Our large-scale genetic and behavioral analyses of the invasive tawny crazy ant provide several new insights into the biological invasion and social system of this ant in its native and invasive (U.S.A.) ranges. Genetic data suggest this ant species experienced a genetic bottleneck following its introduction in the U.S.A. that led to a significant reduction (60%) in genetic diversity. Population genetic analyses show that N. fulva exhibits a multicolonial organization in its native range, with colonies genetically distinct from each another. In contrast, we found that this species displays a unicolonial system with no clear boundaries between nests in its invasive range. This latter finding is supported by the lack of genetic differentiation among nests within populations as well as between geographic populations, and relatedness coefficients among nestmate workers close to zero in introduced populations. Each invasive nest was headed by several, up to hundreds of singly-mated reproductive queens. Behavioral tests reveal no aggressive behaviors toward conspecifics from different nests, even ones separated by several hundred kilometers. Overall, these results suggest that the entire U.S.A. range of the species forms a single large supercolony spreading more than 2000 km.

Population bottleneck and inbreeding

In our study, we uncovered a loss of genetic diversity between native and introduced populations. Such reduction may be particularly costly for hymenopteran species because of their sex determination system. In these species, the sex of an individual is controlled by a single complementary sex-determining locus (multi-locus CSD is known but rare [47]). Heterozygous individuals at this locus develop into females while homozygous individuals develop into males. Females are diploid heterozygous individuals usually produced by sexual reproduction, whereas males arise from unfertilized eggs through arrhenotokous parthenogenesis, and are therefore haploid (i.e., thus homozygous) individuals [48]. However, diploid individuals, homozygous at this locus, which can result from mating between individuals carrying the same sex allele (matched mating), develop as diploid males. Production of diploid males represents a cost for colonies because they are effectively sterile in most hymenopteran species [48,49,50,51]. Loss of allelic diversity at the sex locus as a result of a population bottleneck significantly increases the chances of matched matings [52]. As one example, in the introduced populations of the red fire ant Solenopsis invicta, colonies produce a higher proportion of diploid males than those from native populations [53, 54].

Some ant species have however evolved unorthodox reproductive modes, which may facilitate invasiveness by acting as pre-adaptations against the genetic loss due to bottlenecks during invasions [55]. In some populations of four invasive ant species, Wasmannia auropunctata, Vollenhovia emeryi, Anoplolepis gracilipes and Paratrechina longicornis, queens are clones of their mothers and males are clones of their fathers, whereas workers arise from classical sexual reproduction [35, 55,56,57]. Male and female gene pools are completely segregated, even those produced by the same mother queen [58, 59]. In these species, a single-mated queen may thus establish an introduced population, producing 100% heterozygous workers. This queen may also produce new queens and males able to mate together inside the nest; yet still maintain heterozygosity in their worker offspring. Clonality was recently recorded in the native range of W. auropunctata in southern South America [60, 61]. This strategy thus circumvents the costs of inbreeding after an introduction event over an unlimited number of generations [55, 59] and act as a pre-adaptive trait to invasion. In our study, the level of heterozygosity is not significantly different between workers and queens of N. fulva, indicating that they are both produced through classic sexual reproduction.

Formation of supercolonies seems to be a common trait of invasive social insects [22, 62], allowing a rapid and efficient monopolization of resources to achieve local dominance, mainly in the introduced range where the competitive pressure exerted by the members of local ant community is lower than in the native range [2, 8, 63, 64]. Supercolonies have been reported numerous times in various invasive ant species, such as Linepithema humile [9], Monomorium pharaonis [65], Pheidole megacephala [32], Anoplolepis gracilipes [66], and Lasius neglectus [67], and has also been reported in invasive populations of the termite Reticulitermes urbis [68]. Interestingly, the sizes of supercolonies apparently varies considerably among species, and occasionally even within species. For example, the invasive big-headed ant, P. megacephala, forms a single large supercolony covering up to 3000 km across northeastern Australia [32]. In contrast, the yellow crazy ant, A. gracilipes, inhabiting a small geographic area in northeastern Borneo, comprises at least six supercolonies [66]. In L. humile, two supercolonies are reported in the invasive range in southern Europe; one supercolony is 6000 km long, while the other is only a few km long [9]. In this same species, the invasive area of California comprises at least five supercolonies ranging in areas from 1 to 1000 km [69], while four supercolonies were uncovered in Japan [27, 70], and several in the southeastern U.S.A. [71]. Overall, these results suggest supercoloniality is a common trait in social insects, but the number and the size of their supercolonies can differ greatly among and within species.

