<table><caption>Tableau 11 : Collection sites for Tapinoma ants. Latitude and longitude are given in decimal World Geodetic System (WGS 84). ♂ represents nesting sites morphologically determined via the male genitalia.</caption><thead><tr><th>N°</th><th>Nesting-sites and abbreviations</th><th>Countries</th><th>X</th><th>Y</th><th>Morphological determination</th><th>Chemical determination</th></tr></thead><tbody><tr><td>1</td><td>Itma</td><td>Itm</td><td>Algeria</td><td>36.74733333</td><td>3.0754</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>2</td><td>Tizi-Ouzou</td><td>Tiz</td><td>Algeria</td><td>36.71666667</td><td>4.05</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>3</td><td>Tlemcen</td><td>Tle</td><td>Algeria</td><td>34.87855</td><td>-1.313116667</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>4</td><td>Tlemcen : Lalla setti</td><td>Lal</td><td>Algeria</td><td>34.87875</td><td>-1.275466667</td><td>T. nigerrimum</td><td>T. israele</td></tr><tr><td>5</td><td>Ajaccio</td><td>Aja</td><td>France</td><td>41.91923333</td><td>8.738616667</td><td>♂ T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>6</td><td>Aubagne</td><td>ub</td><td>France</td><td>43.28065</td><td>5.579866667</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>7</td><td>Auriol</td><td>Aur</td><td>France</td><td>43.35691667</td><td>5.6408</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>8</td><td>Auriol : Sainte Baume</td><td>Bau</td><td>France</td><td>43.38835</td><td>5.6537</td><td>T. erraticum</td><td>T. erraticum</td></tr><tr><td>9</td><td>Azay-sur-Cher</td><td>Aza</td><td>France</td><td>47.32741667</td><td>0.809666667</td><td>T. erraticum</td><td>T. erraticum</td></tr><tr><td>10</td><td>Bastia</td><td>Bas</td><td>France</td><td>42.70213333</td><td>9.45075</td><td>♂ T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>11</td><td>Bléré</td><td>Blé</td><td>France</td><td>47.30955</td><td>0.952916667</td><td>T. erraticum</td><td>T. erraticum</td></tr><tr><td>12</td><td>Bléré</td><td>Blr</td><td>France</td><td>47.30941667</td><td>0.953616667</td><td>T. erraticum</td><td>T. erraticum</td></tr><tr><td>13</td><td>Bléré</td><td>Bré</td><td>France</td><td>47.30925</td><td>0.9532</td><td>T. erraticum</td><td>T. erraticum</td></tr><tr><td>14</td><td>Bléré</td><td>Tou</td><td>France</td><td>47 3094</td><td>0.953583333</td><td>T. erraticum</td><td>T. erraticum</td></tr><tr><td>15</td><td>Bléré</td><td>Cen</td><td>France</td><td>47.30918333</td><td>0.953016667</td><td>T. erraticum</td><td>T. erraticum</td></tr><tr><td>16</td><td>Bléré</td><td>Ind</td><td>France</td><td>47.30921667</td><td>0.952816667</td><td>T. erraticum</td><td>T. erraticum</td></tr><tr><td>17</td><td>Favona</td><td>Fav</td><td>France</td><td>41.7729</td><td>9.396816667</td><td>♂ T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>18</td><td>Fréjus</td><td>Fre</td><td>France</td><td>43.45233333</td><td>6.734833333</td><td>T. madeirense</td><td>T. madeirense</td></tr><tr><td>19</td><td>Fréjus : Saint Aygulf</td><td>Sai</td><td>France</td><td>43.43246667</td><td>6.735183333</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>20</td><td>Frioul island</td><td>Fri</td><td>France</td><td>43.28</td><td>5.297666667</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>21</td><td>Istres</td><td>Ist</td><td>France</td><td>43.51013333</td><td>4.9994</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>22</td><td>Lac Palo</td><td>Lac</td><td>France</td><td>41.94758333</td><td>9.40755</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>23</td><td>Plage de l'Ostriconi</td><td>Ost</td><td>France</td><td>42.66136667</td><td>9.063016667</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>24</td><td>Plage de Piémanson</td><td>Pla</td><td>France</td><td>4.347166667</td><td>4.78475</td><td>♂ T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>25</td><td>Porto-Vecchio</td><td>Por</td><td>France</td><td>41.59098333</td><td>9.279483333</td><td>♂ T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>26</td><td>Propriano</td><td>Pro</td><td>France</td><td>41.67585</td><td>8.9032</td><td>♂ T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>27</td><td>Salin de Giraud</td><td>Sal</td><td>France</td><td>43.37545</td><td>4.741333333</td><td>♂ T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>28</td><td>Santa Giulia</td><td>San</td><td>France</td><td>41.51973333</td><td>9.271666667</td><td>♂ T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>29</td><td>Velaux</td><td>Vel</td><td>France</td><td>43.53098333</td><td>5.250166667</td><td>T. madeirense</td><td>T. madeirense</td></tr><tr><td>30</td><td>Fos-sur-Mer</td><td>Fme</td><td>France</td><td>43.47751667</td><td>4.874733333</td><td>T. madeirense</td><td>T. madeirense</td></tr><tr><td>31</td><td>Fos-sur-Mer</td><td>FoM</td><td>France</td><td>43.47865</td><td>4.8744</td><td>T. madeirense</td><td>T. madeirense</td></tr><tr><td>32</td><td>Fos-sur-Mer</td><td>Fos</td><td>France</td><td>43.47798333</td><td>4.874883333</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>33</td><td>Fos-sur-Mer</td><td>FSM</td><td>France</td><td>43.4729</td><td>4.876083333</td><td>T. madeirense</td><td>T. madeirense</td></tr><tr><td>34</td><td>Gardanne</td><td>Gar</td><td>France</td><td>43.44311667</td><td>5.483016667</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>35</td><td>Mimet</td><td>Mim</td><td>France</td><td>43.44021667</td><td>5.472583333</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>36</td><td>Lower Galilee</td><td>Ira</td><td>Israel</td><td>32.72973333</td><td>35.44328333</td><td>T. israele</td><td>T. israele</td></tr><tr><td>37</td><td>Lower Galilee</td><td>Isr</td><td>Israel</td><td>32.75086667</td><td>35.43805</td><td>T. israele</td><td>T. israele</td></tr><tr><td>38</td><td>Lower Galilee</td><td>Iae</td><td>Israel</td><td>32.74455</td><td>35.48606667</td><td>T. israele</td><td>T. israele</td></tr><tr><td>39</td><td>Ijjoukak</td><td>Ijo</td><td>Morocco</td><td>30.9967</td><td>-8.159883333</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>40</td><td>Marrakech</td><td>Mar</td><td>Morocco</td><td>31.63333333</td><td>-8</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>41</td><td>Funchal</td><td>Fun</td><td>Portugal</td><td>32.664159</td><td>-16.87515</td><td>T. madeirense</td><td>T. madeirense</td></tr><tr><td>42</td><td>Algéciras</td><td>Alg</td><td>Spain</td><td>36.14578333</td><td>-5.448633333</td><td>T. simrothi</td><td>T. simrothi</td></tr><tr><td>43</td><td>Cadiz : Bahia de Cadiz</td><td>Cad</td><td>Spain</td><td>36.5004</td><td>-6.273566667</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>44</td><td>El Pedroso</td><td>Ped</td><td>Spain</td><td>37.84103333</td><td>-5.768416667</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>45</td><td>El puerto de Santa María</td><td>PSM</td><td>Spain</td><td>36.57793333</td><td>-6.214166667</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>46</td><td>Dragado</td><td>Dra</td><td>Spain</td><td>36.56831667</td><td>-6.2223</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>47</td><td>Tadeo</td><td>Tad</td><td>Spain</td><td>36.58045</td><td>-6.226816667</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>48</td><td>La Línea de la Concepción</td><td>Lin</td><td>Spain</td><td>36.16758333</td><td>-5.36665</td><td>T. simrothi</td><td>T. simrothi</td></tr><tr><td>49</td><td>San Pedro de Alcántara</td><td>PDA</td><td>Spain</td><td>36.48356667</td><td>-4.980633333</td><td>T. nigerrimum</td><td>T. nigerrimum</td></tr><tr><td>50</td><td>Sotogrande</td><td>Sot</td><td>Spain</td><td>36.28703333</td><td>-5.301483333</td><td>T. simrothi</td><td>T. simrothi</td></tr><tr><td>51</td><td>Tarifa</td><td>Tar</td><td>Spain</td><td>36.02183333</td><td>-5.615283333</td><td>T. simrothi</td><td>T. simrothi</td></tr></tbody></table>

<table><caption>Tableau 12 : Indices and Key for measurements of Tapinoma species.</caption><thead><tr><th>Tapinoma</th><th>b/a</th><th>Funicle segments</th></tr></thead><tbody><tr><td>israele</td><td>&gt;4</td><td>2 ≥ 3</td></tr><tr><td>madeirense</td><td>&gt; 4</td><td>2 &lt; 3</td></tr><tr><td>erraticum</td><td>~ 3.2</td><td>2 = 3</td></tr><tr><td>nigerrimum</td><td>2 - 2.3</td><td>2 &gt; 3</td></tr><tr><td>simrothi</td><td>1.6 - 2</td><td>2 ≥ 3</td></tr></tbody></table>
the carrier gas. Quantitative data were
obtained by integrating peaks. Gas
chromatography analysis of the 350 worker
profiles yielded five different types of
chromatograms. Cuticular compounds of
workers from two nesting sites of each of the
five profiles were determined by coupled
Gas Chromatography /Mass Spectrometry
(GC-MS) [Aza and Blé for group 1; Isr and
Ira for group 2; Fre and FoM for group 3;
Fri and Sai for group 4; Sot and Tar for
group 5]] as reference nests. Total body
washes of 20 pooled workers from each
reference nest were used for extraction.
Workers were immersed in 100 µl of hexane
for 20 min. Four µl of each extract were run
into an Agilent 6890N GC equipped with
Chrompack CPSil5WCOT apolar capillary
column (25 m × 0.25 mm, 0.12 µm). The
GC was coupled with a 5375 Agilent
Technologies Mass Spectro-meter. Electron
impact mass spectra were obtained with an
ionization voltage of 70 eV. The carrier gas
was helium at 1 ml / min with the injector in
splitless mode. Oven temperature was
isothermal at 70°C for 1 min, followed by
30°C / min to 180°C, then increased at a rate
of 5°C / min to 320°C, and finally held for
15 min. Specific peak identity was
determined with hydrocarbon standards and
by matching diagnostic peaks with those
from published spectra. Subsequently,
quantitative analyses obtained by gas
chromatography were performed with only
the hydrocarbons as variables. CHC peaks

areas were standardized to 100% by
calculating the percentage contribution of
each compound to the cuticular
hydrocarbon blend. Comparison of cuticular
profiles was based on this proportion. A
correction coefficient (K) was applied in
order to adjust the integrated values to the
sensitivity of the detector according to the
number of carbon atoms of molecules. We
determined this coefficient after injection of
the same quantity of 10 n-alkanes (18, 20,
22, 24, 28, 32, 34, 36, 38 and 40 carbon
atoms, respectively) where K = 0.0038x² -
0.1738x + 2.9683 (x is the number of
carbon atoms per molecule of the
substance).

Morphological analyses

Immediately following field
collection and during chemical analyses,
several workers from each nesting site were
morphologically identified (Tableau 11).
Morphological identification and chemical
analyses were performed in blind tests in
different laboratories (respectively Spain and
France). Over the fifty nesting sites, two
kinds of morphological analyses were used,
according to whether or not males were
present in the nest. During field collection,
males were found in eight nesting sites, two
in continental France (Pla and Sal) and six in
Corsica (Aja; Bas; Fav; Por; Pro and San:
Tableau 11). The morphological identification
of these eight nesting sites was based on
male genitalia.
<figure><img src="image_49.png" /><figcaption>Photo 1: Metric characters measured in Tapinoma species: a, maximum depth of clypeal notch as it appears in frontodorsal view. B, distance from deepest point of clypeal notch to most posterior point of clypeus. Example of a head of T. nigerrimum. Photo X. Espadaler.</figcaption></figure>

Identifications of the species from the other nesting sites were based on worker cephalic characteristics: clypeal notch, clypeal seta insertion and antennal segments. Two to five workers per sample were examined. The following characteristics and measurements were used: a.) depth of clypeal notch / distance from deepest point of clypeal notch to most posterior point of clypeus, which is easily seen on clean, dry

specimens and using tangential illumination; b.) relative length of second and third antennal segments (Photo 1 & Tableau 12). We measured mounted, dried specimens. A stereomicroscope Nikon SMZ-U with magnification (up to 112.5X) and dual arm fiber optic illuminator was used to measure the two described variables and get the corresponding indices. Measurements and indices used have not been validated as a
formal statistical and revisionary tool for
*Tapinoma*; however, they have been
routinely used over the past years and have
been confirmed whenever worker-associated
males have been available.

Statistical analyses

Statistical analyses were performed
using hydrocarbons as sole variables,
ignoring fatty acids and all other
compounds. One hundred sixty-eight
cuticular compounds were separated and
identified by GC-MS. Multivariate data
analyses were performed using R language
and environment (R 2.15.1 software). The
standardized peak areas were used to
perform multivariate principal component
analyses (PCA). The PCA were performed on
the principal factors of overall variance. We
also conducted a separate *K-means* cluster
analysis to group together data points (factor
coordinates of the variables) showing similar
cuticular hydrocarbon profiles.

A pairwise matrix of CHC differences
was constructed by measuring the binary
squared Euclidean distance (the total number
of CHCs that are absent in one species but
present in the other, and vice versa), in
order to look at the relationship between
species using Ward's linkage method
(Euclidean distance) (Ward, 1963) with
Statistica 6.0 software.

Morphological analyses

Workers from 39 nesting sites were
morphologically analyzed on the basis of
several workers each using the
morphological indices (Photo 1 & Tableau 12).
These analyses revealed the presence of five
species: *Tapinoma erraticum*; *T. israele*; *T. madeirense*; *T. nigerrimum* and *T. simrothi*.
Workers from 21 nesting sites were identified
as *T. nigerrimum*. Workers from eight
nesting sites, all in France, were identified as
*T. erraticum*. Workers from four nesting
sites, all in Spain, were identified as *T. simrothi*.
Workers from three nesting site, all
in Israel, were identified as *T. israele*, and
workers from five nesting sites, all in France,
as *T. madeirense*. The three workers from
Madeira Island were also morphologically
identified as *T. madeirense*. Workers from
Tle, Lal and Bau nesting sites could not be
identified morphologically due to their
deterioration in transit from France to Spain
(Tableau 12). Field collection yielded eight
nesting sites with males (Aja; Bas; Fav; Pla;
Por; Pro; Sal and San). For these sites,
therefore, identification was based on male
genitalia; all were morphologically identified
as *T. nigerrimum* (Tableau 11).

Results
<figure><img src="image_50.png" /><figcaption>Figure 7:</figcaption></figure>
Cuticular hydrocarbon analysis

Chemical analyses of all 350 worker-
profiles revealed clearly five qualitative and
quantitative CHC profiles (Figure 7), leading
us to expect five *Tapinoma* species over the
50 nesting sites. In order to visualize the
different chemical signatures, multivariate
principal component analysis (PCA) was
performed (Erreur ! Source du renvoi introuvable.)
on the cuticular hydrocarbons found in the
seven workers from each of the 50 nesting
sites (Tableau 11).

The PCA was performed on 53
factors which accounted for 95.1% of overall
variance. The PCA (Erreur ! Source du renvoi

introuvable.) revealed a marked discrimination
on axis 1 between on one side, workers from
group one [dark grey triangles] and, on the
other side, workers from group two [light
grey circles]. The first axis accounted for
33.29% of total variance. The second axis,
which accounted for 26.41% of cumulative
variance, revealed a marked separation
between both sides of the axis, with group 4
on the negative side [black circles] and both
groups 1 and 2 on the positive side. Group 3
was located near the axis intersection, in the
middle of the three other groups. All GC-MS
control individuals from groups 1, 2 and 4
from a given location were assigned to the
same
group.

