Discussion
Here we provide evidence that polygyne fire ant colonies in Texas are
not unicolonial and may engage in low levels of nepotism. Polygyne
colonies did not exchange resources or workers, indicating that they
maintain strict colony boundaries (Fig 3). Polygyne workers also
preferentially fed larval siblings and may have cannibalized
non-siblings during times of stress (Fig 6), suggesting that
within-colony conflict exists. Our behavioral results also corresponded
with higher levels of within-mound relatedness between workers in the
field than those previously reported in North America (Fig 5; but see
Ross, 1993). Our results suggest that polygyne fire ant colonies are
actually multicolonial and may engage in high levels of intraspecific
competition in at least parts of their introduced range (Weeks, Wilson,
& Vinson, 2004).
Counter to our expectations, we detected distinct colony boundaries
between almost all mounds in the field regardless of social form (i.e.,
polygyne mounds were no more likely to share than monogyne mounds; Fig
3) and within-mound relatedness between workers (Appendix S5). The
mounds that did share with each other were likely part of the same
polydomous colony based on genetic results and spatial distance between
mounds (Fig 4). Although our results seem to run counter to previous
assumptions about polygyne fire ants (e.g., Greenberg et al., 1992;
Holway et al., 2002; Morel et al., 1990; Plowes et al., 2007; Porter et
al., 1992; Vander Meer et al., 1990), 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 mounds in Georgia, USA, showed distinct genotypic
frequencies and worker weight profiles, suggesting that workers and
queens are not moving freely between mounds (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 well-developed nestmate recognition (Obin, Morel,
& Vander Meer, 1993). There is also evidence of exploitative
competition between polygyne mounds in the field (Weeks et al., 2004).
Our results confirm that polygyne mounds located near each other are not
completely interconnected (Goodisman et al., 2007; Krushelnycky et al.,
2010; Weeks et al., 2004); in fact, polygyne mounds in our study were
just as isolated as monogyne mounds. Moreover, boundaries appear to be
present at relatively small spatial scales, as many mounds of both
social forms did not exchange resources despite being within 5m, and
sometimes even less than 1m, 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 mounds with distinct
boundaries separated by less than 1m, suggesting mounds 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).
Our laboratory experiment provides further evidence that polygyne fire
ants are not as cooperative as previously assumed, as workers
preferentially fed sibling over non-sibling brood (Fig 6a). Workers may
have even preferentially cannibalized non-sibling brood, because there
was a significantly greater reduction in the number of non-sibling brood
remaining at the end of the experiment compared with sibling brood (Fig
6b). All colonies were kept in standardized laboratory conditions and
fed standardized diets to minimize acquired, environmental
identification cues (Obin et al., 1993), so worker recognition of
sibling over non-sibling larvae is likely based on heritable as opposed
to environmental odor cues. One reason for the greater disappearance of
non-sibling brood could be due to differential brood viability between
families, which can cause “sham nepotism” (Holzer, Kümmerli, Keller,
& Chapuisat, 2006). We believe this scenario is unlikely, however,
given the short time frame and reciprocal nature of our experiment.
Furthermore, queens allowed to found in isolation do not express
differential viability of offspring (Vargo & Ross, 1989). 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 sibling brood than non-sibling
brood. We found no desiccated larvae, however, in or around the
experimental colonies. Instead, we hypothesize that polygyne fire ant
workers preferentially cannibalize less related 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). Any
hereditary predisposition towards nepotism should be under positive
selection in a eusocial system, as preferential care of related
offspring should increase inclusive fitness (Keller, 1997; Wilson, 2008;
Wilson & Hölldobler, 2005). However, extremes in nepotism should be
selected against in polygyne systems (Keller, 1997), as they tend to
create an environment of competition within the colony and diminish
colony fitness. While strong nepotism should be selected against,
slightly nepotistic ants may have an advantage over their less
discriminating counterparts as nepotism toward sexual larvae, regardless
of the strength of the interaction, should increase inclusive fitness
(Nonacs, 1988). This would suggest that eusociality should favor low
levels of nepotism, such as those found in this study, which straddles
the gap between selfless and selfish endeavors.
The lack of sharing between polygyne colonies in the field and evidence
of within-colony nepotism in the laboratory corresponds with high
relatedness coefficients observed in the field. Although relatedness
between workers was lower within polygyne mounds than within monogyne
mounds, relatedness coefficients in polygyne mounds were much higher
(mean and standard errors: 0.269 ± 0.037) than those previously observed
in other introduced populations in the USA (Fig 5; 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 (i.e., many unrelated queens producing workers
within the same mound; but see Ross, 1993), but our results suggest that
workers within polygyne mounds in Texas may even be half-sisters
(expected r for half-sisters = 0.25). One explanation is that the
polygyne mounds that we surveyed contained fewer queens than those
sampled in past studies. Ross (1993) demonstrated that relatedness
between workers within polygyne mounds in Georgia was negatively
correlated with queen number. Geographic variation in colony genetic
structure may also explain the higher within-mound relatedness and
pairwise F ST values in polygyne mounds 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 5).
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. Interestingly, our relatedness coefficients between workers
were much more similar to those reported in native polygyne fire ant
populations (Fig 5; 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, Gilbert, & Folgarait, 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 likely multicolonial and engage in high levels of intraspecific
competition. Our within-mound relatedness coefficients between polygyne
workers were also similar to those reported in Australia (Fig 5;
Henshaw, Kunzmann, Vanderwoude, Sanetra, & Crozier, 2005), so it would
be interesting to determine if Australian polygyne fire ant colonies
behave similarly to those in Texas and in the native range.
This does not explain, however, why some studies have detected colony
boundaries despite very low relatedness between polygyne fire ant mounds
(e.g., Goodisman et al., 2007). Our results suggest that relatedness
alone does not predict sharing between mounds, as several neighboring
mounds had low pairwise F ST and relatedness
values but did not share with each other (Appendix S6). In other ant
species, kinship does not always correlate with cooperation between
nests (Procter et al., 2016). For example, nests of the polygyne antFormica lugubris did 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 imply a free exchange
of workers or resources. 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 mounds in other parts of
their invaded range where within-mound relatedness has been reported to
be much lower to ultimately determine the relationship between sharing
and genetic relatedness.
Our study tests fundamental assumptions about the role of inter- and
intracolonial cooperation in polygyne fire ant abundance. Polygyne fire
ants are highly abundant throughout their invaded range (Porter et al.,
1991), but our results suggest that their high abundance is not due to
higher levels of cooperation between neighboring colonies, at least in
parts of Texas. Moreover, workers appear to preferentially direct care
towards more related brood, indicating some level of nepotism within
multiple-queen colonies. Our results add to a growing body of evidence
that unicoloniality does not always explain invasive ant abundance
(e.g., Eyer et al., 2020; Garnas, Drummond, & Groden, 2014; Zhang et
al., 2019). Other factors such as dietary flexibility, competition with
other species, and abiotic features may be more important to invasions
than intraspecific cooperation in some species. Future research should
further examine the relationship between unicoloniality and ant
invasions by directly quantifying sharing between nests in the field.
Identifying the factors that promote invasive ant success is critical to
understanding and managing their ecological impacts.