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.