**Figure 7:** Chromatograms obtained by injection of total body wash of workers of *T. israele* (Ira), *T. simrothi* (Tar), *T. madeirense* (Fre), *T. nigerrimum* (Fri) and *T. erraticum* (Aza).

Tableau 13 : (next double page) : List of the 168 compounds identified by GC-MS and their percentage contribution (Mean), in the cuticular extract sample of workers from field populations of the five *Tapinoma* species. All nests were summed per site (N<sub>T.erraticum</sub> = 56 ; N<sub>T.israele</sub> = 28 ; N<sub>T.madeirense</sub> = 35 ; N<sub>T.nigerrimum</sub> = 203 ; N<sub>T.simrothi</sub> = 28).
<table><caption>Tableau 13:</caption><thead><tr><th>compound<br>n°</th><th>Compounds</th><th>T.<br>madeirens</th><th>T. israele</th><th>T.<br>nigerrimum</th><th>T.<br>erraticum</th><th>T. simrothi</th></tr></thead><tbody><tr><td>1</td><td>n-Pentacosane</td><td>n-C25</td><td>7.27</td><td>0.90</td><td>12.86</td><td>8.45</td></tr><tr><td>2</td><td>13-Methylpentacosane</td><td>13-Me C25</td><td>4.02</td><td>1.08</td><td>4.69</td><td>12.77</td></tr><tr><td>3</td><td>11-Methylpentacosane</td><td>11-Me C25</td><td>2.50</td><td>1.06</td><td>4.44</td><td>12.77</td></tr><tr><td>4</td><td>7-Methylpentacosane</td><td>7-Me C25</td><td></td><td></td><td>5.62</td><td></td></tr><tr><td>5</td><td>5-Methylpentacosane</td><td>5-Me C25</td><td>8.24</td><td></td><td>2.22</td><td></td></tr><tr><td>6</td><td>3-Methylpentacosane</td><td>3-Me C25</td><td>4.04</td><td>0.62</td><td>5.23</td><td>8.27</td></tr><tr><td>7</td><td>11,15-Dimethylpentacosane</td><td>11,15-DimeC25</td><td></td><td>0.78</td><td></td><td></td></tr><tr><td>8</td><td>7,9-Dimethylpentacosane</td><td>7,9-Dime25</td><td></td><td></td><td>2.06</td><td></td></tr><tr><td>9</td><td>5,13-Dimethylpentacosane</td><td>5,13-DimeC25</td><td>12.70</td><td>0.22</td><td>7.49</td><td></td></tr><tr><td>10</td><td>n-Hexacosane</td><td>n-C26</td><td>1.30</td><td>0.87</td><td>2.25</td><td></td></tr><tr><td>11</td><td>4,11,13-Trimethylpentacosane</td><td>4,11, 13-Trime C25</td><td></td><td></td><td>1.57</td><td>1.60</td></tr><tr><td>12</td><td>4,9,13-Trimethylpentacosane</td><td>4,9,13-TrimeC25</td><td></td><td></td><td></td><td>1.60</td></tr><tr><td>13</td><td>14-Methylhexacosane</td><td>14-Me C26</td><td>2.44</td><td>0.46</td><td></td><td></td></tr><tr><td>14</td><td>13-Methylhexacosane</td><td>13-Me C26</td><td></td><td></td><td></td><td>0.99</td></tr><tr><td>15</td><td>12-Methylhexacosane</td><td>12-Me C26</td><td></td><td>0.31</td><td></td><td>0.99</td></tr><tr><td>16</td><td>11-Methylhexacosane</td><td>11-Me C26</td><td></td><td></td><td></td><td>0.99</td></tr><tr><td>17</td><td>10-Methylhexacosane</td><td>10-Me C26</td><td></td><td>0.26</td><td></td><td>0.99</td></tr><tr><td>18</td><td>9-Methylhexacosane</td><td>9-Me C26</td><td></td><td></td><td></td><td>0.99</td></tr><tr><td>19</td><td>8-Methylhexacosane</td><td>8-Me C26</td><td></td><td></td><td>1.59</td><td>0.99</td></tr><tr><td>20</td><td>5,9,11-Trimethylpentacosane</td><td>5,9,11-Trime C25</td><td>3.56</td><td></td><td></td><td></td></tr><tr><td>21</td><td>12,14-Dimethylhexacosane</td><td>12,14-Dime C26</td><td>1.67</td><td></td><td>0.57</td><td></td></tr><tr><td>22</td><td>12,13-Dimethylhexacosane</td><td>12,13-Dime C26</td><td></td><td></td><td>0.59</td><td></td></tr><tr><td>23</td><td>n-Heptacosene</td><td>C27:1</td><td></td><td>0.83</td><td></td><td></td></tr><tr><td>24</td><td>X,Y-Dimethylhexacosane</td><td>X,Y-Dime C26</td><td></td><td></td><td>0.65</td><td></td></tr><tr><td>25</td><td>8,12-Dimethylhexacosane</td><td>8,12-Dime C26</td><td>1.20</td><td></td><td></td><td>0.99</td></tr><tr><td>26</td><td>6-Methylhexacosane</td><td>6-Me C26</td><td></td><td></td><td>0.63</td><td></td></tr><tr><td>27</td><td>5-Methylhexacosane</td><td>5-Me C26</td><td>0.57</td><td></td><td></td><td></td></tr><tr><td>28</td><td>4-Methylhexacosane</td><td>4-Me C26</td><td>0.32</td><td></td><td></td><td></td></tr><tr><td>29</td><td>3-Methylhexacosane</td><td>3-Me C26</td><td></td><td></td><td></td><td>0.99</td></tr><tr><td>30</td><td>10,16-Dimethylhexacosane</td><td>10,16-DimeC26</td><td></td><td></td><td></td><td>0.80</td></tr><tr><td>31</td><td>10,14-Dimethylhexacosane</td><td>10,14-DimeC26</td><td></td><td></td><td></td><td>0.80</td></tr><tr><td>32</td><td>10,12-Dimethylhexacosane</td><td>10,12-DimeC26</td><td></td><td></td><td></td><td>0.80</td></tr><tr><td>33</td><td>2-Methylhexacosane</td><td>2-Me C26</td><td>0.38</td><td></td><td></td><td>1.76</td></tr><tr><td>34</td><td>6,12-Dimethylhexacosane</td><td>6,12-Dime C26</td><td>0.68</td><td></td><td>0.68</td><td></td></tr><tr><td>35</td><td>5,12-Dimethylhexacosane</td><td>5,12-Dime C26</td><td>0.53</td><td></td><td></td><td></td></tr><tr><td>36</td><td>4,12-Dimethylhexacosane</td><td>4,12-Dime C26</td><td>0.63</td><td></td><td></td><td></td></tr><tr><td>37</td><td>4,6-Dimethylhexacosane</td><td>4,6-Dime C26</td><td></td><td></td><td>0.42</td><td></td></tr><tr><td>38</td><td>n-Heptacosane</td><td>n-C27</td><td>4.52</td><td>16.16</td><td>1.12</td><td>6.44</td></tr><tr><td>39</td><td>15-Methylheptacosane</td><td>15-Me C27</td><td></td><td></td><td></td><td>3.65</td></tr><tr><td>40</td><td>13-Methylheptacosane</td><td>13-Me C27</td><td>3.95</td><td>6.05</td><td></td><td>3.65</td></tr><tr><td>41</td><td>11-Methylheptacosane</td><td>11-Me C27</td><td>3.59</td><td>4.33</td><td></td><td>3.01</td></tr><tr><td>42</td><td>9-Methylheptacosane</td><td>9-Me C27</td><td></td><td></td><td></td><td>3.01</td></tr><tr><td>43</td><td>7-Methylheptacosane</td><td>7-Me C27</td><td>1.35</td><td></td><td>3.30</td><td></td></tr><tr><td>44</td><td>5-Methylheptacosane</td><td>5-Me C27</td><td>1.25</td><td>0.78</td><td>1.23</td><td></td></tr><tr><td>45</td><td>11,15-Dimethylheptacosane</td><td>11,15-Dime C27</td><td></td><td>2.41</td><td></td><td>0.40</td></tr><tr><td>46</td><td>11,13-Dimethylheptacosane</td><td>11,13-Dime C27</td><td></td><td></td><td></td><td>0.40</td></tr><tr><td>47</td><td>7,15-Dimethylheptacosane</td><td>7,15-Dime C27</td><td></td><td></td><td></td><td>7.50</td></tr><tr><td>48</td><td>5,15-Dimethylheptacosane</td><td>5,15-Dime C27</td><td></td><td></td><td></td><td>1.93</td></tr><tr><td>49</td><td>5,13-Dimethylheptacosane</td><td>5,13-Dime C27</td><td>5.97</td><td>0.59</td><td></td><td></td></tr><tr><td>50</td><td>5,12-Dimethylheptacosane</td><td>5,12-Dime C27</td><td></td><td></td><td></td><td>0.18</td></tr><tr><td>51</td><td>5,11-Dimethylheptacosane</td><td>5,11-Dime C27</td><td></td><td></td><td></td><td>0.18</td></tr><tr><td>52</td><td>5,9-Dimethylheptacosane</td><td>5,9-Dime C27</td><td></td><td></td><td></td><td>0.18</td></tr><tr><td>53</td><td>4,16-Dimethylheptacosane</td><td>4,16-Dime C27</td><td></td><td></td><td>0.15</td><td></td></tr><tr><td>54</td><td>4,12-Dimethylheptacosane</td><td>4,12-Dime C27</td><td></td><td></td><td>0.15</td><td></td></tr><tr><td>55</td><td>3-Methylheptacosane</td><td>3-Me C27</td><td>4.23</td><td>6.53</td><td></td><td>2.86</td></tr><tr><td>56</td><td>3,14-Dimethylheptacosane</td><td>3,14-Dime C27</td><td></td><td></td><td></td><td>2.40</td></tr><tr><td>57</td><td>3,12-Dimethylheptacosane</td><td>3,12-Dime C27</td><td></td><td></td><td></td><td>2.40</td></tr><tr><td>58</td><td>3,7-Dimethylheptacosane</td><td>3,7-Dime C27</td><td></td><td></td><td></td><td>2.40</td></tr><tr><td>59</td><td>n-octacosane</td><td>n-C28</td><td>1.19</td><td>1.19</td><td></td><td></td></tr><tr><td>60</td><td>14-Methyloctacosane</td><td>14-Me C28</td><td>1.27</td><td>0.27</td><td>1.23</td><td>0.18</td></tr><tr><td>61</td><td>13-Methyloctacosane</td><td>13-Me C28</td><td></td><td></td><td>0.74</td><td>0.18</td></tr><tr><td>62</td><td>12-Methyloctacosane</td><td>12-Me C28</td><td></td><td>0.27</td><td>0.67</td><td>0.18</td></tr><tr><td>63</td><td>11-Methyloctacosane</td><td>11-Me C28</td><td></td><td></td><td></td><td>0.18</td></tr><tr><td>64</td><td>10-Methyloctacosane</td><td>10-Me C28</td><td></td><td></td><td></td><td>0.18</td></tr><tr><td>65</td><td>9-Methyloctacosane</td><td>9-Me C28</td><td></td><td></td><td></td><td>0.18</td></tr><tr><td>66</td><td>8-Methyloctacosane</td><td>8-Me C28</td><td></td><td>0.35</td><td></td><td>0.18</td></tr><tr><td>67</td><td>5,9,13-Trimethylheptacosane</td><td>5,9,13-Trime C27</td><td>1.11</td><td></td><td></td><td></td></tr><tr><td>68</td><td>14,15-Dimethyloctacosane</td><td>14,15-Dime C28</td><td></td><td></td><td>0.63</td><td></td></tr><tr><td>69</td><td>12,16-Dimethyloctacosane</td><td>12,16-Dime C28</td><td></td><td>0.43</td><td></td><td></td></tr><tr><td>70</td><td>12,14-Dimethyloctacosane</td><td>12,14-Dime C28</td><td>0.67</td><td></td><td></td><td></td></tr><tr><td>71</td><td>10,14-Dimethyloctacosane</td><td>10,14-Dime C28</td><td></td><td>0.41</td><td></td><td></td></tr><tr><td>72</td><td>8,12-Dimethyloctacosane</td><td>8,12-Dimethyl C28</td><td></td><td></td><td>0.71</td><td></td></tr><tr><td>73</td><td>6-Methyloctacosane</td><td>6-Me C28</td><td>0.60</td><td></td><td></td><td></td></tr><tr><td>74</td><td>4-Methyloctacosane</td><td>4-Me C28</td><td>0.34</td><td></td><td></td><td></td></tr><tr><td>75</td><td>n-Nonacosane</td><td>C29:1</td><td></td><td>2.39</td><td>0.1</td><td></td></tr><tr><td>76</td><td>6,14-Dimethyloctacosane</td><td>6,14-Dime C28</td><td>0.35</td><td></td><td></td><td></td></tr><tr><td>77</td><td>5,13-Dimethyloctacosane</td><td>5,13-Dime C28</td><td>0.42</td><td></td><td></td><td></td></tr><tr><td>78</td><td>4,14-Dimethyloctacosane</td><td>4,14-Dime C28</td><td>0.22</td><td></td><td></td><td></td></tr><tr><td>79</td><td>4,12-Dimethyloctacosane</td><td>4,12-Dime C28</td><td>0.22</td><td></td><td></td><td></td></tr><tr><td>80</td><td>n-Nonacosane</td><td>n-C29</td><td>1.16</td><td>3.28</td><td>12.34</td><td>2.20</td></tr><tr><td>81</td><td>15-Methylnonacosane</td><td>15-Me C29</td><td>0.64</td><td>0.29</td><td>2.58</td><td>1.40</td></tr><tr><td>82</td><td>13-Methylnonacosane</td><td>13-Me C29</td><td>0.69</td><td>0.28</td><td>3.98</td><td>1.40</td></tr><tr><td>83</td><td>11-Methylnonacosane</td><td>11-Me C29</td><td>0.62</td><td>0.28</td><td></td><td></td></tr><tr><td>84</td><td>4,8,12-Trimethyloctacosane</td><td>4,8,12-TrimeC28</td><td></td><td></td><td>2.52</td><td>3.73</td></tr></tbody></table>
<table><caption>Tableau 13 (suite) :</caption><thead><tr><th>compound<br>n°</th><th>Compounds</th><th>T.<br>madeirense</th><th>T.<br>israele</th><th>T.<br>nigerrimum</th><th>T.<br>erraticum</th><th>T.<br>simrothi</th></tr></thead><tbody><tr><td>85</td><td>7-Methylnonacosane</td><td>7-Me C29</td><td>0.66</td><td></td><td>1.47</td><td></td></tr><tr><td>86</td><td>2,6,12-Trimethyloctacosane</td><td>2,6,12-Trime C28</td><td></td><td>0.34</td><td></td><td></td></tr><tr><td>87</td><td>13,15-Dimethylnonacosane</td><td>13,15-Dime C29</td><td></td><td></td><td></td><td></td></tr><tr><td>88</td><td>5-Methylnonacosane</td><td>5-Me C29</td><td>0.62</td><td>0.76</td><td>0.99</td><td>0.59</td></tr><tr><td>89</td><td>11,17-Dimethylnonacosane</td><td>11,17-Dime C29</td><td></td><td></td><td>0.49</td><td></td></tr><tr><td>90</td><td>11,15-Dimethylnonacosane</td><td>11,15-Dime C29</td><td></td><td>0.77</td><td></td><td></td></tr><tr><td>91</td><td>11,13-Dimethylnonacosane</td><td>11,13 Dime C29</td><td></td><td></td><td></td><td></td></tr><tr><td>92</td><td>9,17-Dimethylnonacosane</td><td>9,17 Dime C29</td><td></td><td></td><td></td><td>0.15</td></tr><tr><td>93</td><td>9,15-Dimethylnonacosane</td><td>9,15 Dime C29</td><td></td><td></td><td></td><td>0.15</td></tr><tr><td>94</td><td>9,13-Dimethylnonacosane</td><td>9,13 Dime C29</td><td></td><td></td><td>1.32</td><td>0.15</td></tr><tr><td>95</td><td>7,19-Dimethylnonacosane</td><td>7,19-Dime C29</td><td></td><td></td><td></td><td>0.05</td></tr><tr><td>96</td><td>7,15-Dimethylnonacosane</td><td>7,15-Dime C29</td><td></td><td></td><td></td><td>0.05</td></tr><tr><td>97</td><td>3-Methylnonacosane</td><td>3-Me C29</td><td>0.46</td><td>0.39</td><td>5.12</td><td>1.25</td></tr><tr><td>98</td><td>5,17-Dimethylnonacosane</td><td>5,17-Dime C29</td><td></td><td></td><td>1.39</td><td></td></tr><tr><td>99</td><td>5,15-Dimethylnonacosane</td><td>5,15-Dime C29</td><td></td><td></td><td>1.28</td><td>0.61</td></tr><tr><td>100</td><td>5,13-Dimethylnonacosane</td><td>5,13-Dime C29</td><td>1.17</td><td>0.80</td><td></td><td></td></tr><tr><td>101</td><td>5,12-Methylnonacosane</td><td>5,12 Dime C29</td><td></td><td></td><td></td><td>0.07</td></tr><tr><td>102</td><td>5,11-Dimethylnonacosane</td><td>5,11 Dime C29</td><td></td><td></td><td></td><td>0.07</td></tr><tr><td>103</td><td>5,9-Dimethylnonacosane</td><td>5,9 Dime C29</td><td></td><td></td><td></td><td>0.07</td></tr><tr><td>104</td><td>3,17-Dimethylnonacosane</td><td>3,17-Dime C29</td><td></td><td></td><td>4.89</td><td></td></tr><tr><td>105</td><td>11,15,17-Trimethylnonacosane</td><td>11,15,17-Trime C29</td><td></td><td>3.62</td><td></td><td></td></tr><tr><td>106</td><td>16-Methyltriacontane</td><td>16-Me C30</td><td>0.33</td><td></td><td></td><td></td></tr><tr><td>107</td><td>15-Methyltriacontane</td><td>15 Me C30</td><td></td><td></td><td></td><td>1.12</td></tr><tr><td>108</td><td>14-Methyltriacontane</td><td>14-Me C30</td><td>0.32</td><td></td><td></td><td>1.12</td></tr><tr><td>109</td><td>13-Methyltriacontane</td><td>13 Me C30</td><td></td><td></td><td></td><td>1.12</td></tr><tr><td>110</td><td>12-Methyltriacontane</td><td>12-Me C30</td><td></td><td>0.26</td><td></td><td>1.12</td></tr><tr><td>111</td><td>11-Methyltriacontane</td><td>11 Me C30</td><td></td><td></td><td></td><td>1.12</td></tr><tr><td>112</td><td>10-Methyltriacontane</td><td>10-Me C30</td><td></td><td>0.25</td><td></td><td>1.12</td></tr><tr><td>113</td><td>9-Methyltriacontane</td><td>9 Me C30</td><td></td><td></td><td></td><td>1.12</td></tr><tr><td>114</td><td>8-Methyltriacontane</td><td>8-Me C30</td><td></td><td>0.25</td><td>6.32</td><td></td></tr><tr><td>115</td><td>14,20-Dimethyltriacontane</td><td>14,20-Dime C30</td><td></td><td>0.09</td><td></td><td></td></tr><tr><td>116</td><td>12,18-Dimethyltriacontane</td><td>12,18-Dime C30</td><td></td><td>0.09</td><td></td><td></td></tr><tr><td>117</td><td>10,14-Dimethyltriacontane</td><td>10,14-Dime C30</td><td>0.40</td><td></td><td></td><td></td></tr><tr><td>118</td><td>8,12-Dimethyltriacontane</td><td>8,12-Dime C30</td><td>4.91</td><td></td><td>24.31</td><td></td></tr><tr><td>119</td><td>6,14-Dimethyltriacontane</td><td>6,14-Dime C30</td><td>0.40</td><td></td><td></td><td></td></tr><tr><td>120</td><td>4,14-Dimethyltriacontane</td><td>4,14-Dime C30</td><td>0.31</td><td></td><td></td><td></td></tr><tr><td>121</td><td>4,12-Dimethyltriacontane</td><td>4,12-Dime C30</td><td>0.30</td><td></td><td>0.44</td><td></td></tr><tr><td>122</td><td>4,8-Dimethyltriacontane</td><td>4,8-Dime C30</td><td></td><td></td><td>0.43</td><td></td></tr><tr><td>123</td><td>n-Untriacontene</td><td>C31:1</td><td></td><td>0.08</td><td></td><td></td></tr><tr><td>124</td><td>n-Untriacontane</td><td>n-C31</td><td>0.21</td><td>0.19</td><td>1.47</td><td>0.84</td></tr><tr><td>125</td><td>2,8,12-Trimethyltriacontane</td><td>2,8,12-Trime C30</td><td>0.07</td><td></td><td></td><td></td></tr><tr><td>126</td><td>4,8,12-Trimethyltriacontane</td><td>4,8,12-Trime C30</td><td></td><td></td><td>3.03</td><td></td></tr><tr><td>127</td><td>15-Methyluntriacontane</td><td>15-Me C31</td><td>0.07</td><td></td><td>0.83</td><td>1.19</td></tr><tr><td>128</td><td>13-Methyluntriacontane</td><td>13-Me C31</td><td>0.07</td><td>0.70</td><td>0.81</td><td>1.21</td></tr><tr><td>129</td><td>11-Methyluntriacontane</td><td>11-Me C31</td><td>0.07</td><td>0.70</td><td></td><td>0.17</td></tr><tr><td>130</td><td>7-Methyluntriacontane</td><td>7-Me C31</td><td>&gt;0.01</td><td>0.89</td><td></td><td>0.17</td></tr><tr><td>131</td><td>13,24-Dimethyluntriacontane</td><td>13,24-Dime C31</td><td></td><td></td><td>0.43</td><td></td></tr><tr><td>132</td><td>5-Methyluntriacontane</td><td>5-Me C31</td><td>0.42</td><td></td><td>1.89</td><td></td></tr><tr><td>133</td><td>13,15-Dimethyluntriacontane</td><td>13,15-Dime C31</td><td></td><td>4.30</td><td></td><td></td></tr><tr><td>134</td><td>7,15-Dimethyluntriacontane</td><td>7,15-Dime C31</td><td></td><td>0.25</td><td>0.61</td><td>1.86</td></tr><tr><td>135</td><td>7,13-Dimethyluntriacontane</td><td>7,13-Dime C31</td><td>0.63</td><td></td><td></td><td></td></tr><tr><td>136</td><td>7,11-Dimethyluntriacontane</td><td>7,11-Dime C31</td><td>0.51</td><td></td><td></td><td></td></tr><tr><td>137</td><td>5,17-Dimethyluntriacontane</td><td>5,17-Dime C31</td><td></td><td></td><td>3.91</td><td></td></tr><tr><td>138</td><td>5,13-Dimethyluntriacontane</td><td>5,13-Dime C31</td><td>0.45</td><td></td><td></td><td></td></tr><tr><td>139</td><td>11,15,17-Trimethyluntriacontane</td><td>11,15,17-Trime C31</td><td></td><td>3.81</td><td></td><td></td></tr><tr><td>140</td><td>7,11,15-Trimethyluntriacontane</td><td>7,11,15-Trime C31</td><td></td><td></td><td>0.21</td><td></td></tr><tr><td>141</td><td>5,9,13-Trimethyluntriacontane</td><td>5,9,13-Trime C31</td><td>0.25</td><td></td><td>2.79</td><td></td></tr><tr><td>142</td><td>12-Methyldotriacontane</td><td>12-Me C32</td><td></td><td>0.10</td><td></td><td></td></tr><tr><td>143</td><td>11-Methyldotriacontane</td><td>11-Me C32</td><td></td><td>0.10</td><td></td><td></td></tr><tr><td>144</td><td>8,12-Dimethyldotriacontane</td><td>8,12-Dime C32</td><td>0.56</td><td></td><td>3.36</td><td></td></tr><tr><td>145</td><td>8,12,16-Trimethyldotriacontane</td><td>8,12,16-Trime C32</td><td></td><td></td><td>0.11</td><td></td></tr><tr><td>146</td><td>6,10,14-Trimethyldotriacontane</td><td>6,10,14-Trime C32</td><td></td><td></td><td>0.11</td><td></td></tr><tr><td>147</td><td>X,Y-Dimethyldotriacontane</td><td>x,y-Dime C32</td><td></td><td>0.41</td><td></td><td></td></tr><tr><td>148</td><td>n-Tritriacontene</td><td>C33:1</td><td></td><td>0.63</td><td></td><td></td></tr><tr><td>149</td><td>14,18,22-Trimethyldotriacontane</td><td>14,18,22-Trime C32</td><td></td><td>0.34</td><td></td><td></td></tr><tr><td>150</td><td>n-Tritriacontane</td><td>n-C33</td><td></td><td>0.34</td><td></td><td></td></tr><tr><td>151</td><td>13-Methyltriacontane</td><td>13 Me C33</td><td></td><td></td><td></td><td>0.09</td></tr><tr><td>152</td><td>11-Methyltriacontane</td><td>11-Me C33</td><td></td><td>4.55</td><td></td><td>0.09</td></tr><tr><td>153</td><td>13,19-Dimethyltriacontane</td><td>13,19-Dime C33</td><td></td><td>4.03</td><td></td><td></td></tr><tr><td>154</td><td>13,17-Dimethyltriacontane</td><td>13,17-Dime C33</td><td></td><td>4.26</td><td></td><td></td></tr><tr><td>155</td><td>11,15,17-Trimethyltriacontane</td><td>11,15,17-Trime C33</td><td></td><td>6.24</td><td></td><td></td></tr><tr><td>156</td><td>12-Methyltétriacontane</td><td>12-Me C34</td><td></td><td>0.13</td><td></td><td></td></tr><tr><td>157</td><td>11-Methyltétriacontane</td><td>11-Me C34</td><td></td><td>0.13</td><td></td><td></td></tr><tr><td>158</td><td>13-Methylpentatriacontane</td><td>13-Me C35</td><td></td><td>1.04</td><td></td><td></td></tr><tr><td>159</td><td>11-Methylpentatriacontane</td><td>11-Me C35</td><td></td><td>1.02</td><td></td><td>0.06</td></tr><tr><td>160</td><td>13,19-Dimethylpentatriacontane</td><td>13,19-Dime C35</td><td></td><td>1.11</td><td></td><td>&gt;0.01</td></tr><tr><td>161</td><td>13,17-Dimethylpentatriacontane</td><td>13,17-Dime C35</td><td></td><td>1.17</td><td></td><td>&gt;0.01</td></tr><tr><td>162</td><td>5,13-Dimethylpentatriacontane</td><td>5,13-Dime C35</td><td>0.44</td><td></td><td></td><td></td></tr><tr><td>163</td><td>11,15,17-Trimethylpentatriacontane</td><td>11,15,17-Trime C35</td><td></td><td>1.45</td><td></td><td></td></tr><tr><td>164</td><td>11-Methylheptatricontane</td><td>11 Me C37</td><td></td><td></td><td></td><td>&gt;0.01</td></tr><tr><td>165</td><td>15,19-Dimethylheptatricontane</td><td>15,19 Dime C37</td><td></td><td></td><td></td><td>&gt;0.01</td></tr><tr><td>166</td><td>15,17-Dimethylheptatricontane</td><td>15,17 Dime C37</td><td></td><td></td><td></td><td>&gt;0.01</td></tr><tr><td>167</td><td>13,17-Dimethylheptatricontane</td><td>13,19 Dime C37</td><td></td><td></td><td></td><td>&gt;0.01</td></tr><tr><td>168</td><td>13,17-Dimethylheptatricontane</td><td>13,17 Dime C37</td><td></td><td></td><td></td><td>&gt;0.01</td></tr></tbody></table>
The K-means analysis identified four chemical groups.

