Genomic data provide insights into the classification of extant termites.

The higher classification of termites requires substantial revision as the Neoisoptera, the most diverse termite lineage, comprise many paraphyletic and polyphyletic higher taxa. Here, we produce an updated termite classification using genomic-scale analyses. We reconstruct phylogenies under diverse substitution models with ultraconserved elements analyzed as concatenated matrices or within the multi-species coalescence framework. Our classification is further supported by analyses controlling for rogue loci and taxa, and topological tests. We show that the Neoisoptera are composed of seven family-level monophyletic lineages, including the Heterotermitidae Froggatt, Psammotermitidae Holmgren, and Termitogetonidae Holmgren, raised from subfamilial rank. The species-rich Termitidae are composed of 18 subfamily-level monophyletic lineages, including the new subfamilies Crepititermitinae, Cylindrotermitinae, Forficulitermitinae, Neocapritermitinae, Protohamitermitinae, and Promirotermitinae; and the revived Amitermitinae Kemner, Microcerotermitinae Holmgren, and Mirocapritermitinae Kemner. Building an updated taxonomic classification on the foundation of unambiguously supported monophyletic lineages makes it highly resilient to potential destabilization caused by the future availability of novel phylogenetic markers and methods. The taxonomic stability is further guaranteed by the modularity of the new termite classification, designed to accommodate as-yet undescribed species with uncertain affinities to the herein delimited monophyletic lineages in the form of new families or subfamilies.

Inter-clonal competition over queen succession imposes a cost of parthenogenesis on termite colonies.

In social insect colonies, selfish behaviour due to intracolonial conflict among members can result in colony-level costs despite close relatedness. In certain termite species, queens use asexual reproduction for within-colony queen succession but rely on sexual reproduction for worker and alate production, resulting in multiple half-clones of a single primary queen competing for personal reproduction. Our study demonstrates that competition over asexual queen succession among different clone types leads to the overproduction of parthenogenetic offspring, resulting in the production of dysfunctional parthenogenetic alates. By genotyping the queens of 23 field colonies of Reticulitermes speratus, we found that clone variation in the queen population reduces as colonies develop. Field sampling of alates and primary reproductives of incipient colonies showed that overproduced parthenogenetic offspring develop into alates that have significantly smaller body sizes and much lower survivorship than sexually produced alates. Our results indicate that while the production of earlier and more parthenogenetic eggs is advantageous for winning the competition for personal reproduction, it comes at a great cost to the colony. Thus, this study highlights the evolutionary interplay between individual-level and colony-level selection on parthenogenesis by queens.

Solving the 250-year-old mystery of the origin and global spread of the German cockroach, Blattella germanica.

The origin of the German cockroach, Blattella germanica, is enigmatic, in part because it is ubiquitous worldwide in human-built structures but absent from any natural habitats. The first historical records of this species are from ca. 250 years ago (ya) from central Europe (hence its name). However, recent research suggests that the center of diversity of the genus is Asian, where its closest relatives are found. To solve this paradox, we sampled genome-wide markers of 281 cockroaches from 17 countries across six continents. We confirm that B. germanica evolved from the Asian cockroach Blattella asahinai approximately 2,100 ya, probably by adapting to human settlements in India or Myanmar. Our genomic analyses reconstructed two primary global spread routes, one older, westward route to the Middle East coinciding with various Islamic dynasties (~1,200 ya), and another younger eastward route coinciding with the European colonial period (~390 ya). While Europe was not central to the early domestication and spread of the German cockroach, European advances in long-distance transportation and temperature-controlled housing were likely important for the more recent global spread, increasing chances of successful dispersal to and establishment in new regions. The global genetic structure of German cockroaches further supports our model, as it generally aligns with geopolitical boundaries, suggesting regional bridgehead populations established following the advent of international commerce.

Immigration and seasonal bottlenecks: high inbreeding despite high genetic diversity in an oscillating population of Culicoides sonorensis (Diptera: Ceratopogonidae).

