4 | DISCUSSION
Here we provide evidence that polygyne fire ants in Texas can and do discriminate between nestmate and non-nestmate workers and brood. Sharing between nests of both social forms was limited to nests within the same polydomous colony, indicating that fire ants maintain strict colony boundaries regardless of social form. Polygyne workers also preferentially fed larvae from the same natal colony and may have cannibalized non-nestmates during times of stress. One hypothesis for worker control of colony social form is that polygyne workers may habituate the colony to odors unique to b -carrying adults (Gotzek & Ross, 2008). Based on our results, however, other hypotheses for supergene control may be more likely. Finally, within-nest relatedness between polygyne workers in the field was higher than those previously reported in North America (DeHeer & Ross, 1997; Goodisman et al., 2007; Ross, 1993; Ross & Fletcher, 1985; Ross et al., 1996; but see Ross, 1993) and much more similar to those found in native populations (Ross et al., 1996) where polygyne workers display well-developed nestmate recognition (Chirino, Gilbert, & Folgarait, 2012). Past studies have referred to polygyne fire ant populations as unicolonial (e.g., Greenberg, Vinson, & Ellison, 1992; Holway et al., 2002; Morel, Vander Meer, & Lofgren, 1990; Plowes, Dunn, & Gilbert, 2007; Porter et al., 1992; Vander Meer, Obin, & Morel, 1990), but our results suggest a multicolonial structure, which has important implications for the ecology and management of this species.
Counter to our expectations, we detected distinct colony boundaries between almost all nests in the field regardless of social form (i.e., polygyne nests were no more likely to share than monogyne nests; Fig 2a) and within-nest relatedness between workers (Appendix S5). The nests that did share with each other were likely part of the same polydomous colony, as suggested by their low genetic differentiation and small spatial distance between nests (Fig 2). Despite previous assumptions that polygyne fire ant populations are highly interconnected, several other studies have found evidence of boundaries, at least on some level, between polygyne colonies (Goodisman et al., 2007; Krushelnycky et al., 2010; Weeks et al., 2004). For example, polygyne nests in Georgia, USA, showed distinct genotypic frequencies and worker weight profiles, suggesting that workers and queens are not moving freely between nests (Goodisman et al., 2007). Moreover, although polygyne workers do not aggressively attack non-nestmates like their monogyne counterparts (Vander Meer et al., 1990), workers will antennate and occasionally bite non-nestmates in the laboratory, indicating nestmate recognition (Obin, Morel, & Vander Meer, 1993). There is also evidence of exploitative competition between polygyne nests in the field (Weeks et al., 2004).
Moreover, boundaries appear to be present at relatively small spatial scales, as many nests of both social forms did not exchange resources despite being within 5m from each other (Appendix S6). Weeks et al. (2004) found that most labeled polygyne fire ant workers remained within 4m of their colony. In our study, we found nests with distinct boundaries separated by less than 1m, suggesting that nests very close to each other may belong to different colonies. These results imply that fire ants are able to distinguish nestmates from non-nestmates, even when environmental odor cues may be similar from living in close proximity. Heritable and environmental odor cues are thought to be additive in fire ants, but monogyne and polygyne fire ant workers have been shown to distinguish nestmate from non-nestmate despite similar environmental odor cues (Obin et al., 1993; Obin, 1986). It should be noted that nest structure and even nestmate recognition can change seasonally in other ant species (Heller & Gordon, 2006; Katzerke, Neumann, Pirk, Bliss, & Moritz, 2006), so we may have detected colony boundaries at such small spatial scales due to our sampling in the late summer/ early fall. It will be important to verify our results at other times of the year to determine if colony boundaries shift temporally.