<figure><img src="image_51.png" /><figcaption>**Figure 8:** Principal Component Analysis (PCA) was based on the proportion of worker cuticular hydrocarbons from the 51 nesting sites. The projection was performed on two dimensions, where the first axis accounts for 33.29% of the total variance and the</figcaption></figure>

second for 26.41%. K-means cluster analysis distinguishes four groups. Morphological analysis allows us to identify these groups as Tapinoma simrothi [1-grey triangles], T. nigerrimum [2-light grey circles], indistinguishable T.madeirensis and T.erraticum [3-black rectangles] and T. israele [4-black circles]. The areas correspond to the 95% confidence limit of the data.

Group 1 (grey triangles,) was composed of all the workers from Alg; Lin; Sot and Tar (GC-MS controls), all from Spain. Group 2 (light grey circles) was composed of workers from Algeria (Itm; Tiz and Tle),

France (Aub; Aur; Cad; Fos; Fri; Gar; Ist; Lac; Mim; Ost and Sai), Morocco (Ijo and Mar) and Spain (Dra; PDA; Ped; PSM and Tad). Group 3 (dark grey rectangle) was composed of all the workers from Aza; Bau; Blé; Blr;
Bré; Cen; Fre; FSM; FMe; FoM; Ind; Tou and Vel (France). Group 4 (black circles) was composed of all the remaining workers [Israel: lae; Isr; Ira and Algeria: Lal]. Given the perfect concordance between the morphological and chemical discriminations, groups 1, 2 and 4 were identified as *T. simrothi*, *T. nigerrimum*, and *T. israele* respectively. In order to discriminate workers of the remaining species, a new PCA (Figure 9) was performed on worker profiles of all the nesting sites of group 3 from the first PCA. This PCA was based on their 77 CHCs and was performed on the 37 major factors which accounted for 95.2% of the overall variance.

This second PCA (Figure 9) revealed a marked discrimination between the four Fréjus worker profiles and the other nesting sites.

Figure 9: Principal Component Analysis based on the 77 CHCs of worker profiles of the third group of the first PCA. The projection was performed on factorial plane 1-2. Axis 1 accounts for 29.37% of the total variance and axis 2 for 13.52%. K-means cluster analysis distinguishes three groups: *T. madeirense* [3a-grey circles], *T. erraticum* [3b-black circles] and 4 workers from Fréjus [3c-dark grey triangles]. The areas correspond to the 95% confidence limit of the data.

<figure><img src="image_52.png" /></figure>
<figure><img src="image_53.png" /></figure>

Figure 10 : Cluster analysis of cuticular hydrocarbon compositions of workers of T. erraticum, T. madeirense, T. israele, T. nigerrimum and T. simrothi using Ward's linkage method (Euclidean distance) (Ward, 1963).
The first and the second axes, which accounted respectively for 29.37% and for 13.52% of total variance, discriminated between, on one side, workers from group 3a (grey circles) and on the other side, workers from group 3b (black circles). The K-means analysis distinguished three chemical groups. The first group [3a] was composed of workers from southern France [Fre; FSM; FMe; FoM and Vel (grey circles, Figure 9)]. The second group [3b] was composed of workers from southern and central France [Aza; Bau; Blé; Blr; Bré; Cen; Ind and Tou (black circles)]. In the first two groups, all the individuals from a given location were assigned to the same group, except for four workers from Fréjus which formed a separate group [3c, dark grey triangles].

Morphological and chemical discriminations led to groups 3a and 3b being identified *T. madeirense* and *T. erraticum* respectively (Tableau 11). Given their chemical composition and their morphological discrimination, workers from Fréjus were identified *T. madeirense*.

Across the five species, 168 distinct CHCs were found, belonging to five distinct hydrocarbon groups (

Tableau 13). These 168 CHCs were identified as ranging from 25 to 39 carbon atoms. Among the five species, the sequence of occurrence is as follows: mono-methylalkanes > dimethylalkanes > *n*-alkanes > trimethylalkanes > *n*-alkenes (

Tableau 13).