Most population genetic studies concern spatial genetic differentiation, but far fewer aim at analyzing the temporal genetic changes that occur within populations. Vector species, including mosquitoes and biting midges, are often characterized by oscillating adult population densities, which may affect their dispersal, selection, and genetic diversity over time. Here, we used a population of Culicoides sonorensis from a single site in California to investigate short-term (intra-annual) and long-term (inter-annual) temporal variation in genetic diversity over a 3 yr period. This biting midge species is the primary vector of several viruses affecting both wildlife and livestock, thus a better understanding of the population dynamics of this species can help inform epidemiological studies. We found no significant genetic differentiation between months or years, and no correlation between adult populations and the inbreeding coefficient (FIS). However, we show that repeated periods of low adult abundance during cooler winter months resulted in recurring bottleneck events. Interestingly, we also found a high number of private and rare alleles, which suggests both a large, stable population, as well as a constant influx of migrants from nearby populations. Overall, we showed that the high number of migrants maintains a high level of genetic diversity by introducing new alleles, while this increased diversity is counterbalanced by recurrent bottleneck events potentially purging unfit alleles each year. These results highlight the temporal influences on population structure and genetic diversity in C. sonorensis and provide insight into factors effecting genetic variation that may occur in other vector species with fluctuating populations.

Complete chromosome-level genome assembly data from the tawny crazy ant, Nylanderia fulva (Mayr) (Hymenoptera: Formicidae).

The tawny crazy ant, Nylanderia fulva (Mayr) (Hymenoptera: Formicidae) has a native range that extends from northern Argentina to southern Brazil. In the U.S.A. this species has often been misidentified as Nylanderia (Paratrechina) pubens or N. cf. pubens and has likely been present in Florida and Texas for several decades [1]. In the early 2000's explosive population growth in Texas and neighboring states drew renewed taxonomic focus. Genetic analyses [2,3] aided in identifying the pest species as N. fulva. This species poses an invasive threat to native flora and fauna and human structures. In its invasive range it has been reported to displace another invasive species, the red imported fire ant. The specimens used for genome sequencing were obtained from the coastal region of Mississippi. DNA was extracted from pupae. The genome data set was deposited to the National Center for Biotechnology Information as submission ID: SUB10775679, Project ID: PRJNA796544, Accession IDs: SAMN24895442 and JAKFQQ000000000. The organism taxid is 613905, locus tag prefixes are L1K79. The assembly, USDA_Nfulva_1.0, was generated in collaboration with Dovetail Genomics (now Cantata Bio) to yield a chromosome-level assembly of 375 Mb with a 15.67 Mb N50 and 78X coverage and revealing 16 putative chromosomes. This high-quality chromosome-level genome assembly was released prior to publication as a public service to the research community.

Post-embryonic development of the female reproductive system of the Neotropical termite Silvestritermes euamignathus (Isoptera: Termitidae: Syntermitinae).

In termites, the maturation of the female reproductive system is progressive following post-embryonic development. In Silvestritermes euamignathus, the imaginal line is characterized by five nymphal instars that develop into the imago, but it can deviate in some instars to neotenic reproductives. In order to understand the rate of development of the female reproductive system throughout post-embryonic development, we analyzed the morphology of the reproductive system of nymphs, characterizing the stage of development and comparing it with neotenics and primary queens. We also followed embryonic development and compared eggs from neotenics and primary queens from incipient and mature colonies. Our results showed that gonadal development follows the post-embryonic instars, and previtellogenic oocytes are present in third-instar nymphs and are retained for the next two successive nymphal instars. The full maturation of the ovaries with vitellogenic oocytes requires molting to either imagos or neotenics. Eggs from neotenics follow the same embryonic development and each stage presents similar volume when compared to those of mature primary queens. Eggs of primary queens from incipient colonies are greater in volume than those of mature primary queens and of neotenics, suggesting an investment in egg quality rather than quantity during colony foundation.

Bacterial Isolates Derived from Nest Soil Affect the Attraction and Digging Behavior of Workers of the Red Imported Fire Ant, Solenopsis invicta Buren.

Populations of monogyne and polygyne red imported fire ants (RIFA), Solenopsis invicta Buren, are distributed throughout the southern United States. This ant species is hazardous to farm animals and workers, damages infrastructure, and depletes native arthropod populations. Colony expansion is affected by several biotic factors, but the effects of soil microbes on ant behavior related to soil excavation within nest sites have not been investigated. Consequently, we cultured bacteria from RIFA nest soils. The effects of individual bacterial isolates and bacterial cell densities on the choice of digging site as well as digging activity of monogyne and polygyne RIFA worker ants were evaluated in two-choice bioassays. Based on phylogenetic analysis, 17 isolates were selected and tested initially at 5 × 108 cells/mL and 20 workers per assay. Firmicutes (Bacillus, Paenibacillus, Brevibacillus) repelled the ants, but Arthrobacter woluwensis strongly attracted ants. Subsequently, the six isolates having the greatest positive or negative effects on ant behavior were evaluated at a lower bacterial cell and worker ant densities. Ant responses to these bacteria generally decreased as cell densities declined to 5 × 106 cells/mL. Observations of ant behavior during a three-hour, two-choice bioassay revealed that ants generally visited both control and bacteria-treated sand prior to making a digging site choice. Our research results indicate that soil bacteria may mediate ant nest expansion or relocation and foraging tunnel construction. Identification of bacterial metabolites that affect RIFA digging behavior merits additional research because these compounds may provide a basis for novel management strategies that repel RIFA away from sensitive infrastructure or attract fire ants to insecticidal baits.