Despite the lack of aggressiveness within supercolonies, genetic identities of two adjoining supercolonies can be maintained because workers display strong aggression towards allocolonial sexuals and workers at colony boundaries, as observed in the Argentine ant [69, 72, 73]. This aggression towards allocolonial sexuals strongly reduces potential for mating between partners from distinct supercolonies. Thus, gene flow is reduced between abutting supercolonies, resulting in maintenance of genetic distinctiveness even after prolonged contact with one another [9, 21, 69, 74]. Supercolony differentiation has been suggested to come about one of two ways. On the one hand, supercolonial structure may stem from an initial colony differentiation, in which different supercolonies came from multiple introductions. These distinct introductions from genetically and chemically differentiated source populations are more likely to result in distinct supercolonies in the invasive range [75, 76]. For example, the worldwide supercolonies of the Argentine ant originated from at least seven founding events out of the native area in Argentina [75]. The dominant supercolonies of Europe, Japan and California probably arose from the same primary introduction [75] and consist of a single supercolony that globally expanded through secondary introductions, since these populations are not aggressive toward each other [76] and have similar hydrocarbon profiles [77]. On the other hand, supercolony differentiation may occur through divergence after introduction. Queen recruitment, intranidal mating and female dispersal through budding may lead to a reduction of gene flow between geographically separated fragments of the same initial supercolony. Over time, the accumulation of genetic and cuticular hydrocarbon (CHC) differentiation may result in mutual aggression between fragments [78]. In the introduced population of L. humile in Corsica, the clear reduction of gene flow between the island and the mainland supercolonies has led to noticeable chemical and behavioral differentiation [79]. A similar pattern has been reported in a single population in the Californian invasive range, yet coming from the introduction event that gave rise to the other supercolonies [80], and in A. gracilipes in Borneo, in which spatial separation has enhanced genetic and CHC differentiation over time [78]. Eventually, these cases may result in allopatric fragmentation if enough differentiation occurs before both fragments come into contact again. However, no pattern of isolation by distance has been found within supercolonies of other invasive ant species [9, 34, 71, 74], suggesting that gene flow is high enough in these supercolonies to prevent differentiation among geographically distant areas within supercolonies.

In N. fulva, no abrupt genetic transition was discovered across all introduced N. fulva populations studied, suggesting that this species forms a single large supercolony from Texas to Florida. We uncovered a weak pattern of isolation by distance in the introduced range, several orders of magnitude lower than that in the native range. This result may stem from the absence of nuptial flights in N. fulva and its invasion front expansion through budding. These features usually lead to a genetic population viscosity, which may, over time, result in population differentiation. The invasion of N. fulva in the U.S.A. is recent, and may not have had sufficient time to induce genetic differentiation between localities and split the invasive range into distinct supercolonies. Although we cannot exclude that other supercolonies of N. fulva are present but were not sampled, our results suggest that introduced populations in the U.S.A. may come from a single introduction from South America, which then spread through human mediated jump dispersal. The hypothesis of a single introduction is also supported by the positive relatedness (r w-w = 0.16) observed when the global population is taken as reference.

Unicoloniality results in several ecological advantages in terms of colony growth, nest density, productivity and survival, and may favor invasive success outcompeting native species through resource monopolization [2]. But on the other side of the coin, unicoloniality reduces to zero the relatedness between nestmate workers, and, thus, the workers’ indirect benefits from helping. In this context, selfish behaviors are expected to disrupt social cohesion within colonies [81, 82]. For this reason, unicoloniality might represent an evolutionary dead-end; an idea supported by the fact that there is no unicolonial species but only unicolonial populations, and by its scattered distribution along the ant phylogeny [22]. In N. fulva, the relatedness between nestmate workers did not differ from zero in the introduced range, while it varied from 0.29 to 0.86 in the native range. A similar loss of relatedness has also been reported in several supercolonial populations of the species L. humile, L. neglectus, P. megacephala, and S. invicta [2, 32, 83, 84]. However, most of these species were comprised of several genetically distinct supercolonies, with members of the same colony more related to each other than to members of other supercolonies. In this case, it is important to measure relatedness with respect to the local competing population rather than to the global population [22]. Taking the two supercolonies of L. humile in the Southern European range as an example, it is unlikely that two workers separated by thousands of kilometers still compete with each other. Therefore, in most parts of the supercolony, workers most likely compete with colonymates; while selection for altruism should only take place at colony boundaries [22]. In our study, the whole introduced range seems to comprise a single supercolony, even if workers within the introduced range are more related to each other than to workers from native populations, introduced workers do not compete with native workers, making introduced relatedness equivalent to zero. In contrast, several supercolonies of A. gracilipes inhabit the island of Borneo [66]. In this limited area, workers of a given supercolony are more likely to compete against workers from other supercolonies. The relatedness coefficients observed in this species are quite high, making social cohesion sustainable when supercolony size is reduced [35]. Actually, supercolonies of smaller size are uncovered in native populations of the Argentine ant [9, 21, 34, 85, 86] and the little fire ant [61, 87, 88]. In noninvasive species, the turnover of supercolonies suggests the occurrence of local competition [89]. Overall, these outcomes suggest that unicoloniality is not only a derived trait in invasive populations, but might represent a sustainable social strategy when the reduction of relatedness outweighs its ecological advantages.