Cuticular profiles of all *Tapinoma* species present methyl-branched alkanes (mono-, di- and trimethyl) and some straight-chain *n*-alkanes, but only *T. israele* and *T. nigerrimum* present *n*-alkenes.

Cuticular profiles of *Tapinoma erraticum*, *T. israele*, *T. madeirense* and *T. simrothi* (Figure 7) were dominated by monomethylalkanes (respectively 44%, 30.5%, 42% and 68%), while the cuticular profile of *T. nigerrimum* workers was dominated by dimethylalkanes (43%)

Tableau 13 & Figure 7). The profile of the *T. simrothi* worker was more diverse and wide-ranging (70 CHCs ranging from C25 to C39) than that of the four other species. The profile of the *T. israele* worker ranged from C25 to C38 (65 CHCs), while that of the *T. erraticum* worker presented 35 CHCs ranging from C25 to C33 carbon atoms. *T. nigerrimum* worker profile ranged from C27 to C35 (35 CHCs) and that of the *T. madeirense* worker was composed of 62 CHCs ranging from C25 to C35 carbon chain lengths (Tableau 13). Only *n*-C27, *n*-C29 and 13-MeC31 were common to all five species (1.9%).

Ten cuticular hydrocarbons were common to four species (5.9%), 14 were common to three species (8.3%), 33 were common to two species (19.6%) and 108 were found in the profile of workers from one species only (64.3%). On average, workers from two different species had 16 CHCs in common; however *Tapinoma*
israele and *T. madeirense* workers shared 26 compounds (respectively 40% and 42% of their entire profile), whereas *T. simrothi* and *T. nigerrimum* shared only four compounds (C27, C29, 11, 17 dimethyl-C29 and 13me-C31), respectively 5.7% and 11% of the entire profile of each species. *T. erraticum* shared 48.7% of its profile with *T. israele* (19 CHCs), while *T. israele* shared with this species only 29% of its own profile (

Tableau 13). It is possible, using putative
species recognition cues, to correctly identify
each *Tapinoma* species through compounds
which greatly contribute to their
discrimination. *T. israele* can be identified
easily by the presence of C27, C29 and C33
alkene, dimethyl-C33, methyl-C34, and a
suite of 11, 15, 17 trimethyl- C29 / C31 / C33
/ C35. The *T. nigerrimum* CHC profile is
dominated by compounds greatly
contributing to the discrimination of this
species (8, 12 dimethylC30 (24.3%); 7, 9- &
3, 17- dimethylC29 and 4, 8, 12 trimethyl
C28 / C31). *T. simrothi* can be easily
discriminated from the other *Tapinoma*
species by the presence of suites like 9 to 13
methyl- C26 / C30; 5, 13- 5, 12- & 5,9-
dimethyl-C27 and 3, 12- 3, 7- & 3,4-
dimethyl-C27. Four dimethyl-alkanes (7, 5
dimethyl-C25; 12, 13- & 4, 6- dimethyl-C26
and 7, 15 dimethyl-C27) greatly contributed
to the discrimination of *T. erraticum*. *T.
madeirense* can be identified by using both
methyl-alkane (5 & 4 methyl-C26 and 6 & 4
methyl-C28) and dimethyl- alkane (5, 12- &

4, 12 dimethylC26; 6, 14- & 5, 13- dimethyl-
C28; 4, 14- & 4, 12 dimethyl-C28; 7, 13- 7,
11- dimethyl-C31 and 5, 13 dimethyl-C31) (

Tableau 13).

In order to better understand the
contribution of the five hydrocarbon groups
in the species determination, a PCA based
only on dimethyl- and trimethyl- alkanes was
performed. The same discrimination (PCA
not shown) between the five species was
obtained irrespective of the presence or not
of the alkenes, alkanes and methyl-alkanes.
The K-means analysis identified the same four
chemical groups. Moreover, the exclusion of
the dimethyl- and / or trimethyl- alkanes
significantly decreases the capacity to
distinguish species.

A hierarchical cluster analysis was
performed on all the qualitative cuticular
hydrocarbon dissimilarities (Figure 10). This
cluster was constructed by measuring the
binary squared Euclidean distance, in order
to look at the relationship between species
using Ward's linkage method. This cluster
shows a first separation between, on the one
hand *Tapinoma simrothi* and *T. israele* and,
on the other hand, *T. madeirense*, *T.
erraticum* and *T. nigerrimum*. A second
separation distinguishes *T. nigerrimum* from
*T. erraticum* and *T. madeirense*.

Cuticular hydrocarbons from the
three workers from Madeira Island which
arrived alive at the laboratory were analyzed
by gas chromate-graphy. Their profiles were
qualitatively identical to the profile of *T. madeirense*.

Discussion

High correlation and reliability between
CHCs and morphological analyses for species
identification has previously been shown, for
example, with the cryptic *Pachycondyla
villosa* complex (Lucas *et al.*, 2002) or the
European *Tetramorium* sibling species
(Schlick-Steiner *et al.*, 2006). CHCs can
discriminate sibling species (Cremer *et al.*,
2008), cryptic species (Lucas *et al.*, 2002), or
even hybrids (Vander Meer, 1986), like any
phenotypic character that is an expression of
DNA (Seifert, 2009). Here, based on multiple
comparisons, both chemical and
morphological, we concluded that all the
groups studied are distinct *Tapinoma* species.
Chemical analyses support and corroborate
the morphological separation of the total
nesting sites into five groups and allow us to
identify them as *T. israele*, *T. erraticum*, *T.
nigerrimum*, *T. simrothi* and *T. madeirense*.
These differences lend support to the species
status of the five studied taxa.

Our study shows that the CHCs are
extremely different among all five species.
The species can be reliably separated by
principal component analysis of their
cuticular hydrocarbons, since considerable
interspecific dissimilarities were found in the
composition of the five *Tapinoma* CHC
profiles. Recently, MORRISON & WITTE

(MORRISON & WITTE, 2011) showed strong
differences in chemical recognition cues
between two species of *Lasius*. *Lasius niger*
and *L. platythorax* shared 17 compounds
(i.e., 26.6% of the *L. niger* CHCs and 34%
of those of *L. platythorax*). Here, we found
that *T. israele* shared with *T. nigerrimum*
only ten compounds (i.e., 15.4% of the *T.*
*israele* CHCs and 28.6% of those of *T.*
*nigerrimum*) and *T. erraticum* shared with *T.*
*simrothi* 13 compounds (i.e., 33.33% of the
*T. erraticum* CHCs and 18.6% of those of *T.*
*simrothi*). Maximum interspecific
dissimilarities were found between *T.*
*simrothi* sharing 5.7% of its profile and *T.*
*nigerrimum* sharing 11.4% of its profile, with
only four compounds in common. All five
*Tapinoma* species share only three CHCs and
only *T. nigerrimum* and *T. israele* have *n*-alkenes. Of the three shared CHCs, two are
odd-numbered *n*-alkanes (C27 and C29) which are typically dominant in ant CHC
profiles (Martin & Drijfhout, 2009). They
may be used as chemical communication
signals, but their ubiquity means that they
could be general indicators only (Martin &
Drijfhout, 2009). As previously shown by
MARTIN & al. (Martin *et al.*, 2008a), the
exclusion of the methyl-alkane, alkane and
alkene did not significantly reduce the
capacity of distinguish species. However, the
exclusion of the dimethyl and / or trimethyl-
alkanes in the PCA significantly decreases the
discrimination, suggesting that it is these
compounds that contain the maximum
information.

Moreover, VAN WILGENBURG & al. (van Wilgenburg et al., 2011) showed that ant species have on average 18.7 ± 9.84 different homologous alkenes and methyl branched alkane series within their profile; *Tapinoma* species have on average 53.6 ± 16.02 different CHCs. These closely-related *Tapinoma* species appear to differ strongly in their CHCs, as with *Formica fusca* and *F. lemani* (Martin et al., 2008a) or others (Akino et al., 2002; Lucas et al., 2005), suggesting that major changes in their composition may have occurred during speciation events (van Wilgenburg et al., 2011).

In addition to being very different chemically, these closely-related *Tapinoma* species seem to produce a wide range of CHCs, leading to diverse biophysical properties. Across the 78 ant species included in their review, MARTIN & DRIJFHOUT (MARTIN & DRIJFHOUT, 2009) found 993 different compounds; these five *Tapinoma* species presented more than 16% of the total of these compounds. The image of the ant as a chemical factory is fully reinforced by the *Tapinoma* genus.

Because of their species-specificity, CHCs
were first considered for chemotaxonomy in
1970 (Jackson & Baker, 1970). They may
help to delimit a number of different species
and can be used as an additional taxonomic
tool in combination with morphological

methods. Cluster analysis was based here on
the presence / absence of hydrocarbons
rather than on their proportions, because
conspecific colony profiles differ in CHC
ratios. In the light of the chemical cluster
analysis, *T. simrothi* seems to be closer to *T.
israele* and *T. erraticum* to *T. madeirense*,
with *T. nigerrimum* in between. This result
converges with morphological characteristics.
However, a cluster tree based on the
presence or absence of compounds must be
taken with precaution. Using CHC profile
information is potentially problematic when
major changes in species composition have
occurred at speciation events, as with several
*Formica* (Martin *et al.*, 2008a) or with our
*Tapinoma* species. For example, these five
*Tapinoma* species belonging to the same
genus would not be clustered together in the
dendrogram of MARTIN & DRIJFHOUT
(MARTIN & DRIJFHOUT, 2009) because they
do not all produce *n*-alkenes. When such
speciation events are involved, it is preferable
to use CHC profiles only for intra-genus
phylogeny. Chemical cuticular profiles are
known to be influenced by genetic and
environmental factors (Crosland, 1989; van
Zweden *et al.*, 2009; Wagner *et al.*, 2001). If
adaptations to abiotic parameters such as
humidity or temperature (Wagner *et al.*,
2001) were the driving force for chemical
differentiation between species, we would
expect longer-chained hydrocarbons with the
more xero- and thermophilic species
(*Tapinoma simrothi* and *T. israele*), as they
offer better protection against desiccation (Gibbs, 1998). *Tapinoma erraticum* presented shorter-chained hydrocarbons than *T. simrothi* or *T. israele* (Tableau 13).

Chemical profiles of species can be altered by a close contact with the prey profiles (Liang & Silverman, 2000) or by environmental factors. In most cases, the changes remain small or moderate, mainly affecting the proportions of some of the compounds (Morrison & Witte, 2011). But in our study, the chemical similarity within *Tapinoma* species is remarkable. The effect of environmental factors and diet seems negligible in spite of the obvious environmental gradient induced by the fact that some of these populations are separated by 900 kilometers (*T. erraticum*), 1300 km (*T. nigerrimum*) and even more than 3000 km (*T. israele*). At shorter distances, the same stability of CHCs has been shown, for example, in *Lasius niger* in western Europe [France and Belgium (Lenoir *et al.*, 2009); Germany (Dinter *et al.*, 2002) and Denmark (Dreier & D'Ettorre, 2009)], in *Formica lemani* [England, Finland and Ireland (Martin *et al.*, 2009; Seppa *et al.*, 2011)], and in *Formica exsecta* (Martin *et al.*, 2008a).

Although the pronounced interspecific dissimilarities found here may well result from ecological differences, this cannot explain the intraspecific similarities. In addition to passive mechanisms, adaptations to specific niches might have been expected

to lead to chemical differences between *T. israele* from Algeria and from Israel, or between *T. nigerrimum* from Morocco and from France. If the parameters principally influencing CHCs were environmental, species would consequently have been more similar within sites, where they share the same environment. If CHCs serve mainly protective functions, selection should drive the species' profiles apart, but we did not find intermediate chemical phenotypes.

Among all the nesting sites, 58% of the nesting sites were identified as *Tapinoma nigerrimum*. This species seems to be well spread around the Mediterranean Basin. Despite the major search made, *T. simrothi* nesting-sites were not found in southern mainland France and Corsica. This absence of *T. simrothi* in southern mainland France and Corsica could be explained by misidentification, insufficient field collection or the disappearance of the species.

The presence of the nesting site from Lalla Setti (Lal) permits us to demonstrate the existence of *T. israele* in Algeria. Despite the major search made in France and Spain, no *T. israele* nesting-sites were found.

The findings from our work clearly show how using chemical and morphological techniques together facilitates the study of these species, by allowing precise differentiation and identification. The chemistry of the CHC bouquet is shown to be an excellent tool for taxonomists, being species-stable over thousands of kilometers.
## ACKNOWLEDGEMENTS

We thank M. BOUKHEMZA and N. BOUKHEMZA - ZEMMOURI (University of Tizi-Ouzou), A. PAYAN, E. BERVILLE, P. PONEL (IMBE), J. OFER (Israel), A. IONESCU (Tel-Aviv University, Israel), C. PLATNER (University of Bielefeld, Germany) and Y. BEN MORDECHAI (Israel) for providing preserved and/or live ant samples. We are very grateful to J-P. CHRISTIDÈS (IRBI) and to S. GREFF (IMBE) for their help and advice with the FID and the GC-MS. We thank M. SWEETKO for English language revision. This work was supported by the Provence-Alpes-Côtes d'Azur Region. X.E. is currently supported by MCYT-FEDER CGL2010-18182. We thank S.J. MARTIN and an anonymous referee for their constructive comments.

<figure><img src="image_54.png" /></figure>
**III. Expansion de la fourmi d'Argentine : supercolonies et fusion.**

*Article B:*

Variation in the level of aggression, chemical
and genetic distance among three
supercolonies of the Argentine ant in
Europe

O. BLIGHT,\*¹ L. BERVILLE,\*¹ V. VOGEL,†¹ A. HEFETZ,‡M. RENUCCI,§ J.

ORGEAS,\* E. PROVOST§ AND L. KELLER†

Molecular Ecology (2012) 21, 4106–4121

Received 8 March 2011; revision received 15 March 2012; accepted 24 March 2012

*Article C:*

A Peaceful zone bordering two Argentine
ant (*Linepithema humile*) supercolonies

L. BERVILLE,\*¹ O. BLIGHT,\*¹ A. HEFETZ,‡M. RENUCCI,§ AND E. PROVOST§

Chemoecology DOI: 10.1007/s00049-013-0135-0

<footnote>†These authors contributed equally to this study.</footnote>

<footnote>†These authors contributed equally to this study.</footnote>

<footnote>\*Aix-Marseille Université, CNRS§ Institut Méditerranéen de Biodiversité et d'Ecologie marine et continentale (UMR 7263 CNRS-IRD), Europôle Méditerranéen de l'Arbois BP 80, 13545 Aix-en-Provence Cedex 4, France.</footnote>

<footnote>†Department of Ecology and Evolution, University of Lausanne, Biophore, CH-1015 Lausanne, Switzerland.</footnote>

<footnote>‡Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978. Israel.</footnote>
<figure><img src="image_55.png" /><figcaption>Carte 2 : Carte des 33 populations de L. humile prélevées par Giraud et al. (Giraud et al., 2002). Les cercles blancs représentent les populations appartenant à la supercolonie catalane. Les cercles noirs représentent les populations appartenant à la supercolonie principale. L'échelle représentait par le trait est de 100km.</figcaption></figure>

<figure><img src="image_56.png" /><figcaption>Carte 3 : Carte des 6 populations de L. humile prélevées par Blight et al. (Blight et al., 2010b). Les cercles rouges représentent les populations appartenant à la supercolonie principale. Le cercle blanc représente la population appartenant à la supercolonie catalane. Les cercles bleus représentent les populations appartenant à la supercolonie corse. OL: Olmeto, CA: Cannelle, BI: Biguglia, DA: Danio, GE: Gerona, SFG: Sant Feliu de Guixols.</figcaption></figure>
## Avant-propos :

Alors qu'elle passe inaperçue dans son milieu naturel (Suarez et al., 1999), la fourmi d'Argentine (*Linepithema humile*) étend sa domination depuis plus de 120 ans dans toutes les régions du globe (Wetterer et al., 2009) à climat de type méditerranéen (Wetterer et al., 2009) et également de type subtropical et tempéré-doux (Silverman & Brightwell, 2008). Profitant de l'explosion des relations internationales humaines, elle colonise l'Amérique du Nord (NEWELL & BARBER, 1913), puis l'Europe (Wetterer & Wetterer, 2006), l'Afrique (Skaife, 1955), Océanie (Passfield, 1968) et enfin l'Asie (Sugiyama, 2000).

