources, the extent and type of individual investment in multiqueen colonies is not well understood. We compared singlyand group-founded laboratory colonies of the desert leaf-cutter ant Acromyrmex versicolor to investigate how cooperation affects colony survival, foraging success, and worker production. Under laboratory conditions, single foundresses were significantly less likely to initiate a successful symbiotic fungus garden, which inevitably led to colony starvation and death. If gardens were initiated successfully, however, there was no difference in the growth trajectories and foraging patterns between colony types. Cooperation in this species may more likely be maintained by survival benefits than by growth rate differences, which may be constrained in groups by individual and colony-level costs.

Transitions in behaviour across a continuous distribution of organisms can provide valuable information on how variation in behaviour is maintained. We used analyses developed for interspecific hybrid zones to examine geographic variation in colony founding strategy in the desert seed-harvester ant, Messor pergandei. Newly mated females initiate new colonies either alone (haplometrosis) or cooperatively with other foundresses (pleometrosis). The incidence of these founding strategies were surveyed across the species' range and found to occur in geographically distinct regions joined by a narrow transition zone. Foundresses collected from haplometrotic sites were more likely to display aggression and found solitary nests than foundresses from pleometrotic sites, suggesting that geographical variation in metrosis is due to genotypic divergence. Foundresses from transitional sites were generally not aggressive and tended to co-found nests in the laboratory, yet rarely formed associations in the field. Such an abrupt shift in behaviour indicates that variation in colony founding strategy is maintained by selection rather than the result of secondary contact of neutral characters. Level of aggression displays a wider cline than founding strategy and is likely under selection only when accompanied by active strategy preference. Copyright 1998 The Association for the Study of Animal Behaviour. Copyright 1998 The Association for the Study of Animal Behaviour.

Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via adaptive introgression or cause near instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation highlighting issues of current interest and debate. In secondary contact zones it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky Muller model of hybrid incompatibilities requires a broader interpretation. Finally although the incidence of reinforcement remains uncertain this and other interactions in areas of sympatry may have knock on effects on speciation both within and outside regions of hybridization. © 2013 European Society For Evolutionary Biology.

We report the first described non-plant family of TRIMs (terminal-repeat retrotransposons in miniature), which are small nonautonomous LTR retrotransposons, from the whole-genome sequence of the red harvester ant, Pogonomyrmex barbatus (Hymenoptera: Myrmicinae). Members of this retrotransposon family, named PbTRIM, have typical features of plant TRIMs in length and structure, although they share no overall sequence similarity. PbTRIM elements and their solo-LTRs are abundant in the host genome and exhibit an uneven distribution pattern. Elements are preferentially inserted into TA-rich regions with ATAT as the most common pattern of target site duplication (TSD). PbTRIM is most likely mobile as indicated by the young age of many complete elements, the high degree of sequence similarity among elements at different genomic locations, the abundance of elements in the host genome, and the presence of 4-bp target site duplications (TSDs) flanking the elements and solo-LTRs. Many PbTRIM elements and their solo-LTRs are located within or near genes, suggesting their potential roles in restructuring the host genes and genome. Database search, PCR and sequencing analysis revealed the presence of homologous PbTRIM elements in other ant species. The high sequence similarity between elements from distantly related ant species, the incongruence between the phylogenies of PbTRIM and its hosts, and the patchy distribution of the retroelement within the Myrmicinae subfamily indicate possible horizontal transfer events of the retroelement.

Parents can influence the phenotypes of their offspring via a number of mechanisms. In harvester ants, whether female progeny develop into workers or daughter queens is strongly influenced by the age and temperature conditions experienced by their mother, which is associated with variation in maternal ecdysteroid deposition in fertilized eggs. In many insects, juvenile hormone (JH) is antagonistic to ecdysteroid release, suggesting that seasonal and age-based variation in maternal JH titers may explain maternal effects on offspring size and reproductive caste. To test this hypothesis, we artificially increased maternal JH titers with methoprene, a JH analog, in laboratory colonies of two Pogonomyrmex populations exhibiting genetic caste determination. Increasing maternal JH resulted in a 50% increase in worker body size, as well as a sharp reduction in total number of progeny reared, but did not alter the genotype of progeny reared to adulthood. The intergenerational effect of JH manipulation was not mediated by a reduction in ecdysteroid deposition into eggs; instead, changes in egg size, trophic egg availability or brood/worker ratio may have altered the nutritional environment of developing larvae. Egg ecdysteroid content was significantly negatively correlated with natural variation in worker body size, however, suggesting that there are multiple independent routes by which queens can modify offspring phenotypes.