Short and long-term costs of inbreeding in the lifelong-partnership in a termite.

Social life and lifelong partner commitments are expected to favor thorough partner choice, as an ill-suited partnership may have long-term consequences, adversely affecting the parents and spanning several cohorts of offspring. Here, we used ~1400 termite incipient colonies to estimate the short- and long-term costs of inbreeding upon the survival of the parents over a 15-month period, their productivity, and the resistance of their offspring toward pathogen pressure. We observed that foundation success was not influenced by the relatedness of partners, but by their levels of microbial load. We showed faster growth in inbred colonies with low levels of microbial load, revealing a potential tradeoff between pathogen defense and offspring production. Yet, inbreeding takes its toll later in colony development when offspring from incipient colonies face pathogen pressure. Although the success of a lifetime partnership is initially determined by the partner's health, the cost of inbreeding in incipient colonies favors outbred colonies reaching maturity.

Development of microsatellite markers for population genetics of biting midges and a potential tool for species identification of Culicoides sonorensis Wirth & Jones.

BACKGROUND: Proper vector surveillance relies on the ability to identify species of interest accurately and efficiently, though this can be difficult in groups containing cryptic species. Culicoides Latreille is a genus of small biting flies responsible for the transmission of numerous pathogens to a multitude of vertebrates. Regarding pathogen transmission, the C. variipennis species complex is of particular interest in North America. Of the six species within this group, only C. sonorensis Wirth & Jones is a proven vector of bluetongue virus and epizootic hemorrhagic disease virus. Unfortunately, subtle morphological differences, cryptic species, and mitonuclear discordance make species identification in the C. variipennis complex challenging. Recently, single-nucleotide polymorphism (SNP) analysis enabled discrimination between the species of this group; however, this demanding approach is not practical for vector surveillance. METHODS: The aim of the current study was to develop a reliable and affordable way of distinguishing between the species within the C. variipennis complex, especially C. sonorensis. Twenty-five putative microsatellite markers were identified using the C. sonorensis genome and tested for amplification within five species of the C. variipennis complex. Machine learning was then used to determine which markers best explain the genetic differentiation between species. This led to the development of a subset of four and seven markers, which were also tested for species differentiation. RESULTS: A total of 21 microsatellite markers were successfully amplified in the species tested. Clustering analyses of all of these markers recovered the same species-level identification as the previous SNP data. Additionally, the subset of seven markers was equally capable of accurately distinguishing between the members of the C. variipennis complex as the 21 microsatellite markers. Finally, one microsatellite marker (C508) was found to be species-specific, only amplifying in the vector species C. sonorensis among the samples tested. CONCLUSIONS: These microsatellites provide an affordable way to distinguish between the sibling species of the C. variipennis complex and could lead to a better understanding of the species dynamics within this group. Additionally, after further testing, marker C508 may allow for the identification of C. sonorensis with a single-tube assay, potentially providing a powerful new tool for vector surveillance in North America.

RNA meets toxicology: efficacy indicators from the experimental design of RNAi studies for insect pest management.

RNA interference (RNAi) selectively targets genes and silences their expression in vivo, causing developmental defects, mortality and altered behavior. Consequently, RNAi has emerged as a promising research area for insect pest management. However, it is not yet a viable alternative over conventional pesticides despite several theoretical advantages in safety and specificity. As a first step toward a more standardized approach, a machine learning algorithm was used to identify factors that predict trial efficacy. Current research on RNAi for pest management is highly variable and relatively unstandardized. The applied random forest model was able to reliably predict mortality ranges based on bioassay parameters with 72.6% accuracy. Response time and target gene were the most important variables in the model, followed by applied dose, double-stranded RNA (dsRNA) construct size and target species, further supported by generalized linear mixed effect modeling. Our results identified informative trends, supporting the idea that basic principles of toxicology apply to RNAi bioassays and provide initial guidelines standardizing future research similar to studies of traditional insecticides. We advocate for training that integrates genetic, organismal, and toxicological approaches to accelerate the development of RNAi as an effective tool for pest management. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Species delimitation and mitonuclear discordance within a species complex of biting midges.

