"The Reference Genome Of The Kidnapper Ant, Polyergus Mexicanus".

Polyergus kidnapper ants are widely distributed, but relatively uncommon, throughout the Holarctic, spanning an elevational range from sea level to over 3000 m. These species are well known for their obligate social parasitism with various Formica ant species, which they kidnap in dramatic, highly coordinated raids. Kidnapped Formica larvae and pupae become integrated into the Polyergus colony where they develop into adults and perform nearly all of the necessary colony tasks for the benefit of their captors. In California, Polyergus mexicanus is the most widely distributed Polyergus, but recent evidence has identified substantial genetic polymorphism within this species, including genetically divergent lineages associated with the use of different Formica host species. Given its unique behavior and genetic diversity, Polyergus mexicanus plays a critical role in maintaining ecosystem balance by influencing the population dynamics and genetic diversity of its host ant species, Formica, highlighting its conservation value and importance in the context of biodiversity preservation. Here, we present a high-quality genome assembly of P. mexicanus from a sample collected in Plumas County, CA, USA, in the foothills of the central Sierra Nevada. This genome assembly consists of 364 scaffolds spanning 252.31 Mb, with contig N50 of 481,250 kb, scaffold N50 of 10.36 Mb, and BUSCO completeness of 95.4%. We also assembled the genome of the Wolbachia endosymbiont of P. mexicanus - a single, circular contig spanning 1.23 Mb. These genome sequences provide essential resources for future studies of conservation genetics, population genetics, speciation, and behavioral ecology in this charismatic social insect.

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

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

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

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

Reference genome of the bicolored carpenter ant, Camponotus vicinus.

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

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

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

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

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

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

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

The Ant Who Cried Wolf? Short-Term Repeated Exposure to Alarm Pheromone Reduces Behavioral Response in Argentine Ants.

In this study we test whether Argentine ants (Linepithema humile) progressively reduce their response to a salient stimulus (alarm pheromone) with increased exposure over time. First, we used a two-chamber olfactometer to demonstrate three focal behaviors of Argentine ants that indicate an alarmed state in response to conspecific alarm pheromone and pure synthetic iridomyrmecin (a dominant component of L. humile alarm pheromone). We then measured how these behaviors changed after repeated exposure to conspecific alarm pheromone from live ants. In addition, we investigate whether there is a difference in the ants' behavioral response after "short" (3 min) versus "long" (6 min) intervals between treatments. Our results show that Argentine ants do exhibit reduced responses to their own alarm pheromone, temporarily ceasing their response to it after four or five exposures, and this pattern holds whether exposure is repeated after "short" or "long" intervals. We suggest alarm pheromones may be perceived as false alarms unless threatening stimuli warrant a continued state of alarm. These results should be kept in mind while developing pheromone-based integrated pest management strategies.

Correction: A Species delimitation approach to uncover cryptic species in the South American fire ant decapitating flies (Diptera: Phoridae: Pseudacteon).

[This corrects the article DOI: 10.1371/journal.pone.0236086.].

Transcriptomic signatures of cold adaptation and heat stress in the winter ant (Prenolepis imparis).

Climate change is a serious threat to biodiversity; it is therefore important to understand how animals will react to this stress. Ectotherms, such as ants, are especially sensitive to the climate as the environmental temperature influences myriad aspects of their biology, from optimal foraging time to developmental rate. In this study, we conducted an RNA-seq analysis to identify stress-induced genes in the winter ant (Prenolepis imparis). We quantified gene expression during heat and cold stress relative to a control temperature. From each of our conditions, we sequenced the transcriptome of three individuals. Our de novo assembly included 13,324 contigs that were annotated against the nr and SwissProt databases. We performed gene ontology and enrichment analyses to gain insight into the physiological processes involved in the stress response. We identified a total of 643 differentially expressed genes across both treatments. Of these, only seven genes were differentially expressed in the cold-stressed ants, which could indicate that the temperature we chose for trials did not induce a strong stress response, perhaps due to the cold adaptations of this species. Conversely, we found a strong response to heat: 426 upregulated genes and 210 downregulated genes. Of these, ten were expressed at a greater than ten-fold change relative to the control. The transcripts we could identify included those encoding for protein folding genes, heat shock proteins, histones, and Ca2+ ion transport. One of these transcripts, hsc70-4L was found to be under positive selection. We also characterized the functional categories of differentially expressed genes. These candidate genes may be functionally conserved and relevant for related species that will deal with rapid climate change.