La principale raison du succès écologique des fourmis d'Argentine résulte de son changement de structure sociale quand elle envahit un nouveau territoire (Suarez et al., 1999). En zone envahie, *L. humile* met en place une supercolonie qui englobe toutes les colonies au sein d'un gigantesque territoire (Giraud et al., 2002). Cette organisation sociale est très spécifique des fourmis invasives et est appelée unicolonialité (Wilson, 1971). L'unicolonialité est caractérisée par de constants échanges d'individus entre les différents nids sans aucune agressivité. Les ouvrières, le couvain ou des reines peuvent ainsi être transférés d'un nid à l'autre sans susciter de réaction hostile. Ce phénomène, observé par NEWELL & BARBER (NEWELL & BARBER, 1913) dès 1913, a été confirmé à de multiples reprises en Californie (Suarez et al., 1999; Tsutsui et al., 2000), même lorsque les nids ne sont pas physiquement connectés. En France, cette tolérance se retrouve entre les populations prélevées dans l'Hérault et dans l'Aude (Kaufmann & Passera, 1991), ainsi que celles prélevées dans le Var et les Pyrénées-Orientales (Passera & Aron, 2005).

Chaque fois qu'un individu d'une colonie est confronté à un autre individu, il compare la signature coloniale de ce dernier avec la sienne. Si les deux individus portent une signature différente alors il y a une confrontation qui finit souvent par un combat à mort ; si deux individus portent la même signature alors ils appartiennent à la même colonie. Cette reconnaissance est basée sur la variation quantitative et/ou qualitative de composés odorants présents à la surface de la cuticule des fourmis. Chaque individu synthétise grâce à des glandes phéromonales et des œnocytes des hydrocarbures qui se retrouvent sur la cuticule (Wicker-Thomas & Chertemps, 2010). Ces hydrocarbures forment la signature coloniale ; celle-ci est la résultante du brassage des signatures individuelles, qui sont homogénéisées par des échanges trophallactiques, des contacts antennaires, des frottements corporels et/ou des léchages. Ce brassage est défini par le terme allemand « Gestalt (Crozier & Dix, 1979)».

Cette variation quantitative et/ou qualitative dans la signature chimique peut avoir différentes origines : 1- génétique (Tsutsui et al., 2003; Tsutsui et al., 2000), 2-
environnementale (Liang & Silverman, 2000) ou 3- subissant l'action conjointe des deux
(Vonshak et al., 2009).

En 2002, GIRAUD et al. (Giraud et al., 2002) effectuent des prélèvements dans le sud de l'Europe le long des côtes de la Méditerranée et de l'Atlantique depuis le Nord-ouest de l'Espagne, jusqu'au Golfe de Gênes en Italie (Carte 2). Des tests d'agressivité dyadiques (une ouvrière contre une ouvrière), en laboratoire, révèlent alors l'existence de deux supercolonies. Au sein de chaque supercolonie les ouvrières se reconnaissent et ne s'agressent pas, mais entre les deux supercolonies, les ouvrières se battent à mort. Une première supercolonie localisée en Catalogne sera nommée « supercolonie catalane », la seconde supercolonie localisée sur plus de 6 000km sera nommée « supercolonie principale ». Ces auteurs (Giraud et al., 2002) montrent également que ces populations introduites présentent une perte limitée de diversité génétique.

En Corse à Calvi, les fourmis d'Argentine sont présentes depuis plus de 55 ans (Bernard, 1960). Dans l'optique de connaître la répartition des fourmis d'Argentine en Corse (Blight et al., 2009) ainsi que leur intégration dans le patron des supercolonies européennes, BLIGHT et al. ont effectué des prélèvements sur le littoral provençal et corse (Carte 3) (Blight et al., 2010b). Ils ont constaté la présence de populations appartenant à la supercolonie principale mais aussi d'une troisième supercolonie. Cette troisième supercolonie est aussi présente sur le littoral de Provence.

Les travaux de BLIGHT et al. et de GIRAUD et al. ont soulevé de nombreuses problématiques (Blight et al., 2010b) (Giraud et al., 2002). Comment les populations de *L. humile* françaises s'incorporent-elles dans la trame des supercolonies européennes ? Quelle est l'origine de la supercolonie corse ? Observe-t-on des différences dans la structure sociale des colonies sur une petite ou moyenne échelle ? Afin de répondre à ces questions, nous avons effectué une nouvelle étude, utilisant l'outil génétique, en plus de l'outil chimique, couplé à une étude comportementale. Cette étude réalisée en collaboration a porté sur un plus grand nombre de populations. Les résultats sont rapportés ci-après (Blight et al., 2012). Ces travaux ont soulevé de nouvelles questions et ont permis de mettre en évidence une zone géographique située dans la zone de la presqu'île de Giens et de l'île de Porquerolles, où les deux supercolonies pourraient former un front. En situation d'invasion, la fusion des colonies faiblement agressives pourrait conduire à la formation de supercolonies. Au point de contact entre les supercolonies principale et corse y - a-t-il des confrontations ? Une fusion ?

VASQUEZ et SILVERMAN ont montré en laboratoire que des transferts libres d'ouvrières entre deux supercolonies faiblement agressives étaient possibles (Vasquez & Silverman, 2008). Est-ce possible *in natura* ? S'il y a fusion, la nouvelle colonie sera-t-elle agressive vis-à-vis des deux supercolonies d'origines ?
Molecular Ecology (2012) 21, 4106–4121

doi: 10.1111/j.1365-294X.2012.05668.x

# Variation in the level of aggression, chemical and genetic distance among three supercolonies of the Argentine ant in Europe

## Abstract

In their invasive ranges, Argentine ant populations often form one geographically vast supercolony, genetically and chemically uniform within which there is no intraspecific aggression. Here we present regional patterns of intraspecific aggression, cuticular hydrocarbons (CHCs) and population genetics of 18 nesting sites across Corsica and the French mainland. Aggression tests confirm the presence of a third European supercolony, the Corsican supercolony, which exhibits moderate to high levels of aggression, depending on nesting sites, with the Main supercolony, and invariably high levels of aggression with the Catalonian supercolony. The chemical analyses corroborated the behavioural data, with workers of the Corsican supercolony showing moderate differences in CHCs compared to workers of the European Main supercolony and strong differences compared to workers of the Catalonian supercolony. Interestingly, there were also clear genetic differences between workers of the Catalonian supercolony and the two other supercolonies at both nuclear and mitochondrial markers, but only very weak genetic differentiation between nesting sites of the Corsican and Main supercolonies (FST = 0.06). A detailed comparison of the genetic composition of supercolonies also revealed that, if one of the last two supercolonies derived from the other, it is the Main supercolony that derived from the Corsican supercolony rather than the reverse. Overall, these findings highlight the importance of conducting more qualitative and quantitative analyses of the level of aggression between supercolonies, which has to be correlated with genetic and chemical data.

**Keywords:** aggression, cuticular hydrocarbons, Linepithema humile, population genetics, supercolonies
Introduction

The ability to recognize self from
non-self is a central characteristic of social
insects and is crucial for maintaining social
structure (Hölldobler & Wilson, 1990; Smith
& Breed, 1995). Social insects have a highly
developed recognition system that enables
them, on the one hand, to behave
altruistically towards nestmates and, on the
other hand, to act aggressively towards
non-nestmates, thus protecting the colony
from colony resource exploitation by
conspecific intruders. Nestmate recognition
is based on a distinct colony odour (label),
which in social insects, including ants, bees,
wasps and termites, is composed of
cuticular hydrocarbons (CHCs) (Hefetz,
2007; Lahav et al., 1999; Provost et al.,
2008). A mismatch between the CHCs of
intruder and resident workers generally
leads to aggression.

Most ant species are multicolonial,
with well-developed nestmate discrimina-
tion whereby workers aggressively defend
territories against either conspecifics or
heterospecific colonies (Hölldobler &
Wilson, 1990). However, in some species,
there is greatly reduced aggression
between colonies. An extreme case of such
reduced levels of aggression occurs in
unicolonial species that are characterized by
the presence of supercolonies consisting of

large networks of nests exhibiting no
aggression within supercolonies but high
aggression between supercolonies (Errard et
al., 2005; Giraud et al., 2002; Le Breton et
al., 2004; Pedersen et al., 2006; Thomas et
al., 2010; Tsutsui et al., 2000). A peculiarity
of supercolonies is that they range over
spatial scales that are so large that direct
interactions among workers of all nests are
impossible (Pedersen et al., 2006).
Unicolonial species are ecologically among
the most successful organisms, with very
high invasive potential (Holway et al.,
2002a).

The Argentine ant (Linepithema humile) provides a good example of the invasion success of unicolonial species. This invader has successfully spread across many zones of the globe with Mediterranean and subtropical climates, through the intensification of human activities over the last century (Suarez et al., 2001). Its unicolonial structure affords it major ecological advantages in terms of colonization ability (Giraud et al., 2002; Holway, 1999; Tsutsui et al., 2000), resource exploitation (Holway & Case, 2001) and interspecific competition (Holway, 1999; Human & Gordon, 1996), allowing Argentine ant populations to reach high densities of workers (Holway et al., 2002a; Holway et al., 1998). In their introduced range, Argentine ant
populations often form one geogra-phically
vast supercolony, genetically and chemically
uniform within which there is no
intraspecific aggression (Giraud *et al.*,
2002; Sunamura *et al.*, 2009b; Tsutsui *et
al.*, 2000). Studies on the Argentine ant in
its introduced range have described a large
number of supercolonies worldwide
(Giraud *et al.*, 2002; Suhr *et al.*, 2009;
Sunamura *et al.*, 2009b; Tsutsui *et al.*,
2000). Interestingly, several supercolonies
extending over hundreds of kilometers in
the USA, Europe, Australia and Hawaii
have been found to be chemically and
genetically similar (Brandt *et al.*, 2009;
Vogel *et al.*, 2010) and exhibit no mutual
aggression (Sunamura *et al.*, 2009a;
Sunamura *et al.*, 2009b; van Wilgenburg *et
al.*, 2010), suggesting that they belong to a
single global supercolony. Frequent
movements and introductions of Argentine
ant propagules in distant parts of the global
supercolony may facilitate its persistence
by avoiding genetic and chemical changes
resulting from isolation by distance (van
Wilgenburg *et al.*, 2010). While it was
thought that there are two supercolonies
in Europe (Giraud *et al.*, 2002), a recent
study identified a third supercolony, the
Corsican supercolony, which elicited
moderate levels of agression toward the
Main European supercolony and was
chemically closer to the Main European
supercolony than to the Catalonian
supercolony (Blight *et al.*, 2010b). On the

basis of these results, the authors suggested
that the Corsican supercolony may
represent a third European supercolony
originating either from the Main European
supercolony or from an independent
introduction event. Unfortunately, no
information is available on the genetic
composition of the Corsican
supercolony. Moreover, a
misidentification was made on the identity
of the Main and the Corsican supercolonies
with the control nesting site previously
used for the Main supercolony actually
belonging to the Corsican supercolony (see
results of this study).

To get a better understanding of the
process by which invasive ant supercolonies
are segregated and their evolutionary
trajectories, we first conducted additional
behavioural tests to obtain quantitative data
on the level of aggression between putative
nesting sites from the Corsican and Main
supercolonies. In addition to the six
previously studied nesting sites (Blight *et al.*,
2010b), we investigated 12 additional nesting
sites, seven from Corsica and five from
continental France. Moreover, to determine
whether the Corsican supercolony may
possibly originate from the Main or
Catalonian supercolonies, we conducted a
genetic analysis where we compared the
genotypes of workers from each of the three
supercolonies at 12 microsatellites loci and
one fragment of the mitchondrial
cytochrome b gene (Vogel *et al.*, 2010).
Finally, we complemented these
studies by analysing the CHCs of workers
collected from all the studied sites. This
allowed us to compare the behavioural,
genetic and chemical differentiation of
individuals from each of the three
supercolonies.

**Materials & methods**

*Study context and field collection*

We collected nests of *Linepithema humile* from 18 sites in southern Europe. Sixteen colony fragments were collected in France: 10 along the Corsican coast (Ajaccio - AJA, Bigulia - BIG, Calvi -CAL, Campomoro - CAM, Canella - CAN, Liamone - LIA, Olmeto - OL, Porto Vecchio - PVE, Santa Giulia - SGI, and Tizzano - TIZ) and six on the coast of continental France (Anthéor - ANT, Ceyreste - CEY, Danio - DAN, Les Embiez - EMB, Fréjus - FRE and Théoule - THE). The remaining two nesting sites were collected in Spain, one in Caldès d'Estrac (CDE) and the other in Sant Feliù de Guixols (SFG).

Unfortunately, only one of these
nests (CDE) was found to actually belong to
the Catalonian supercolony (see results),
hence requiring to use a single nesting site for
all the tests including the Catalonian
supercolony. However, the inclusion of a
single nesting site for the Catalonian
supercolony has only limited consequences
for this study whose primary aim is to

compare the Main and Corsican
supercolonies. In that respect, it is important
to note that the previous studies have shown
that aggression is invariably high between
nests of the Catalonian supercolony and
nests of both the Corsican and Main
supercolonies (Blight et al., 2010b; Giraud et
al., 2002; Jaquiy et al., 2005). The level of
aggression observed in the present study was
the same as that observed in those studies.
Moreover, the hydrocarbon profiles of the
workers from CDE were typical of those of
nests of the Catalonian supercolony (Blight
et al., 2010b). Finally, the genetic data on
individuals of the nesting site CDE are also
completely congruent with those obtained
with other nests of the Catalonian
supercolony (Giraud et al., 2002; Vogel et
al., 2010). In particular, the alleles of the
microsatellites loci Lhum-18 and Lhum-28
and the haplotype H9 characteristic of the
Catalonian supercolony (Jaquiy et al.,
2005; Vogel et al., 2010) were all detected
in the nesting site of CDE (see result section).
Thus, although we acknowledge a problem
of pseudoreplication in the statistical
analyses, we believe that the main results of
this study are not influenced by the inclusion
of a single nest of the Catalonian
supercolony.

Colony fragments were collected in
October 2008 with a minimum of 10 000
workers and 20 queens. These were kept in
a plastic nest container (360 x 240 x 110
mm) filled with their original substrate. In
addition to the arthropods caught with the
original substrate that served as a protein
source, the colonies were supplied with
honey and water ad libitum. Colony
fragments were maintained in the laboratory
at 23 ± 2 °C with soil moisture. From each
of the 18 nesting sites, we also froze some
workers immediately upon collection for
chemical analyses and stored others in
alcohol for the genetic analyses.

## *Behavioural assays*

We assessed the level of worker-
worker aggression between all nesting sites
using combinations of pairwise encounters
consisting of confrontations between two
groups of 100 workers that were competing
for food and nesting space (Blight *et al.*,
2010b). Before each assay, workers from
each of the two nesting sites were placed in
closed artificial nests (60 x 45 x 50 mm) with
their own nest material and moist cotton for
1 h, for acclimatization and construction of
small galleries.

