4 ǀ Discussion
Our new population and colony genetic data corroborate earlier estimates (Vidal et al., 2021) that colonies of the ant Cardiocondyla elegans in Southern France typically harbor a single, multiply mated queen and that a large percentage of mating episodes involve siblings. Workers promote outbreeding by transferring female sexuals between nests. We have previously hypothesized that during the mating period in summer female sexuals are carried repeatedly from nest to nest (Vidal et al., 2021). While first related workers will carry them away from their natal nests, later unrelated workers will move them from a first unrelated recipient colony to another. Our data appear to support this hypothesis. First, the percentage of carriers and female sexuals that were less closely related than 0.25 decreased from mid to late August 2019. Second, in contrast to our previous study (Vidal et al., 2021), the average relatedness between carrier and carried female sexual was significantly lower than mean nestmate relatedness in both years, probably because in 2019 and 2020 more sexuals were collected late in the mating season. Third, genotyping the offspring of carried female sexuals showed that several of them had mated with six or seven genetically distinguishable males, which suggests the “visit” of more than one unrelated colony.
A large fraction of field-collected female sexuals died without producing workers when housed in artificial nests in the laboratory. Dissections of a small number of corpses, in which the ovaries had not yet decayed, revealed the absence of sperm in the spermatheca, suggesting that also other unproductive individuals had been virgins. Their higher mortality matches the finding that in social insects mating and reproduction increase life expectancy (e.g., Hartmann & Heinze, 2003; Schrempf et al., 2005), in contrast to many solitary insects, in which virgins outlive reproductively active, mated females (e.g., Service, 1989; Chapman et al., 1998; Limberger et al., 2021). A positive association between fecundity and longevity appears to be a trait associated with the evolution of perennial insect societies (e.g., Parker, 2010; Rodrigues & Flatt, 2016, Jaimes-Niño et al., 2022).
Contrary to our expectation colony growth rates were highest in colonies with a singly or doubly mated queen, while colonies with queens with higher mating frequencies were much less productive. Multiple mating has been shown to increase colony growth rate in the western harvester ant (Pogonomyrmex occidentalis; Wiernasz et al., 2004), increase the resistance to pathogens in bees and wasps (e.g., Shykoff & Schmid-Hempel, 1991; Seeley & Tarpy, 2007; Saga et al., 2020) and a more efficient division of labor (e.g., Oldroyd & Fewell, 2007). As in our study colony growth rate was monitored only for about one year and no sexuals were produced during this time we cannot exclude that multiple mating in C. elegans might have a beneficial effect later in the life of a colony or under more variable environmental conditions in the field.
Laboratory-reared queens that were only given the chance to mate with their brothers produced similar numbers of offspring during the first three months after hibernation as single- or double-mated queens. This means that outbreeding does not immediately lead to a higher fitness than a single round of inbreeding. In honeybees and many other Hymenoptera with single locus complementary sex determination (sl-CSD) inbreeding results in the production of sterile or inviable diploid males (e.g., van Wilgenburg et al., 2006; Darvill et al., 2012). WhileCardiocondyla appears to have a different mechanism of sex determination, in which sister-brother and even mother-son mating does not result in the production of diploid males (Schrempf et al., 2006; Schmidt et al., 2013), repeated sib-mating nevertheless results in decreased lifespan and reproductive success (Schrempf et al., 2006). That a single round of sib-mating does not immediately result in inbreeding depression in C. elegans does therefore not exclude any possible negative effects of inbreeding over several generations.
The significant initial costs of multiple mating and the efforts associated with assisted outbreeding raise the question of why C. elegans has evolved the carrying of female sexuals and polyandry. Female sexuals may mate “for convenience” when resistance to courting males is too costly, as has been suggested for the polyandrous queens ofPlagiolepis pygmaea (Trontti et al., 2007). However, previous observations of mating behavior in Cardiocondyla clearly show that female sexuals are well capable of defending themselves even against the most vicious courtship by much larger males (e.g., Heinze et al., 2021). Instead, female sexuals of C. elegans might copulate repeatedly “to pay rent” for hibernation in an alien colony. Colonies should not tolerate alien queens, which already mated with alien males and are no longer available for mating with the workers’ brothers, as this would pose the risk of usurpation and queen replacement without fitness benefits. If the adopted queen mated with the resident males this might increase the potential reproductive success of the latter and therefore the inclusive fitness of the workers. Such a hypothesis might also explain why workers carry alien female sexuals from their nest to other, unrelated recipient colonies – doing so they disperse the sperm of their brothers without risking that their own queen is later replaced, in a way passing the buck like in the “Black Peter” or “Old Maid” card game, in which players try to win by getting rid of an unmatchable card. In any case, by exporting female sexuals to other nests, colonies increase the likelihood that their sexual offspring survive hibernation. Temporary flooding of nests on the banks of Loire during winter results in high colony mortality (Lenoir et al., 2007) and assisted dispersal may be a reasonable risk-averse strategy in unpredictable environments.
ACKNOWLEDGMENTS
The study was supported by DFG (He 1623/42).
CONFLICT OF INTEREST
None declared.
AUTHOR CONTRIBUTIONS
Mathilde Vidal: Investigation and data analysis (lead)
Jürgen Heinze: Conceptualization (lead); investigation and data analysis (supporting); writing–original
draft (lead)
ETHICAL APPROVAL
The ants were collected with the permit TREL1902817S/132 issued by Access and Benefit-Sharing Clearing-House (ABSCH 2019). No other permits were required for the study.
DATA AVAILABILITY STATEMENT
Raw data, individual genotypes, pairwise relatedness estimates and colony growth data are available at https://doi.org/10.6084/m9.figshare.19982081.