Caste differentiation and division of labour are the hallmarks of insect societies and at the root of their ecological success. Kin selection predicts that caste determination should result from environmentally induced differences in gene expression, a prediction largely supported by empirical data. However, two exceptional cases of genetically determined caste differentiation have recently been found in harvester ants. Here we show that genetic caste determination evolved in these populations after complex hybridization events. We identified four distinct genetic lineages, each consisting of unique blends of the genomes of the parental species, presumably Pogonomyrmex barbatus and P. rugosus. Crosses between lineages H1 and H2 and between J1 and J2 give rise to workers, whereas queens develop from within-lineage matings. Although historical gene flow is evident, genetic exchange among lineages and between lineages and the parental species no longer occurs. This unusual system of caste determination seems to be evolutionarily stable.

The reproductive interests of males and females are not always aligned, leading to sexual conflict over parental investment, rate of reproduction and mate choice. Traits that increase the genetic interests of one sex often occur at the expense of the other, selecting for counter-adaptations leading to antagonistic coevolution. Reproductive conflict is not limited to intraspecific interactions; interspecific hybridization can produce pronounced sexual conflict between males and females of different species, but it is unclear whether such conflict can drive sexually antagonistic coevolution between reproductively isolated genomes. We tested for hybridization-driven sexually antagonistic adaptations in queens and males of the socially hybridogenetic ‘J’ lineages of Pogonomyrmex harvester ants, whose mating system promotes hybridization in queens but selects against it in males. We conducted no-choice mating assays to compare patterns of mating behaviour and sperm transfer between inter- and intra-lineage pairings. There was no evidence for mate discrimination on the basis of pair type, and the total quantity of sperm transferred did not differ between intra- and inter-lineage pairs; however, further dissection of the sperm transfer process into distinct mechanistic components revealed significant, and opposing, cryptic manipulation of copulatory investment by both sexes. Males of both lineages increased their rate of sperm transfer to high-fitness intra-lineage mates, with a stronger response in the rarer lineage for whom mating mistakes are the most likely. By contrast, the total duration of copulation for intra-lineage mating pairs was significantly shorter than for inter-lineage crosses, suggesting that queens respond to prevent excessive sperm loading by prematurely terminating copulation. These findings demonstrate that sexual conflict can lead to antagonistic coevolution in both intra-genomic and inter-genomic contexts. Indeed, the resolution of sexual conflict may be a key determinant of the long-term evolutionary potential of host-dependent reproductive strategies, counteracting the inherent instabilities arising from such systems.

We investigated undertaking behaviour in the desert leaf-cutter ant to determine whether colonies show undertaking specialization, how task specialization is regulated and the consequences of specialization on colony performance. Task specialization has been hypothesized to be a result of internal physiological or genetic factors that govern worker behaviour. On the other hand, it has been suggested that task specialization could be a result of spatial structuring of workers allowing only a subset to have the opportunity to perform certain tasks. To test between these two hypotheses, we experimentally introduced dead ants into laboratory colonies with individually marked workers, and recorded all encounters and undertaking behaviours. Workers demonstrated individual specialization, in that some workers removed the dead ant more frequently than expected from encounter rate. Although individual workers differed in their opportunity to perform undertaking, this did not account for the specialization. These results suggest that undertaking specialization is governed by internal differences among workers. In addition, undertaking specialists removed corpses more successfully, and in the largest colony, more quickly, than nonspecialist workers, indicating that internally governed specialization is an important part of overall colony efficiency. Copyright 1999 The Association for the Study of Animal Behaviour.

SummaryCaste differentiation and division of labor are the hallmarks of social insect colonies [1, 2]. The current dogma for female caste differentiation is that female eggs are totipotent, with morphological and physiological differences between queens and workers stemming from a developmental switch during the larval stage controlled by nutritional and other environmental factors (e.g., [3–8]). In this study, we tested whether maternal effects influence caste differentiation in Pogonomyrmex harvester ants. By conducting crossfostering experiments we identified two key factors in the process of caste determination. New queens were produced only from eggs laid by queens exposed to cold. Moreover, there was a strong age effect, with development into queens occurring only in eggs laid by queens that were at least two years old. Biochemical analyses further revealed that the level of ecdysteroids was significantly lower in eggs developing into queens than workers. By contrast, we found no significant effect of colony size or worker exposure to cold, suggesting that the trigger for caste differentiation may be independent of the quantity and quality of resources provided to larvae. Altogether these data demonstrate that the developmental fate of female brood is strongly influenced by maternal effects in ants of the genus Pogonomyrmex.