The nests were connected via plastic
tubes to a common foraging arena (60 x 90
x 50 mm) provided with honey in the
center; following acclimatization, the
workers were allowed access to the foraging
arena. We monitored behavioural
interactions among workers for 20 min,
using the following classification of
interactions: (1) touch (contacts including
prolonged antennation); (2) avoidance

(contacts followed by at least one ant
running away); (3) aggression (<2 s, lunging,
biting); (4) asymmetrical intense aggression
(>2 s, at least one aggressor and one non-
aggressive individual, lunging, biting, pulling
legs or antennae) and (5) symmetrical
intense fighting (all individuals are
aggressors; gaster flexion; death a few
seconds after the beginning of fights). Scores
3, 4 and 5 were considered as aggressive
interactions. The highest score observed
during the 20 min for each assay was
recorded. To minimize the effect of
pseudoreplication, aggression scores were
averaged across replicates and used as the
aggression index of the nest pair. Twenty-
four hours after the beginning of the
experiment, the trial was stopped to count
dead and living workers. Three replicates
were conducted for each of the 153 pairs of
nesting sites and 16 within-nest
confrontations (control). Due to limiting
number of individuals, we were not able to
conduct three replicates for all
confrontations involving the CAL and BIG
nesting sites, as well the within-nest
confrontations in these nesting sites.
Individuals were used only once per
behavioural test. The supercolony affiliation
of each nesting site was determined
according to the above bioassay. Two
nesting sites exhibiting aggression (score
above 2) were classified as belonging to
different supercolonies, whereas those with
indices of 1 or 2 were classified as belonging
to the same supercolony. Differences in
levels of aggression and mortality rates were
compared with the non-parametric *Mann–
Whitney U test*.

Cuticular hydrocarbon analyses

We first identified by gas chromatography /
mass spectrometry (GC / MS) the cuticular
compounds of a pool of 30 workers from
one nesting site of each of the three
European supercolonies (CDE for the
Catalonian supercolony, CEY for the Main
European supercolony and FRE for the
Corsican supercolony). For extractions, we
immersed 30 workers of each nesting site for
1 h in 100 µL of hexane. After extract
concentration, 2 mL of each sample were
injected into an Agilent 6890N GC equipped
with Chrompack CPSil5WCOT apolar
capillary column (fused silica, 25 m x 0.25
mm; thickness of stationary phase, 0.12 µm).
The injector was in splitless mode with
helium carrying gas flow at 1 mL/min. Initial
oven temperature was held at 70 °C for 1
min and then increased at a rate of 30 °C /
min to 180 °C, followed by 5 °C/min to 320
°C, and a final hold of 15 min. The GC was
coupled with a 5375 Agilent Technologies
Mass Spectrometer, using 70 eV electron
impact ionization. Compounds were
identified on the basis of their mass spectra
in comparison with standard com-pounds,
when available, and published spectra.
Second, we quantified CHCs of 10 individual

workers from each of the 18 nesting sites by
individually immersing them in 10 µL of
hexane for 15 min. The samples were
analysed using capillary gas chromatography
(Varian 3900 Gas chroma-tograph),
equipped with a flame ionization detector
and a Chrompack CPSil5WCOT apolar
capillary column (fused silica, 25 m x 0.25
mm; thickness of stationary phase, 0.12 µm).
Oven temperature was programmed from
100 to 220 °C at the rate of 10 °C/min, then
from 220 to 320 °C at the rate of 3 °C/min,
with a final hold of 10 min. Comparison of
cuticular profiles was based on the
proportions of 51 major CHCs displayed on
a chromatogram by using Star 5.5 (Varian)
software. Integrated values were adjusted by
applying the correction coefficient K =
0.0038x²)0.1738X + 2.9683, where x is the
number of carbon atoms, which corrects
variations in detector sensitivity as a function
of carbon chain length. This coefficient was
determined after injection of the same
quantity of 14 n-alkanes with chain-lengths
of 18, 20, 22, 24, 28, 32, 34, 36, 38, 40, 42,
44, 46 and 48 carbon atoms, respectively.

Between nesting-site variability in profile
composition was assessed using multivariate
principal component analysis (PCA; Statistica
v.6, Statsoft 2001) based on the relative
proportion of the CHCs identified. In total,
we identified 130 peaks but only used the 51
peaks that accounted for at least 0.5% of the
overall peak area of the whole profile in one
of the nesting sites. As the resulting data
matrix contained many zeros (CHCs present
in a profile but absent from another),
standard multivariate procedures were not
applicable. We thus attributed a very small
value (0.001%) to eliminate the zeros. We
also conducted a separate *K-means* cluster
analysis to group together data points
showing similar cuticular hydrocarbon
profiles (Hartigan & Wong, 1979). The
number of groups to be obtained is
unknown a priori, and the choice is
somewhat arbitrary. To select an optimum
number, we used the ratio of inter- to
intragroup variance of an increasing number
of groups, considering a stopping rule when
the gain was <0.10.

Genetic analyses

DNA was extracted from 10 workers
per nesting site (total 180) using the
Puregene DNA Isolation Kit (Gentra Systems)
and analysed at 12 microsatellite loci: Lhum-
3, Lhum-11, Lhum-13, Lhum-19, Lhum-28,
Lhum-33, Lhum-35, Lhum-39, Lhum-52,
Lhum-62, Lihu-S3 and LihuT1(Krieger &
Keller, 1999; Tsutsui et al., 2000). PCR
products were separated on polyacrylamide
gels and run on an ABI 377 XL automated
sequencer (Applied Biosystems) with
subsequent scoring of alleles using
GENEMAPPER 4.0 (Applied Biosystems).
One fragment of the mitochondrial DNA
was also amplified for one worker per
nesting site [524 bp of the cytochrome b

gene, PEDERSEN et al. (Pedersen et al.,
2006)]. After PCR products were checked on
a 1.5% agarose gel, they were purified using
the QIAquick purification kit (Qiagen,
Valencia, CA, USA) and directly double-
stranded sequenced by the automated
sequencer using the BigDye terminator
ready-reaction kit. Sequence data were
edited and compiled using LASERGENE 7.1.0
(Dnastar Inc., USA).

To confirm the assignment of nesting
sites to the Main, the Corsican and the
Catalonian super-colonies, we used a
Bayesian clustering method. Bayesian
clustering methods were used to determine,
without a priori assumptions, the number of
genetic groups in a data set and subsequently
to assign individuals or groups of individuals
to these genetic groups. This approach has
been recognized as efficient for the
identification of supercolonies, because the
clustering of nests is highly congruent with
aggression tests (Jaquiery et al., 2005;
Pedersen et al., 2006; Vogel et al., 2010).
We used the clustering method implemented
in STRUCTURE 2.1 (Pritchard et al., 2000).
This method infers the number of different
clusters (K) that best suit a data set and
assigns individuals to the inferred clusters. All
simulations performed in STRUCTURE
featured 500 000 runs following a burn-in
period consisting of 100 000 runs. Ten
separate runs were performed for each value
of K tested (between 1 and 15). We ensured
accurate estimates of the simulation values
by checking that model parameters equilibrated before the end of the burn-in phase and that posterior probabilities were consistent across all ten runs. Then, we determined the appropriate value of K for each data set using the DK method of EVANNO et al. (Evanno et al., 2005). For the analyses, each nesting site was considered as a group, and the maximum number of genetically divergent groups (K) was set at 18, the number of nesting sites included in the data set. Each analysis was repeated 10 times to ensure consistency of results between different runs. To prevent the high level of genetic differentiation owing to the presence of Catalonian samples from obscuring the structure of the two supercolonies of interest here, we performed all the above analyses both with and without the Catalonian nesting site (CDE).

To determine the genetic structure of the 18 nesting sites forming the three supercolonies, we assessed genetic differentiation with three different approaches. First, pairwise PST we computed with FSTAT 2.9.3.2 (Goudet, 1995) between all pairs of supercolonies and nesting sites. For these pairwise comparisons, significance levels for multiple tests were adjusted according to the BOFERONI corrections. Second, to compare the level of structure between and within the three supercolonies, a hierarchical analysis was performed with HIERFSTAT (Goudet, 2005). The different levels considered were the

individuals (Ind), within nesting sites (Nest) and nesting sites within the supercolonies (Sc). Significance of FNest/Sc was tested in HIERFSTAT(de Meeus & Goudet, 2007) with 10 000 permutations of individuals between nests within supercolonies and FSc/Pop with 10 000 permutations of nests between supercolonies. This analysis was performed with and without the nesting site belonging to the Catalonian supercolony (CDE). Finally, to infer the relationship between the nesting sites, we performed a principal component analyses (PCA) on the microsatellite data using the program PCAGEN 1.2 (Goudet, 2000). The per cent inertia of each principal component axis and its respective P value were estimated by performing 10 000 randomizations of multilocus genotypes. This analysis was also performed with and without the nesting site belonging to the Catalonian supercolony (CDE).

To investigate the relationship between the different nesting sites sampled and the worldwide supercolonies described in VOGEL et al. (Vogel et al., 2010), we performed a principal component analyses (PCA) on the microsatellite data using the program PCAGEN 1.2 (Goudet, 2000) by using all the nesting sites of the present study and those of VOGEL et al. (Vogel et al., 2010). To obtain more detailed information on the relationship between the Main and Corsican supercolonies, we conducted the same analysis by considering only
supercolonies harbou-ring the H1 haplotype and excluding the South Africa population because it was clearly differentiated from all other supercolonies with the H1 haplotype (Figure 16a). The same set of 12 microsatellites was used for all analyses and all the analyses performed by the same person (V.V.) in the same laboratory (Lausanne). The per cent inertia of each principal component axis and its respective P value were estimated by performing 10 000 randomizations of multilocus genotypes.

To test the hypothesis that the Corsican supercolony derived from the Main supercolony, we measured the allelic richness for each nesting site and each supercolony, correcting the observed number of alleles to the estimated value for the minimum common sample size using rarefaction statistics (Petit et al., 1998). Statistical significance of differences in allelic richness (corrected number of alleles) per locus between the Main and Corsican nesting sites were assessed in two-sided permutation tests applying 15 000 randomizations. We also estimated the allelic nestedness by estimating, for each pair of nesting sites, the percentage of alleles found in one nesting site that were also present in the other nesting site and vice versa. This analysis was based on the principle that nesting sites deriving from secondary introductions should contain a subset of the alleles present in the nesting site of origin.

*Association between levels of aggression, chemical and genetic distances*

To test for an association between the level of aggression, chemical distance (Euclidian distance based on CHCs) and genetic distance (pairwise FST) between nesting sites, we performed Mantel tests by using the non-parametric Spearman test with XLSTAT 2011. We determined statistical significance by per-forming 10 000 random permutations. Mantel tests were performed both with and without the Catalonian supercolony.

## Results

### *Behavioural tests*

The tests of aggression between workers from all nesting sites confirmed the existence of three supercolonies in southern Europe, the Catalonian supercolony and the Main supercolony already described by GIRAUD et al. (Giraud et al., 2002), as well as the Corsican supercolony identified by BLIGHT et al. (Blight et al., 2010b) (Tableau 14). This Corsican supercolony was composed of eight nesting sites in Corsica (AJA, BIG, CAM, LIA, OL, PVE, SGI and TIZ), three in continental France (ANT, FRE and THE) and one in Spain (SFG). The Main supercolony contained two nesting sites in Corsica (CAL and CAN) and three in continental France (CEY, DAN and EMB) (Carte 4).
<table><caption>Tableau 14 : Indices of aggression (left part) and percentages of mortality after 24 h (right part) for each nest pair across replicates and within-nest confrontations. Values in bold indicate pairs of nesting-sites assigned to the Main and Corsican supercolonies. AJA, Ajaccio; ANT, Anthéor; BIG, Biguglia; CAL, Calvi; CAM, Campomoro; CAN, Cannella; CDE, Caldès d'Estrac; CEY, Ceyreste; DAN, Danio; EMB, Les Embiez; FRE, Fréjus; LIA, Liamone; OL, Olmeto; PVE, Porto-Vecchio; SFG, Sant Feliù de Guixols; SGI, Santa Giulia; THE, Théoule; TIZ, Tizzano.</caption><thead><tr><th></th><th>OL</th><th>AJA</th><th>LIA</th><th>BIG</th><th>PVE</th><th>SGI</th><th>TIZ</th><th>CAM</th><th>FRE</th><th>ANT</th><th>THE</th><th>SFG</th><th>CAL</th><th>CAN</th><th>CEY</th><th>EMB</th><th>DAN</th><th>CDE</th></tr></thead><tbody><tr><td>OL</td><td>1-0</td><td>3.7</td><td>4.7</td><td>4.2</td><td>3.2</td><td>1.8</td><td>5.9</td><td>2.8</td><td>4.7</td><td>6.2</td><td>3.8</td><td>4.4</td><td>19.5</td><td>15.9</td><td>12.9</td><td>31.7</td><td>49</td><td>80.1</td></tr><tr><td>AJA</td><td>1</td><td>1-1.5</td><td>8.8</td><td>3.1</td><td>1.7</td><td>4</td><td>5.2</td><td>5</td><td>2.8</td><td>6.4</td><td>3.8</td><td>4.8</td><td>3.6</td><td>24.3</td><td>21</td><td>16.3</td><td>17</td><td>75.8</td></tr><tr><td>LIA</td><td>1.33</td><td>1</td><td>1-6.1</td><td>5</td><td>4.6</td><td>3.8</td><td>4.6</td><td>1.8</td><td>2.8</td><td>2.3</td><td>2.9</td><td>5.6</td><td>9.9</td><td>3.7</td><td>11.2</td><td>9.2</td><td>11.9</td><td>70</td></tr><tr><td>BIG</td><td>1</td><td>2</td><td>1</td><td>-</td><td>5.7</td><td>6.4</td><td>3.1</td><td>1.5</td><td>3.6</td><td>2.5</td><td>4.1</td><td>3.9</td><td>12.3</td><td>2.3</td><td>8.8</td><td>26.5</td><td>17.7</td><td>64.2</td></tr><tr><td>PVE</td><td>1</td><td>1</td><td>1.33</td><td>1.33</td><td>1-5.1</td><td>4.5</td><td>1.5</td><td>1.2</td><td>4.8</td><td>8.8</td><td>3.9</td><td>3.1</td><td>4.7</td><td>5.1</td><td>5.6</td><td>16.8</td><td>24.1</td><td>57.3</td></tr><tr><td>SGI</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1-2.4</td><td>3.8</td><td>3.7</td><td>4.5</td><td>5.5</td><td>3.8</td><td>5.2</td><td>4.7</td><td>11.7</td><td>4.6</td><td>36.6</td><td>32.7</td><td>65.3</td></tr><tr><td>TIZ</td><td>1</td><td>1.33</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1-1.1</td><td>3.4</td><td>2.6</td><td>6.4</td><td>3</td><td>1.1</td><td>4.6</td><td>10</td><td>20.5</td><td>22.4</td><td>23</td><td>70.8</td></tr><tr><td>CA</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1-0.8</td><td>2.5</td><td>9</td><td>1.3</td><td>2.2</td><td>16</td><td>17.3</td><td>10.1</td><td>19</td><td>18.9</td><td>68.6</td></tr><tr><td>M</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1-0.5</td><td>6.3</td><td>2.1</td><td>4.4</td><td>5.3</td><td>6.7</td><td>3.6</td><td>7.6</td><td>4.8</td><td>72.4</td></tr><tr><td>FRE</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1-1.5</td><td>3.7</td><td>10.8</td><td>1.6</td><td>8.9</td><td>8.3</td><td>18.7</td><td>29.2</td><td>69.5</td></tr><tr><td>ANT</td><td>1.67</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1-1.5</td><td>3.7</td><td>10.8</td><td>1.6</td><td>8.9</td><td>8.3</td><td>18.7</td><td>29.2</td><td>72</td></tr><tr><td>THE</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1.33</td><td>1-0.5</td><td>3.6</td><td>3.6</td><td>5.2</td><td>4.3</td><td>17.9</td><td>18.6</td><td>72</td></tr><tr><td>SFG</td><td>2</td><td>1.33</td><td>2</td><td>1</td><td>1</td><td>1.33</td><td>1.33</td><td>1</td><td>1</td><td>3.67</td><td>1</td><td>1-0.5</td><td>3.9</td><td>4.9</td><td>6.6</td><td>13.9</td><td>12.7</td><td>74</td></tr><tr><td>CAL</td><td>4</td><td>4</td><td>3</td><td>4</td><td>3</td><td>4</td><td>3.5</td><td>3.5</td><td>3</td><td>3</td><td>3</td><td>4</td><td>-</td><td>3</td><td>7.2</td><td>2</td><td>2</td><td>61</td></tr><tr><td>CAN</td><td>4.67</td><td>4.33</td><td>3.33</td><td>3</td><td>3.33</td><td>3.67</td><td>3.67</td><td>4</td><td>3</td><td>4.5</td><td>4</td><td>4</td><td>1</td><td>1-2.5</td><td>5</td><td>3.5</td><td>5</td><td>68.1</td></tr><tr><td>CEY</td><td>4.33</td><td>4.33</td><td>4</td><td>3.67</td><td>3.67</td><td>3.67</td><td>4</td><td>4</td><td>3.67</td><td>3.67</td><td>3.67</td><td>4</td><td>1.5</td><td>1</td><td>1-1.5</td><td>1.6</td><td>1.9</td><td>72.1</td></tr><tr><td>EMB</td><td>4.67</td><td>4</td><td>4</td><td>4.33</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4.33</td><td>4</td><td>3.67</td><td>1</td><td>1</td><td>1</td><td>1-3.6</td><td>3.2</td><td>74.8</td></tr><tr><td>DAN</td><td>4.67</td><td>4</td><td>4</td><td>4.33</td><td>3.67</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4.33</td><td>4</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1-2.7</td><td>71.3</td></tr><tr><td>CDE</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>1-4.1</td></tr></tbody></table>

<figure><img src="image_57.png" /></figure>
Carte 4: Geographical location of the 18 studied nesting sites of Linepithema humile in southern Europe, and their affiliation to the three supercolonies based on the behavioural data; the Catalonian supercolony (square), the Corsican supercolony (dots) and the Main supercolony (triangles).