The inability to distinguish between species can be a serious problem in groups responsible for pathogen transmission. Culicoides biting midges transmit many pathogenic agents infecting wildlife and livestock. In North America, the C. variipennis species complex contains three currently recognized species, only one of which is a known vector, but limited species-specific characters have hindered vector surveillance. Here, genomic data were used to investigate population structure and genetic differentiation within this species complex. Single nucleotide polymorphism data were generated for 206 individuals originating from 17 locations throughout the United States and Canada. Clustering analyses suggest the occurrence of two additional cryptic species within this complex. All five species were significantly differentiated in both sympatry and allopatry. Evidence of hybridization was detected in three different species pairings indicating incomplete reproductive isolation. Additionally, COI sequences were used to identify the hybrid parentage of these individuals, which illuminated discordance between the divergence of the mitochondrial and nuclear datasets.

Cloning and Functional Characterization of a Double-Stranded RNA-Degrading Nuclease in the Tawny Crazy Ant (Nylanderia fulva).

RNA interference is a powerful tool that post-transcriptionally silences target genes. However, silencing efficacy varies greatly among different insect species. Recently, we attempted to knock down some housekeeping genes in the tawny crazy ant (Nylanderia fulva), a relatively new invasive species in the southern United States, but only achieved relatively low silencing efficiency when dsRNA was orally administered. Here, we detected divalent cation-dependent, dsRNA-degrading activity in the midgut fluid of worker ants in ex vivo assays. To determine whether dsRNA degradation could contribute to low effectiveness of oral RNAi in N. fulva, we cloned its sole dsRNase gene (NfdsRNase). The deduced amino acid sequence contained a signal peptide and an endonuclease domain. Sequence alignment indicated a high degree of similarity with well-characterized dsRNases, particularly the six key residues at active sites. We also identified dsRNase homologs from five other ant species and found a tight phylogenetic relationship among ant dsRNases. NfdsRNase is expressed predominantly in the abdomen of worker ants. Oral delivery of dsRNA of NfdsRNase significantly reduced the expression of NfdsRNase transcripts, and substantially suppressed dsRNA-degrading activity of worker ants' midgut fluids as well. Our data suggest that dsRNA stability in the alimentary tract is an important factor for gene silencing efficiency in N. fulva, and that blocking NfdsRNase in gut lumen could potentially improve RNAi, a novel pest management tactic in control of N. fulva and other ant species.

Assessing colony elimination in multicolonial ants: Estimating field efficacy of insecticidal baits against the invasive dark rover ant (Brachymyrmex patagonicus).

BACKGROUND: A frequent goal of pest management strategies targeting social insects is total colony elimination. Insecticidal baits are highly effective at controlling social insect pests, although their ability to provide total colony elimination has only been well studied in a few species. Genetically testing colony elimination in many urban pest ants can be challenging due to indistinct colony boundaries observed in unicolonial, invasive species; however, some pest ants, such as the dark rover ant (Brachymyrmex patagonicus), maintain strict colony borders through aggression towards non-nestmates. Each of these distinct colonies can be identified using molecular markers, allowing for the tracking of individual colonies pre- and post-treatment to measure colony density. While counting the number of foraging workers to assess treatment efficacy may suffice in some cases, it offers little insight into the colony-level impacts of a treatment. RESULTS: Using microsatellite markers, distinct rover ant colonies were identified and tracked around residential structures before and after the application of an imidacloprid bait. The number of foraging ants at the treated structures was reduced by an average of 83.0% over a 28-day observation period. Baiting also significantly reduced the total number of colonies present. At the treatment structures, only ~25% of the original colonies remained at the end of the study. Colonies with foraging trails <1.5 m from a bait station had a higher chance of being eliminated. CONCLUSION: Using insecticidal baits against B. patagonicus can be highly effective at colony elimination; however, with such small foraging ranges and high colony densities, proper placement is required to ensure enough bait is properly positioned to treat all colonies affecting a structure. © 2022 Society of Chemical Industry.

Consistent signatures of urban adaptation in a native, urban invader ant Tapinoma sessile.