A Species delimitation approach to uncover cryptic species in the South American fire ant decapitating flies (Diptera: Phoridae: Pseudacteon).

South American fire ant decapitating flies in the genus Pseudacteon (Diptera: Phoridae) are potential biocontrol agents of the invasive fire ants Solenopsis invicta and S. richteri in the United States and other regions of the world due to their high host specificity and the direct and indirect damage to their host ants. Despite their importance and the fact that several flies have already been released in the US, little is known about the genetic variability and phylogenetic relationships of Pseudacteon flies parasitizing South American fire ants in the Solenopsis saevissima species-group. A species delimitation analysis was conducted using a distance-based method (ABGD) and two tree-based methods (GMYC and mPTP) using COI sequences of 103 specimens belonging to 20 of the 22 Pseudacteon species known from southern South America. Additionally, phylogenetic relationships between the already described and new candidate species were inferred using mitochondrial (COI) and nuclear (wingless) sequences. The species delimitation analysis suggests that species richness in these flies has been previously underestimated, due to the existence of putative cryptic species within nominal Pseudacteon obtusus, P. pradei, P. tricuspis, P. cultellatus, and P. nudicornis. Geographic distribution and host fire ant species seem to support cryptic lineages, though additional morphological data are needed to corroborate these results. All phylogenetic analyses reveal that South American fire ant decapitating flies are grouped into two main clades, with Pseudacteon convexicauda sister and well differentiated relative to these clades. Neither host nor geographic association appeared to be related to the differentiation of these two main clades within South American fire ant decapitating flies. This work provides information that will allow testing whether the putative cryptic phorid fly species show differences in their effectiveness as biocontrol agents against the highly invasive imported fire ants.

Intraspecific variation in thermal acclimation and tolerance between populations of the winter ant, Prenolepis imparis.

Thermal phenotypic plasticity, otherwise known as acclimation, plays an essential role in how organisms respond to short-term temperature changes. Plasticity buffers the impact of harmful temperature changes; therefore, understanding variation in plasticity in natural populations is crucial for understanding how species will respond to the changing climate. However, very few studies have examined patterns of phenotypic plasticity among populations, especially among ant populations. Considering that this intraspecies variation can provide insight into adaptive variation in populations, the goal of this study was to quantify the short-term acclimation ability and thermal tolerance of several populations of the winter ant, Prenolepis imparis. We tested for correlations between thermal plasticity and thermal tolerance, elevation, and body size. We characterized the thermal environment both above and below ground for several populations distributed across different elevations within California, USA. In addition, we measured the short-term acclimation ability and thermal tolerance of those populations. To measure thermal tolerance, we used chill-coma recovery time (CCRT) and knockdown time as indicators of cold and heat tolerance, respectively. Short-term phenotypic plasticity was assessed by calculating acclimation capacity using CCRT and knockdown time after exposure to both high and low temperatures. We found that several populations displayed different chill-coma recovery times and a few displayed different heat knockdown times, and that the acclimation capacities of cold and heat tolerance differed among most populations. The high-elevation populations displayed increased tolerance to the cold (faster CCRT) and greater plasticity. For high-temperature tolerance, we found heat tolerance was not associated with altitude; instead, greater tolerance to the heat was correlated with increased plasticity at higher temperatures. These current findings provide insight into thermal adaptation and factors that contribute to phenotypic diversity by revealing physiological variance among populations.

The evolution of species recognition labels in insects.

The evolution of pre-zygotic reproductive isolation is a key step in the process of speciation. In many organisms, particularly insects, chemical labels are used as pheromones for species-specific mate recognition. Although an enormous body of knowledge exists regarding the patterns of pheromone chemical ecology, much less is known about the evolutionary processes that underlie the origin of new mating pheromones. Here, we examine case studies that have illuminated the origins of species-specific mating pheromones and suggest future directions for productive research. This article is part of the theme issue 'Signal detection theory in recognition systems: from evolving models to experimental tests'.