No aggression was recorded between
workers from the same nesting site (Main
super-colony: mean ± SE = 1 ± 0, n = 4;
Corsican super-colony: mean ± SE = 1 ± 0, n
= 11; Catalonian supercolony: 1, n = 1) and
from sites of the same supercolony (Main
supercolony: 1.05 ± 0.05, n = 10; Corsican
super-colony: 1.05 ± 0.03, n = 66). The level
of aggression between sites of the Corsican
and Main supercolonies varied from
moderate to high (mean ± SE = 3.80 ±
0.05, n = 60) and was significantly higher
than the level of aggression within the Main
and Corsican supercolonies (both P < 0.0001,
Mann-Whitney U tests).
By contrast,
aggression was invariably high when workers
of these two supercolonies were paired with
workers of the Catalonian supercolony
(Corsican vs. Catalonian supercolonies: 5 ±
0, n = 12; Main vs. Catalonian supercolonies:
5 ± 0, n = 5). In both cases, the level of
aggression was higher than between the Main
and Corsican supercolonies (both P < 0.0001,
Mann-Whitney U tests).

Overall, there was considerable
variability among workers in the level of
aggression in encoun-ters between sites of the
Corsican and Main super-colonies with some
workers eliciting fights, whereas others
moved freely from one nest to the other. In

complete contrast, all workers were highly
aggressive when workers of either the Main
or the Corsican supercolonies were paired
with workers of the Catalonian
supercolony. These differences in the level
of aggression resulted in differences in
mortality rate (Tableau 14). The percentage of
dead workers was 2.2 ± 0.4% (n = 16) for
confrontations within nesting sites, 4.1 ±
0.2% (n = 66) for confrontations within
the Corsican supercolony, 3.4 ± 0.8% (n =
10) for confrontations within the Main
supercolony, 13.7 ± 1.3% (n = 60) for
confrontations between the Main and
Corsican supercolonies, 70 ± 1.7% (n = 12)
for confrontations between the Corsican
and Catalonian supercolonies and 69.5 ±
2.4% (n = 5) for confrontations between
the Main and Catalonian supercolonies. The
mortality rates were significantly higher in
confrontations between the Main –
Catalonian and Corsican – Catalonian
supercolonies than those between Main –
Corsican supercolonies (both P < 0.0001,
Mann-Whitney U tests).

Cuticular hydrocarbon analysis

Based on the pooled samples of workers
from the three nesting sites representing the
three supercolonies, we identified 135 CHCs
ranging in chain length from 25 to 51
carbon atoms. Cuticular profiles of
Argentine ant workers were dominated by
methyl-branched alkanes (mono-, di- and
tri-methyl), although some straightchain *n*-alkanes and *n*-alkenes were present (Tableau 16, p138). The profile of workers from the Catalonian supercolony was more diverse and wide ranging than the profiles of workers from the two other supercolonies. We identified 97 hydrocarbons ranging from 25 to 51 carbon chain lengths in the profile of workers from the Catalonian supercolony (Figure 11A), 49 hydro-carbons ranging from 25 to 40 carbon atoms in the profile of workers from the Main supercolony (Figure 11B) and 55 hydrocarbons ranging from 25 to 41 carbon atoms in the profile of workers from the Corsican supercolony (Figure 11C, Annexe : Tableau 16). Only 16 CHCs were common to the three supercolonies. The number of hydrocarbons in common between workers of different supercolonies was 18 for the Catalonian and Main supercolonies, 20 for the Catalonian and Corsican supercolonies, and 44 for the Main and Corsican supercolonies. However, in cases in which several compounds eluted as a single peak, there were occasions where one of the components comprising the peak was common to two or more of the supercolonies but not the other coeluting compounds.

The PCA, based on the proportions of the 51 major CHCs of the 10 individuals from each of the 18 nesting sites, separated the nesting site from the Catalonian supercolony from the others on PC1 that accounted for 39.99% of the total variance (Figure 12).

For the remaining nesting sites, discrimination was less pronounced and, projected on PC2, accounted for 12.50% of the total variance. The *k*-means analysis identified three chemical groups that matched perfectly with the three supercolonies identified by the behavioural tests (1 000 permutations were used to assess an optimum number of three groups). All individuals from a given location were assigned to the same group, except for one worker from CAL and one from TIZ, which were assigned to other groups.

Figure 11 : Gas chromatograms of total body wash of 30 workers of Linepithema humile from (A) Caldès d'Estrac (Catalonian supercolony), (B) Ceyreste (Main European supercolony), (C) Fréjus (Corsican supercolony). Peak numbers correspond to the list of the identified cuticular hydrocarbons in Table S1.
<figure><img src="image_58.png" /><figcaption>Figure 11</figcaption></figure>
Figure 12 : Principal component analysis (PCA) of worker cuticular hydrocarbons (CHCs) from the 18 nesting sites.
The PCA was based on the relative proportion of 51 CHCs. Axis 1 accounts for 39.99% of total variance and axis 2
for 12.50%. Results of K-means cluster analysis, group 1 (squares): Calvi, Cannella, Ceyreste, Danio, Les Embiez;
group 2 (circles): Anthéor, Ajaccio, Biguglia, Campomoro, Fréjus, Liamone, Olmeto, Porto-Vecchio, Sant Feliù de
Guixols, Santa Giulia, Tizzano, Théoule; and group 3 (triangles): Caldès d'Estrac. The ellipses correspond to the
95% confidence limit of the data.

<figure><img src="image_59.png" /><figcaption>Figure 12 :</figcaption></figure>
<figure><img src="image_60.png" /><figcaption>Figure 14 A: Assignment of individuals into genetic groups resulting from the analyses performed with the software STRUCTURE when individuals of all nesting sites were included and when K = 2 (panel A), when individuals of all nesting sites were included except those of the Catalonian nesting site (CDE).</figcaption></figure>

<figure><img src="image_61.png" /></figure>

## Genetic analyses *

The three supercolonies could not be clearly distinguished on the basis of the genetic data. The Bayesian analysis performed with STRUCTURE clearly distinguished the CDE nesting site (Catalonian supercolony) from the others.

The most probable number of genetic groups was K = 2 (Figure 13A, B). All the individuals sampled in CDE belonged to the first genetic group, with an average proportion of membership of 1.00, while the other nesting sites were assigned to the second genetic group, with an average
proportion of membership always higher
than 0.97 (Figure 13).

To determine whether the inclusion
of CDE affected the analysis, we conducted
the same analysis without the individuals
from CDE. This second analysis gave very
ambiguous results. According to method
developed by EVANNO et al. (Evanno et al.,
2005), the most probable number of genetic
groups was four (Figure 13D; K = 4).
However, this clustering made no sense
because individuals of a given nesting site
were not consistently assigned to the same
genetic group. Instead, they were typically
assigned to each of the four genetic groups (

Figure 15B as an example of one of the
run).

This probably reflects the fact that
STRUCTURE does not permit to identify the
true number of genetic groups when K = 1.
Indeed the analyses of LnP(D) curve (Figure
13C) reveal that the K for which the LnP(D)
was highest and exhibited the lowest
variation between runs was K = 1 suggesting
that the individuals of the 17 nesting sites
included in the analysis form a single genetic
group. When forcing K = 2, the results were
similarly very ambiguous. Individuals from
each of the 17 nesting sites were assigned to
both genetic groups (with pro-portion
varying between 0.3 and 0.7

<caption>Figure 15C).</caption>

Figure 15 BC : Assignment of individuals into genetic
groups resulting from the analyses performed with the
software STRUCTURE when individuals of all nesting
sites were included and when K = 4 (panel B) and
when individuals of all nesting sites were included
except those of the Catalonian nesting site (CDE) and
when K =4 (panel C).

<figure><img src="image_62.png" /><figcaption>C. All nesting sites except the Catalonian one; K=2</figcaption></figure>

<figure><img src="image_63.png" /></figure>
<table><caption>Tableau 15 : Proportions of shared alleles between the three European supercolonies of Linepithema humile (left part) and FST values (right part). Values in bold indicate pairs of nesting-sites assigned to the Main and Corsican supercolonies. AJA, Ajaccio; ANT, Anthéor; BIG, Biguglia; CAL, Calvi; CAM, Campomoro; CAN, Cannella; CDE, Caldès d'Estrac; CEY, Ceyreste; DAN, Danio; EMB, Les Embiez; FRE, Fréjus; LIA, Liamone; OL, Olmeto; PVE, Porto-Vecchio; SFG, Sant Feliù de Guixols; SGI, Santa Giulia; THE, Théoule; TIZ, Tizzano.</caption><thead><tr><th>Nesting-sites</th><th>OL</th><th>AJA</th><th>LIA</th><th>BIG</th><th>PVE</th><th>SGI</th><th>TIZ</th><th>CAM</th><th>FRE</th><th>ANT</th><th>THE</th><th>SFG</th><th>CAL</th><th>CAN</th><th>CEY</th><th>EMB</th><th>DAN</th><th>CDE</th></tr></thead><tbody><tr><td>OL</td><td>-</td><td>0,04</td><td>0,07</td><td>0,08</td><td>0,03</td><td>0,12</td><td>0,03</td><td>0,08</td><td>0,09</td><td>0,07</td><td>0,04</td><td>0,11</td><td><strong>0,27</strong></td><td><strong>0,16</strong></td><td><strong>0,13</strong></td><td><strong>0,18</strong></td><td><strong>0,11*</strong></td><td>0,48</td></tr><tr><td>AJA</td><td>93/78</td><td>-</td><td>0,01</td><td>0,05</td><td>0,00</td><td>0,02</td><td>0,09</td><td>0,06</td><td>-0,01</td><td>0,04</td><td>0,00</td><td>0,12</td><td><strong>0,23</strong></td><td><strong>0,12</strong></td><td><strong>0,09</strong></td><td><strong>0,20</strong></td><td><strong>0,16</strong></td><td>0,47</td></tr><tr><td>LIA</td><td>89/80</td><td>88/93</td><td>-</td><td>0,07</td><td>0,05</td><td>0,08</td><td>0,07</td><td>0,09</td><td>0,05</td><td>0,02</td><td>0,01</td><td>0,12</td><td><strong>0,18</strong></td><td><strong>0,06</strong></td><td><strong>0,04</strong></td><td><strong>0,13</strong></td><td><strong>0,12</strong></td><td>0,44</td></tr><tr><td>BIG</td><td>93/78</td><td>88/88</td><td>87/81</td><td>-</td><td>0,09</td><td>0,11</td><td>0,10</td><td>0,10</td><td>0,04</td><td>0,05*</td><td>0,02</td><td>0,09</td><td><strong>0,22</strong></td><td><strong>0,08</strong></td><td><strong>0,12*</strong></td><td><strong>0,17</strong></td><td><strong>0,19</strong></td><td>0,43</td></tr><tr><td>PVE</td><td>85/82</td><td>81/93</td><td>87/93</td><td>78/89</td><td>-</td><td>0,08</td><td>0,05</td><td>0,01*</td><td>0,06*</td><td>0,06*</td><td>0,04*</td><td>0,13*</td><td><strong>0,27*</strong></td><td><strong>0,13</strong></td><td><strong>0,10</strong></td><td><strong>0,19</strong></td><td><strong>0,15</strong></td><td>0,47*</td></tr><tr><td>SGI</td><td>81/92</td><td>75/100</td><td>77/96</td><td>72/96</td><td>82/96</td><td>-</td><td>0,15</td><td>0,10*</td><td>0,11*</td><td>0,13*</td><td>0,08*</td><td>0,10</td><td><strong>0,21*</strong></td><td><strong>0,12</strong></td><td><strong>0,10</strong></td><td><strong>0,16</strong></td><td><strong>0,21*</strong></td><td>0,50</td></tr><tr><td>TIZ</td><td>85/82</td><td>75/86</td><td>77/82</td><td>81/93</td><td>79/79</td><td>83/71</td><td>-</td><td>0,04</td><td>0,13</td><td>0,09</td><td>0,05</td><td>0,10*</td><td><strong>0,28*</strong></td><td><strong>0,11</strong></td><td><strong>0,04</strong></td><td><strong>0,06</strong></td><td><strong>0,04*</strong></td><td>0,50</td></tr><tr><td>CAM</td><td>81/92</td><td>75/100</td><td>77/96</td><td>75/100</td><td>82/96</td><td>92/92</td><td>75/88</td><td>-</td><td>0,13</td><td>0,09*</td><td>0,05*</td><td>0,08*</td><td><strong>0,26*</strong></td><td><strong>0,10*</strong></td><td><strong>0,05*</strong></td><td><strong>0,15*</strong></td><td><strong>0,16*</strong></td><td>0,48*</td></tr><tr><td>FRE</td><td>89/80</td><td>84/90</td><td>87/87</td><td>88/93</td><td>86/80</td><td>92/73</td><td>93/87</td><td>96/77</td><td>-</td><td>0,08*</td><td>0,04</td><td>0,14</td><td><strong>0,31</strong></td><td><strong>0,16</strong></td><td><strong>0,15</strong></td><td><strong>0,24</strong></td><td><strong>0,17*</strong></td><td>0,48*</td></tr><tr><td>ANT</td><td>89/77</td><td>88/90</td><td>93/90</td><td>88/90</td><td>89/81</td><td>96/74</td><td>89/81</td><td>96/74</td><td>90/87</td><td>-</td><td>0,01*</td><td>0,13*</td><td><strong>0,25*</strong></td><td><strong>0,13*</strong></td><td><strong>0,11</strong></td><td><strong>0,18</strong></td><td><strong>0,21*</strong></td><td>0,42*</td></tr><tr><td>THE</td><td>93/78</td><td>91/91</td><td>90/84</td><td>91/91</td><td>89/78</td><td>96/72</td><td>89/78</td><td>100/75</td><td>93/88</td><td>90/88</td><td>-</td><td>0,09</td><td><strong>0,14</strong></td><td><strong>0,02*</strong></td><td><strong>0,04*</strong></td><td><strong>0,14</strong></td><td><strong>0,12</strong></td><td>0,41</td></tr><tr><td>SFG</td><td>85/68</td><td>81/76</td><td>87/76</td><td>88/82</td><td>93/76</td><td>96/68</td><td>86/71</td><td>96/68</td><td>87/76</td><td>87/79</td><td>84/79</td><td>-</td><td><strong>0,24</strong></td><td><strong>0,12</strong></td><td><strong>0,06*</strong></td><td><strong>0,09*</strong></td><td><strong>0,13</strong></td><td>0,44*</td></tr><tr><td>CAL</td><td><strong>67/82</strong></td><td><strong>63/91</strong></td><td><strong>67/91</strong></td><td><strong>56/82</strong></td><td><strong>68/86</strong></td><td><strong>67/73</strong></td><td><strong>61/77</strong></td><td><strong>71/77</strong></td><td><strong>63/86</strong></td><td><strong>61/86</strong></td><td><strong>59/86</strong></td><td><strong>56/86</strong></td><td>-</td><td>0,11*</td><td>0,21*</td><td>0,27</td><td>0,32</td><td>0,49*</td></tr><tr><td>CAN</td><td><strong>74/77</strong></td><td><strong>72/88</strong></td><td><strong>77/88</strong></td><td><strong>72/88</strong></td><td><strong>79/85</strong></td><td><strong>79/73</strong></td><td><strong>71/77</strong></td><td><strong>83/77</strong></td><td><strong>80/92</strong></td><td><strong>74/88</strong></td><td><strong>75/92</strong></td><td><strong>68/88</strong></td><td>82/69</td><td>-</td><td>0,06</td><td>0,14</td><td>0,15*</td><td>0,47</td></tr><tr><td>CEY</td><td><strong>74/74</strong></td><td><strong>69/81</strong></td><td><strong>70/78</strong></td><td><strong>69/81</strong></td><td><strong>71/74</strong></td><td><strong>79/70</strong></td><td><strong>79/81</strong></td><td><strong>83/74</strong></td><td><strong>70/78</strong></td><td><strong>71/81</strong></td><td><strong>66/78</strong></td><td><strong>65/81</strong></td><td>86/70</td><td>73/70</td><td>-</td><td>0,04</td><td>0,10</td><td>0,50*</td></tr><tr><td>EMB</td><td><strong>74/83</strong></td><td><strong>66/88</strong></td><td><strong>67/83</strong></td><td><strong>69/92</strong></td><td><strong>71/83</strong></td><td><strong>83/83</strong></td><td><strong>75/88</strong></td><td><strong>83/83</strong></td><td><strong>70/88</strong></td><td><strong>71/92</strong></td><td><strong>66/88</strong></td><td><strong>65/92</strong></td><td>82/75</td><td>69/75</td><td>78/88</td><td>-</td><td>0,13*</td><td>0,52*</td></tr><tr><td>DAN</td><td><strong>78/78</strong></td><td><strong>75/89</strong></td><td><strong>77/85</strong></td><td><strong>78/93</strong></td><td><strong>79/81</strong></td><td><strong>83/74</strong></td><td><strong>82/85</strong></td><td><strong>88/78</strong></td><td><strong>77/85</strong></td><td><strong>74/85</strong></td><td><strong>75/89</strong></td><td><strong>68/85</strong></td><td>73/59</td><td>73/70</td><td>74/74</td><td>83/74</td><td>-</td><td>0,55*</td></tr><tr><td>CDE</td><td>41/35</td><td>34/35</td><td>40/39</td><td>41/42</td><td>43/39</td><td>38/29</td><td>39/35</td><td>42/32</td><td>43/42</td><td>39/39</td><td>41/42</td><td>41/45</td><td>50/35</td><td>50/42</td><td>33/29</td><td>38/29</td><td>37/32</td><td>-</td></tr></tbody></table>