Biological invasions are becoming more prevalent due to the rise of global trade and expansion of urban areas. Ants are among the most prolific invaders with many exhibiting a multiqueen colony structure, dependent colony foundation and reduced internest aggression. Although these characteristics are generally associated with the invasions of exotic ants, they may also facilitate the spread of native ants into novel habitats. Native to diverse habitats across North America, the odorous house ant Tapinoma sessile has become abundant in urban environments throughout the United States. Natural colonies typically have a small workforce, inhabit a single nest, and are headed by a single queen, whereas urban colonies tend to be several orders of magnitude larger, inhabit multiple nests (i.e., polydomy) and are headed by multiple queens (i.e., polygyny). Here, we explore and compare the population genetic and breeding structure of T. sessile within and between urban and natural environments in several localities across its distribution range. We found the social structure of a colony to be a plastic trait in both habitats, although extreme polygyny was confined to urban habitats. Additionally, polydomous colonies were only present in urban habitats, suggesting T. sessile can only achieve supercoloniality within urbanized areas. Finally, we identified strong differentiation between urban and natural populations in each locality and continent-wide, indicating cities may restrict gene flow and exert intense selection pressure. Overall, our study highlights urbanization's influence in charting the evolutionary course for species.

Exotic Ants of the Asia-Pacific: Invasion, National Response, and Ongoing Needs.

Human activity has facilitated the introduction of many exotic species via global trade. Asia-Pacific countries comprise one of the most economically and trade-active regions in the world, which makes it an area that is highly vulnerable to invasive species, including ants. There are currently over 60 exotic ant species in the Asia-Pacific, with the red imported fire ant, Solenopsis invicta, among the most destructive. Exotic ants pose many economic and ecological problems for the region. Countries in the Asia-Pacific have dealt with the problem of exotic ants in very different ways, and there has been an overall lack of preparedness. To improve the management of risks associated with invasive ants, we recommend that countries take action across the biosecurity spectrum, spanning prevention, containment, and quarantine. The creation of an Asia-Pacific network for management of invasive ants should help prevent their introduction and mitigate their impacts.

Distinct colony boundaries and larval discrimination in polygyne red imported fire ants (Solenopsis invicta).

Evaluating the factors that promote invasive ant abundance is critical to assess their ecological impact and inform their management. Many invasive ant species show reduced nestmate recognition and an absence of boundaries between unrelated nests, which allow populations to achieve greater densities due to reduced intraspecific competition. We examined nestmate discrimination and colony boundaries in introduced populations of the red imported fire ant (Solenopsis invicta; hereafter, fire ant). Fire ants occur in two social forms: monogyne (colonies with a single egg-laying queen) and polygyne (colonies with multiple egg-laying queens). In contrast with monogyne nests, polygyne nests are thought to be interconnected due to the reduced antagonism between non-nestmate polygyne workers, perhaps because polygyne workers habituate the colony to an odour unique to Gp-9b -carrying adults. However, colony boundaries and nestmate discrimination are poorly documented, particularly for worker-brood interactions. To delimit boundaries between field colonies, we correlated the exchange of a 15 N-glycine tracer dissolved in a sucrose solution with social form. We also evaluated nestmate discrimination between polygyne workers and larvae in the laboratory. Counter to our expectations, polygyne colonies behaved identically to monogyne colonies, suggesting both social forms maintain strict colony boundaries. Polygyne workers also preferentially fed larval nestmates and may have selectively cannibalized non-nestmates. The levels of relatedness among workers in polygyne colonies was higher than those previously reported in North America (mean ± standard error: 0.269 ± 0.037). Our study highlights the importance of combining genetic analyses with direct quantification of resource exchange to better understand the factors influencing ant invasions.

Colonization by the Red Imported Fire Ant, Solenopsis invicta, Modifies Soil Bacterial Communities.

The long-standing association between insects and microorganisms has been especially crucial to the evolutionary and ecological success of social insect groups. Notably, research on the interaction of the two social forms (monogyne and polygyne) of the red imported fire ant (RIFA), Solenopsis invicta Buren, with microbes in its soil habitat is presently limited. In this study, we characterized bacterial microbiomes associated with RIFA nest soils and native (RIFA-negative) soils to better understand the effects of colonization of RIFA on soil microbial communities. Bacterial community fingerprints of 16S rRNA amplicons using denaturing gradient gel electrophoresis revealed significant differences in the structure of the bacterial communities between RIFA-positive and RIFA-negative soils at 0 and 10 cm depths. Illumina sequencing of 16S rRNA amplicons provided fine-scale analysis to test for effects of RIFA colonization, RIFA social form, and soil depth on the composition of the bacterial microbiomes of the soil and RIFA workers. Our results showed the bacterial community structure of RIFA-colonized soils to be significantly different from native soil communities and to evidence elevated abundances of several taxa, including Actinobacteria. Colony social form was not found to be a significant factor in nest or RIFA worker microbiome compositions. RIFA workers and nest soils were determined to have markedly different bacterial communities, with RIFA worker microbiomes being characterized by high abundances of a Bartonella-like endosymbiont and Entomoplasmataceae. Cloning and sequencing of the 16S rRNA gene revealed the Bartonella sp. to be a novel bacterium.