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

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

Behavioural variation and plasticity along an invasive ant introduction pathway.

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

Genome Sequencing of Museum Specimens Reveals Rapid Changes in the Genetic Composition of Honey Bees in California.

The western honey bee, Apis mellifera, is an enormously influential pollinator in both natural and managed ecosystems. In North America, this species has been introduced numerous times from a variety of different source populations in Europe and Africa. Since then, feral populations have expanded into many different environments across their broad introduced range. Here, we used whole genome sequencing of historical museum specimens and newly collected modern populations from California (USA) to analyze the impact of demography and selection on introduced populations during the past 105 years. We find that populations from both northern and southern California exhibit pronounced genetic changes, but have changed in different ways. In northern populations, honey bees underwent a substantial shift from western European to eastern European ancestry since the 1960s, whereas southern populations are dominated by the introgression of Africanized genomes during the past two decades. Additionally, we identify an isolated island population that has experienced comparatively little change over a large time span. Fine-scale comparison of different populations and time points also revealed SNPs that differ in frequency, highlighting a number of genes that may be important for recent adaptations in these introduced populations.

Genetic and chemical divergence among host races of a socially parasitic ant.

Host-parasite associations facilitate the action of reciprocal selection and can drive rapid evolutionary change. When multiple host species are available to a single parasite, parallel specialization on different hosts may promote the action of diversifying natural selection and divergence via host race formation. Here, we examine a population of the kidnapper ant (Polyergus mexicanus) that is an obligate social parasite of three sympatric ant species: Formica accreta, F. argentea, and F. subaenescens (formerly F. fusca). Behavioral and ecological observations of P. mexicanus have shown that individual colonies parasitize only one species of host and that new Polyergus queens maintain host fidelity when establishing new colonies. To successfully adapt to a particular host, Polyergus ants may mimic or camouflage themselves with the species-specific chemical cues (cuticular hydrocarbons) that their hosts use to ascertain colony membership. To investigate the extent of host specialization, we collected both genetic and chemical data from P. mexicanus that parasitize each of the three different Formica species in sympatry. We show that host-associated genetic structure exists for both maternally inherited mitochondrial DNA data and biparentally inherited microsatellite markers. We also show that P. mexicanus can be distinguished by chemical profile according to host due to partial matching with their host. Our results support the hypothesis that host race formation is occurring among lineages of P. mexicanus that use different Formica hosts. Thus, this system may represent a promising model for illuminating the early steps of divergence, accumulation of reproductive isolation, and speciation.

The Complex Demographic History and Evolutionary Origin of the Western Honey Bee, Apis Mellifera.

The western honey bee, Apis mellifera, provides critical pollination services to agricultural crops worldwide. However, despite substantial interest and prior investigation, the early evolution and subsequent diversification of this important pollinator remain uncertain. The primary hypotheses place the origin of A. mellifera in either Asia or Africa, with subsequent radiations proceeding from one of these regions. Here, we use two publicly available whole-genome data sets plus newly sequenced genomes and apply multiple population genetic analysis methods to investigate the patterns of ancestry and admixture in native honey bee populations from Europe, Africa, and the Middle East. The combination of these data sets is critical to the analyses, as each contributes samples from geographic locations lacking in the other, thereby producing the most complete set of honey bee populations available to date. We find evidence supporting an origin of A. mellifera in the Middle East or North Eastern Africa, with the A and Y lineages representing the earliest branching lineages. This finding has similarities with multiple contradictory hypotheses and represents a disentangling of genetic relationships, geographic proximity, and secondary contact to produce a more accurate picture of the origins of A. mellifera. We also investigate how previous studies came to their various conclusions based on incomplete sampling of populations, and illustrate the importance of complete sampling in understanding evolutionary processes. These results provide fundamental knowledge about genetic diversity within Old World honey bee populations and offer insight into the complex history of an important pollinator.

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

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

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

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