Our laboratory experiment provides further evidence that polygyne fire ants are able to discriminate between nestmates and non-nestmates, as workers preferentially fed brood from the same natal colony (Fig 4a). Workers may have even preferentially cannibalized non-nestmate brood, because there was a significantly greater reduction in the number of non-nestmate than nestmate brood remaining at the end of the experiment (Fig 4b). Larvae were given to the colonies by placing them outside of the nest dishes and allowing the workers to bring them into the nest, so it is also possible that workers collected greater numbers of nestmate than non-nestmate brood. We found no desiccated larvae, however, in or around the experimental colonies. Instead, we hypothesize that polygyne fire ant workers preferentially cannibalize non-nestmate brood in times of stress. High levels of cannibalism are known from this species (Sorensen, Busch, & Vinson, 1983; Tschinkel, 1993) and often occur when resources are in short supply (e.g., a lack of proteinaceous food). All colonies were kept in standardized laboratory conditions and fed standardized diets to minimize acquired, environmental identification cues (Obin et al., 1993). As such, heritable odor cues may have played a more important role in worker recognition because nestmate and non-nestmate brood came from two different mothers. Workers that preferentially care for closely related nestmates consequently increase their own inclusive fitness (Hamilton, 1964; Helanterä et al., 2009), but the potential for nepotism must be further investigated using laboratory colonies with multiple queens (rather than individuals as in our experiment). Our results suggest, however, that the presence alone of the b allele of the supergene does not determine sharing between workers and larvae in polygyne fire ants.
We also detected greater relatedness within polygyne colonies than previously reported in North America, which may be one reason why we observed distinct boundaries between field colonies. Although relatedness between workers was lower within polygyne nests than within monogyne nests, relatedness coefficients in polygyne nests were much higher (mean and standard errors: 0.269 ± 0.037) than those previously observed in other introduced populations in the USA (Fig 3; DeHeer & Ross, 1997; Goodisman et al., 2007; Ross, 1993; Ross & Fletcher, 1985; Ross et al., 1996). Past studies have reported values that were not significantly different from zero due to the reproduction of many unrelated queens within the same nest (but see Ross, 1993), but our results suggest that workers within polygyne nests in Texas may even be half-sisters (expected r for half-sisters = 0.25). Interestingly, our relatedness coefficients between workers were more similar to those reported in native polygyne fire ant populations (Fig 3; Ross et al., 1996). In these native populations, polygyne colonies are multicolonial; nestmate queens are highly related (Ross et al., 1996), workers recognize nestmate from non-nestmate (Chirino et al., 2012), and colony densities are 4-7 times lower than those observed throughout North America (Porter, Williams, Patterson, & Fowler, 1997). Although we did not measure relatedness between nestmate queens, our behavioral results in the field and in the laboratory support the conclusion that polygyne fire ants in Texas likely function similarly to native conspecifics, in that colonies are multicolonial and could engage in high levels of intraspecific competition. It is important to note that our within-nest relatedness coefficients between polygyne workers were also similar to those reported in Australia and Taiwan (Fig 3; Henshaw, Kunzmann, Vanderwoude, Sanetra, & Crozier, 2005; Yang, Shoemaker, Wu, and Shih, 2008), so it would be interesting to determine if other introduced populations of polygyne fire ant behave similarly to those in Texas and in the native range.
One explanation for why we observed higher relatedness coefficients than those previously documented in the USA could be that the polygyne nests that we surveyed contained fewer queens than those sampled in past studies. Ross (1993) demonstrated that relatedness between workers within polygyne nests in Georgia was negatively correlated with queen number. Geographic variation in colony genetic structure, perhaps due to variation in queen number, may also explain the higher within-nest relatedness and pairwise F ST values in polygyne nests compared with those in other states (DeHeer & Ross, 1997; Goodisman et al., 2007; Ross & Fletcher, 1985; Ross et al., 1996; but see Ross, 1993). Much of the population genetics data of introduced polygyne fire ants in the USA has focused on one or a few geographic regions (DeHeer & Ross, 1997; Ross, 1993; Ross & Fletcher, 1985; Ross & Keller, 1995; Ross et al., 1996). Although fire ant populations in Texas have been shown to vary genetically from other parts of the country (Shoemaker, Deheer, Krieger, & Ross, 2006), only a few studies have examined colony genetic structure in Texas (Chen, Lu, Skow, & Vinson, 2003; Ross, Vargo, Keller, & Trager, 1993; Ross et al., 1996), and none that we know of have reported within-colony relatedness between workers (Fig 3). It is also possible that colony genetic structure has changed over time. For example, relatedness was almost twice as high in older compared with younger populations (i.e., over 100 years old vs. 17 years old) in the polygyne ant Formica fusca (Hannonen, Helanterä, & Sundström, 2004). Past studies of polygyne fire ant queens in Texas reported a near zero relatedness between co-occurring queens (Chen et al., 2003; Ross et al., 1996), which should result in similarly low relatedness between workers, but it is possible that within-colony relatedness has increased over the past 20 years. The ecological impact of polygyne fire ants weakened significantly over a 10-year period in parts of Texas (Morrison, 2002), which may have corresponded with a change in genetic structure.
