Genetic analyses
Genetic analyses were used to infer the social structure (i.e., monogyne
or polygyne) and genetic relatedness within and between each fire ant
mound analyzed (Fig 1). For each mound, eight workers were randomly
selected for DNA extraction. DNA was extracted from individual workers
following a modified Gentra-PureGene protocol (Gentra Systems, Inc.
Minneapolis, MN, USA).
To determine the social form of each mound, we pooled the DNA extracted
from the eight individual workers per mound and screened this pooled
sample for the presence of the Gp-9b allele,
exclusively present in polygyne colonies (Krieger & Ross, 2002; Ross &
Keller, 1998). A PCR reaction was performed on each pooled sample using
the specific primer pair 24bS and 25bAS (Valles & Porter, 2003). This
primer pair amplifies a 423 bp amplicon, and a successful amplification
denotes the presence of the Gp-9b allele,
thereby characterizing the workers as polygyne. Amplifications were
performed according to the protocol described in Valles and Porter
(2003) and visualized on a 1% agarose gel. In all, we identified 38
monogyne and 35 polygyne mounds across all six fire ant sites using theGp-9 method, of which 11 monogyne and 8 polygyne mounds were
treated with the isotope tracer.
In addition, five microsatellite markers previously developed forS. invicta (Sol11, Sol20 , Sol42 ,Sol49 and Sol55 ; Krieger & Keller, 1997) were amplified
for each of the eight individual workers per mound. The allelic
polymorphism of these five microsatellites was previously shown to be
suitable to delimit colonies of S. invicta and infer their colony
structure. The microsatellites were genotyped using the M13-tailed
primer method (Boutin-Ganache, Raposo, Raymond, & Deschepper, 2001),
consisting of 5’-fluorescently labeled tails with 6-FAM, VIC, PET or NED
dyes to facilitate multiplexing. DNA amplifications were performed in a
volume of 15 µL including 0.25-1.0 U of MyTaq™ HS DNA polymerase
(Bioline), 2 µL of MyTaq™ 5x reaction buffer (Bioline), 0.08 µL of each
primers, 0.08 of each M13 dye and 1 µL of the DNA template. PCR
reactions were carried out using a Bio-Rad thermocycler T100 (Bio-Rad,
Pleasanton, CA, USA). PCR products were sized against LIZ500 internal
standard on an ABI 3500 genetic analyzer (Applied Biosystems, Foster
City, CA, USA). Allele calling was performed using Geneious software
v.9.1 (Kearse et al., 2012).
For every mound, the social structure result obtained with theGp-9b method was confirmed using microsatellite
markers, inferring whether all workers from a mound could be assigned to
a single queen (carrying one of the two alleles of the mother queen at
each microsatellite marker studied). Polygyny was deduced when more than
one worker per colony could not be unambiguously assigned to a single
queen (see Appendix S2 for results). In addition, we compared the
relatedness coefficients (r ) between monogyne and polygyne mounds
(as identified using Gp-9 ) using analysis of variance (ANOVA) to
verify that relatedness coefficients were significantly lower in
polygyne versus monogyne mounds (i.e., suggesting the reproduction of
several unrelated queens) and to determine any differences by site. We
also used t-tests to establish if relatedness coefficients were
significantly different from 0 for polygyne mounds (i.e., multiple
unrelated queens producing workers within a single mound) and 0.75 for
monogyne mounds (i.e., one singly-mated queen producing workers within a
mound). Relatedness coefficients were calculated using the program
COANCESTRY v.1.0 (Wang, 2011), according to the algorithm described by
Queller and Goodnight (1989). Relatedness coefficients were weighted
equally and standard errors (SE) were obtained by jackknifing over
colonies.
Colony spatial structure was investigated for the six sites to determine
whether distinct mounds of S. invicta, especially those collected
within 5 m of each other, consisted of a single colony (i.e.,
polydomy) or separate colonies. To answer this question, genotypic
frequencies at all mounds were compared using a log-likelihood (G)-based
test of differentiation using GENEPOP ON THE WEB (Rousset, 2008).
Bonferroni’s correction was applied to account for multiple comparisons
of all pairs (adjusted P -value < 0.0008). Significance
was determined using a Fisher’s combined probability test.
Colony clustering was visualized for each site by plotting individuals
on a principal component analysis (PCA) using the adegenet R
package (Jombart, 2008). The clustering of mounds into distinct colonies
was also represented by Bayesian assignments of individuals into genetic
clusters (i.e., colonies; K) using STRUCTURE v.2.3.4 (Pritchard,
Stephens, & Donnelly, 2000). For each site, STRUCTURE simulations were
run with values of K from 1 to the total number of mounds encountered in
each site and repeated 10 times for each value of K. Each run included a
5 × 104 burn‐in period followed by 1 ×
105 iterations of the MCMC. The most likely number of
groupings was evaluated using the ΔK method (Evanno, Regnaut, & Goudet,
2005) implemented in Structure Harvester v.0.6.8 (Earl, 2012).
Additional details and results for clustering analysis can be found in
Appendix S2 and S3.