Development of a Set of Microsatellite Markers to Investigate Sexually Antagonistic Selection in the Invasive Ant Nylanderia fulva.

Sexually antagonistic selection (SAS) occurs when distinct alleles are differentially selected in each sex. In the invasive tawny crazy ant, Nylanderia fulva, a genomic region is under SAS, while the rest of the genome is randomly selected in males and females. In this study, we designed a suite of 15 microsatellite markers to study the origin and evolution of SAS in N. fulva. These SAS markers were polymorphic, with allelic frequencies that are highly different between males and females. All haploid males carry only a subset of the alleles present in the population, while females are reliably heterozygous, with one allele from the male gene pool and a different allele inherited from their mother. In addition, we identified six polymorphic markers not associated with SAS and six markers yielding consistent, yet monomorphic, amplification in the introduced range of this species. Reaction condition optimizations allowed all retained markers to be co-amplified in four PCR mixes. The SAS markers may be used to test for the strength and the extent of the genomic regions under SAS in both the native and introduced ranges of N. fulva, while the set of non-SAS loci may be used to assess the invasion route of this species. Overall, the application of these microsatellite markers will yield insights into the origin and evolution of SAS within and among species of the genus Nylanderia.

Extensive human-mediated jump dispersal within and across the native and introduced ranges of the invasive termite Reticulitermes flavipes.

As native ranges are often geographically structured, invasive species originating from a single source population only carry a fraction of the genetic diversity present in their native range. The invasion process is thus often associated with a drastic loss of genetic diversity resulting from a founder event. However, the fraction of diversity brought to the invasive range may vary under different invasion histories, increasing with the size of the propagule, the number of reintroduction events, and/or the total genetic diversity represented by the various source populations in a multiple-introduction scenario. In this study, we generated a SNP data set for the invasive termite Reticulitermes flavipes from 23 native populations in the eastern United States and six introduced populations throughout the world. Using population genetic analyses and approximate Bayesian computation random forest, we investigated its worldwide invasion history. We found a complex invasion pathway with multiple events out of the native range and bridgehead introductions from the introduced population in France. Our data suggest that extensive long-distance jump dispersal appears common in both the native and introduced ranges of this species, probably through human transportation. Overall, our results show that similar to multiple introduction events into the invasive range, admixture in the native range prior to invasion can potentially favour invasion success by increasing the genetic diversity that is later transferred to the introduced range.

Divide and conquer: Multicolonial structure, nestmate recognition, and antagonistic behaviors in dense populations of the invasive ant Brachymyrmex patagonicus.

The ecological success of ants has made them abundant in most environments, yet inter- and intraspecific competition usually limit nest density for a given population. Most invasive ant populations circumvent this limitation through a supercolonial structure, eliminating intraspecific competition through a loss of nestmate recognition and lack of aggression toward non-nestmates. Native to South America, Brachymyrmex patagonicus has recently invaded many locations worldwide, with invasive populations described as extremely large and dense. Yet, in contrast with most invasive ants, this species exhibits a multicolonial structure, whereby each colony occupies a single nest. Here, we investigated the interplay between genetic diversity, chemical recognition, and aggressive behaviors in an invasive population of B. patagonicus. We found that, in its invasive range, this species reaches a high nest density with individual colonies located every 2.5 m and that colony boundaries are maintained through aggression toward non-nestmates. This recognition and antagonism toward non-nestmates is mediated by chemical differentiation between colonies, as different colonies exhibit distinct chemical profiles. We highlighted that the level of aggression between colonies is correlated with their degree of genetic difference, but not their overall chemical differentiation. This may suggest that only a few chemical compounds influence nestmate recognition in this species or that weak chemical differences are sufficient to elicit aggression. Overall, this study demonstrates that invasive ant populations can reach high densities despite a multicolonial structure with strong aggression between colonies, raising questions about the factors underlying their ecological success and mitigating negative consequences of competitive interactions.