Relatedness alone does not predict sharing between nests, however, as some studies have detected colony boundaries despite very low relatedness between polygyne fire ant nests (e.g., Goodisman et al., 2007). Likewise, several neighboring nests in our study had low pairwiseF ST and relatedness values but did not share with each other (Appendix S6). In other ant species, kinship does not always correlate with a lack of boundaries between nests (Procter et al., 2016). For example, nests of the polygyne ant Formica lugubrisdid not share workers or resources with each other despite high genetic relatedness (Procter et al., 2016). Similarly, Argentine ants (L. humile ) did not freely exchange workers between all nests within a single supercolony, even though there were no detectable genetic differences between nests (Heller et al., 2008). Likewise, gene flow was limited, and some workers were unexpectedly aggressive towards each other within the same supercolony in the unicolonial ant Formica pressilabris , suggesting that supercolonies do not always function as a single unit (Hakala, Ittonen, Seppä, & Helanterä, 2020). This highlights the importance of quantifying colony boundaries using several different methods (Ellis et al., 2017), as genetic relationships do not always reflect the levels of worker and resource sharing. Low relatedness among nestmate workers may also result from extreme polygyny, where workers originate from numerous unrelated queens (Keller 1995). In this case, each polygyne colony can contain as much genetic diversity as the background population, with nestmate workers being as related to each other than to any random worker within this population, leading to a zero relatedness within the colony (Queller and Goodnight 1989). Future research should examine the exchange of resources between polygyne fire ant nests in other parts of their invaded range where within-nest relatedness has been reported to be positive (i.e., Australia and Taiwan) or close to zero (i.e., Georgia, USA) to ultimately determine the relationship between sharing and genetic relatedness.
Our study tests fundamental assumptions about polygyne fire ant behavior and suggests that workers can discriminate between nestmate and non-nestmate. Polygyne fire ants are often more abundant than the monogyne form throughout their invaded range (Porter et al., 1991), but our results suggest that their high abundance is not due to a lack of boundaries between neighboring colonies, at least in parts of Texas. Past research has found that the “social chromosome” determines worker acceptance of queens (DeHeer & Ross, 1997; Gotzek & Ross, 2008; Ross & Keller, 2002) and worker aggression towards non-nestmates (Vander Meer et al., 1990), but our results show that supergene control does not extend to sharing between nests or sharing between workers and brood. Our study also has important implications for fire ant management, as it is often assumed that fields with polygyne fire ants may require less insecticidal bait due to high interconnectedness and sharing between nests (i.e., horizontal transfer). Although this is an effective management strategy in the unicolonial Argentine ant (L. humile ; Buczkowski & Wossler, 2019), our results suggest that polygyne fire ants are multicolonial and should be managed similarly to their monogyne counterparts. Overall, our findings call for caution in assuming that a lack of clear aggression and genetic differentiation among nests always denotes a collapse of colony boundaries in ants. By directly quantifying sharing between nests in the field, future research can trace and measure the flow of workers and resources among non-aggressive nests. Identifying the network of connectivity among nests within supercolonies will surely provide insights into the factors promoting invasive ant success and will improve our understanding and managing of their ecological impacts.