<figure><img src="image_64.png" /><figcaption>Figure 16 : Principal component analysis (PCA) based on microsatellite allele frequencies calculated on nesting sites. (A) The 18 nesting sites of the Main, the Corsican and the Catalonian supercolonies of the present study (represented by dots) were included in the analysis. (B) Analysis based exclusively on the nesting sites of the Main and Corsican supercolonies</figcaption></figure>
Further analyses revealed a similar pattern with a clear genetic differentiation of the Catalonian supercolony but no clear differentiation between the Corsican and Main supercolonies. Overall, the Catalonian supercolony was highly differentiated from the two other supercolonies (FST: Cat-Main = 0.47, P < 0.05; FST: Cat-Corsica = 0.43, P < 0.05) while the Main and Corsican supercolonies were only weakly differentiated (FST: Corsica-Main = 0.06, P < 0.05). Accordingly, the measures of genetic differentiation between pairs of nesting sites (Tableau 15) were invariably high between the Catalonian nesting sites and those of the two other supercolonies (FST values ranging between 0.41 and 0.55) while differentiation between pairs of nesting sites from the Main and Corsican supercolonies was much smaller (between 0.04 and 0.31) and similar to values between nesting sites within supercolonies (between 0.01 and 0.32).

A hierarchical analysis revealed a similar pattern with a relatively important genetic differentiation between supercolonies when the Catalonian nesting site was considered (FSc/Pop = 0.16, P < 0.001) but a much lower differentiation when it was excluded from the analysis (FSc/Pop = 0.04, P < 0.001). When only the Main and Corsican super-colonies were considered, the differentiation between nesting sites within supercolonies (FNest/Sc = 0.10, P < 0.001) was in the same order

of magnitude as the differentiation between supercolonies (FSc / Pop = 0.04, P < 0.001).

Finally, the PCA based on the nesting sites clearly distinguished the Catalonian nesting site from those of the other supercolonies (Figure 16a; PC1 = 39%, P <0.001) while those of the Main and Corsican supercolonies were not discriminated (PC2 = 16%, P = 0.62). The PCA based exclusively on the nesting sites of the Corsican and Main supercolonies also did not discriminate the two supercolonies with the first axis being non-significant (Figure 16b; PC1 = 25%, P = 0.24; PC2 = 23%, P < 0.01).

Altogether, these data show that there is only very weak differentiation between the Main and Corsican supercolonies which stands in strong contrast to the Catalonian supercolony that is strongly differentiated from both the Main and Corsican supercolonies.

To assess the relationship among the supercolonies identified in this study as well as the relationship between these supercolonies and those found worldwide, we performed a PCA based on the alleles frequencies of the 12 microsatellites. This analysis revealed high similarity of the Main and Corsican supercolonies that constituted a genetic cluster with the supercolonies from California, Australia, New Zealand, Hawaii and Japan (the S2 supercolony) (Figure 17C). By contrast, the Catalonian
supercolony was genetically very distinct
from both the Main and Corsican
supercolonies.

Analyses of the mitochondrial DNA
were highly congruent with the
microsatellite data. The analysis of the
cytochrome b genetic marker confirmed
that the individuals from the Main and
Corsican supercolonies exclusively
harboured the H1 haplotype (GenBank:
FJ466647.1) This is the haplo-type which is
also found in the other supercolonies with
which the Main and Corsican supercolonies
cluster(Vogel et al., 2010). By contrast, the
CDE nesting site had the H9 haplotype that
was previously found to characterize
nesting sites of Catalonian
supercolony(Vogel et al., 2010) (GenBank:
FJ466655.1).

A PCA of microsatellite genotypes
comprising only the supercolonies
genetically similar to the Main and Corsican
supercolonies confirmed their high similarity
(Figure 17D). There was actually some
overlap in the distribution of nesting sites of
these two supercolonies along the two main
axes which accounted for a high propor-
tion of the overall variation (PC1 = 38%, P
< 0.001; PC2 = 20%, P < 0.001). The
genetic composition of the S2 Japanese
supercolony was also very similar to that of
ants from the nesting sites of the Corsican
supercolony and notably to SGI and SFG. In
this respect, it is important to point out a
misidentification made in a previous study

on the classification of the SFG nesting site.
On the basis of information on the spatial
distribution of the Main supercolony,
BLIGHT et al. (Blight et al., 2010b) assumed
that the SFG nesting site belonged to the
Main European super-colony. However, the
genetic data of the study reveal that SFG
and the nesting sites to which it was
behaviourally and chemically clustered were
closer to the S2 Japanese supercolony than
to the Main supercolony described by
VOGEL et al. (Vogel et al., 2010). Later
aggression tests with nests from Port-Leucate
[known to be from the Main supercolony
(Jaquiery et al., 2005) the error
(unpublished data). As a consequence, SFG
actually belongs to the Corsican
supercolony. Consequently, all the nesting
sites identified as the Corsican supercolony
by BLIGHT et al. (Blight et al., 2010b)
actually belong to the Main supercolony,
and inversely all nesting sites of the Main
super-colony belong to the Corsican
supercolonie.

To further investigate the genetic
composition between the Main, Corsican
and Catalonian supercolonies and test
whether one may derive from one of the
two others, we measured the allelic richness
and compared the proportion of alleles
shared between nesting sites (allelic
nestedness).
Figure 17: Principal component analysis (PCA) based on microsatellite allele frequencies calculated on nesting sites. (C) Analysis of the 18 nesting sites of the present study in relation to all the nests of the worldwide supercolonies studied in Vogel et al. (Vogel et al., 2010). (D) Analysis of the nesting sites of the Main and Corsican supercolonies in relation to the nests of the worldwide supercolonies studied in Vogel et al. (Vogel et al., 2010) that formed a cluster with those two supercolonies in the PCA presented in panel C (PC1 > 0). In panels C and D, we distinguished the nests of the European main supercolonies from the present study from those issued from the Vogel et al. (Vogel et al., 2010) study by using dots and squares, respectively. The percentage of variance explained is given for each axis in brackets.

<figure><img src="image_65.png" /></figure>

Figure 18: Allelic richness at 12 microsatellite loci estimated for each nesting site (A) and each supercolony (B). Open squares represent the total number of alleles while the closed squares correspond to the estimated number of alleles corrected for differences in sample size. The way the alleles are distributed among the supercolonies is presented in panel.

<figure><img src="image_66.png" /></figure>
Support for one supercolony originating from another supercolony would come from the derived supercolony having a high proportion of alleles being also present in the source supercolony. This analysis confirmed that the Catalonian supercolony is clearly distinct from the two other supercolonies (figure 18). Of the 36 alleles in common between super-colonies, 13 were present in the three supercolonies, 20 were shared between the Main and Corsican supercolonies and only three and one alleles were common to the Catalonian and Corsican super-colonies, and Catalonian and Main supercolonies, respectively. The great divergence of the Catalonian supercolony was also reflected by a lower proportion of alleles being in common between nesting sites of this supercolony and those of the two other supercolonies, compared to nesting sites of the Main and Corsican supercolonies (Tableau 15).

A comparison of the Corsican and Main supercolonies revealed a higher allelic richness for the Corsican supercolony (both when corrected and not corrected by sampling size, P < 0.05, see figure 18). Accordingly, 73% of the alleles found in the Corsican supercolony were also present in the Main supercolony, while 80% of the alleles detected in the Main supercolony also occurred in the Corsican supercolony (Figure 19). The comparison of allelic richness among nesting sites revealed a similar pattern with nesting sites of the Corsican supercolony

having a higher number of alleles in 83% of the pairwise comparisons (P = 0.01, figure 18). These data, together with the high genetic similarity between the Corsican and Main European supercolonies, are compatible with the Main supercolony originating from the Corsican supercolony.

<figure><img src="image_67.png" /></figure>

Figure 19 : Allelic richness at 12 microsatellite loci estimated for each nesting site. The total number of alleles found per supercolony is given in brackets.

Association among behavioural, chemical and genetic data

There was a high correlation between the aggression level and genetic distance ($r^2$ = 0.41, P < 0.0001), the aggression level and chemical distance, ($r^2$ = 0.60, P < 0.0001) as well as the genetic and chemical distances ($r^2$ = 0.76, P < 0.0001) (Figure 20). When the Catalonian supercolony was excluded from the analyses all correlations decreased but remained significant (aggression-genetic distance: $r^2$ = 0.24, P < 0.01; aggression-chemical distance: $r^2$ = 0.45, P < 0.001; genetic-chemical distances: $r^2$ = 0.26, P < 0.001). Importantly, however, the inspection of Figure 20 reveals that the association was nonlinear and that there
was a strong effect of supercolony. The high
levels of aggression, and high chemical and
genetic distances were invariably found for
the comparison between the Catalonian
supercolony and the two other supercolonies.
A second cloud of points corresponding to
inter-mediate levels of aggression, chemical
and genetic distances characterized the
comparison between the Main and Corsican
supercolonies. Finally, the within Main and
Corsican supercolony comparisons revealed
similarly low levels of aggression (1-2) and
genetic and chemical distances. Overall, the
differentiation between the Main and
Corsican supercolonies was well marked for
aggression tests and less marked for genetic
and chemical distances with many Corsican-
Main comparisons intermingled with the
within supercolony comparisons.

Discussion

This study confirms the existence of a
distinct Corsican supercolony with a
distribution range including at least Corsica,
south-eastern France and one location in
Spain. Members of this supercolony were
aggressive towards ants from the other two
supercolonies studied. Aggression between
workers from the Corsican and Main
European supercolonies varied from
moderate to high, while aggression was
systematically high when workers from the
Catalonian supercolony were involved. This
behavioural pattern was confirmed by

chemical data. Cuticular hydro-carbon
proximity of members of the Corsican
super-colony to members of the Main
supercolony was greater than to those of
the Catalonian supercolony. Surprisingly,
despite the noteworthy concordance
between chemical and behavioural groups,
there was no clear genetic differentiation
between the Main and Corsican
supercolonies (FST = 0.06). This level of
genetic differentiation corresponds to the
differentiation observed between nesting
sites from a single supercolony. Previous
studies showed that the genetic
differentiation of nesting sites within a
supercolony varies between 0 and 0.1
depending of the supercolonies and the
scale at which the study is
performed(Giraud et al., 2002; Jaquiery et
al., 2005; Pedersen et al., 2006; Sunamura
et al., 2009b; Thomas et al., 2006; Tsutsui
& Case, 2001; Vogel et al., 2009) while the
genetic differentiation between
supercolonies usually exceed 0.2 (Jaquiery
et al., 2005; Pedersen et al., 2006;
Sunamura et al., 2009b; Thomas et al.,
2006; Vogel et al., 2009; Vogel et al.,
2010). In line with a very weak genetic
differentiation, the STRUCTURE analysis
did not discriminate between the two
supercolonies.
<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

Figure 20 : The associations of pairwise genetic (FST), chemical and behavioural distances between the three European supercolonies of Linepithema humile. Light-blue circles represent comparisons within the Main supercolony (n = 10), dark-blue circles represent comparisons within the Corsican supercolony (n = 66), open black circles represent comparisons between the Main and Corsican supercolonies (n = 60) and open red circles represent comparisons between the Catalonian and both the Main and Corsican supercolonies (n = 17).
This is also supported by the central
position of nesting sites from the Main and
Corsican supercolonies on the PCA including
peaceful-nests belonging to the worldwide
supercolony and considered as genetically
and chemically similar (Brandt et al., 2009;
Sunamura et al., 2009a; van Wilgenburg et
al., 2010; Vogel et al., 2010) (Figure 17).

The process of nestmate recognition
by which the Corsican supercolony is
behaviourally segregated from the Main
supercolony remains unclear. There are a
number of factors influencing the expression
and variation of the Argentine ant chemical
phenotype, including environmental and
genetic factors (Giraud et al., 2002; Liang &
Silverman, 2000; Suarez et al., 2008; Tsutsui
et al., 2000). Environment has been
hypothesized to influence nestmate
recognition cues in the Argentine ant,
because colonymates reared on different
diets in laboratory experiments became
mutually aggressive (Buczkowski et al.,
2005; Liang & Silverman, 2000; Silverman &
Liang, 2001). However, it seems unlikely that
such differences in diet could explain the
chemical differ-rences and the aggressiveness
between nesting sites of the Corsican and
Main supercolonies in view of the wide
dietbreadth experienced by workers of
opportunistic species such as the Argentine
ant(Vonshak et al., 2009) and the
geographic overlap in the distribution of the
two supercolonies.

Alternatively, the difference in
chemical profiles and behaviour between
workers of the Main and Corsican
supercolonies could have a genetic basis.
Consistent with a predominant role of
genetic factors in nestmate recognition in
Argentine ants, chemical and genetic
distances were highly correlated (r² = 0.76,
P < 0.0001). However, when the Catalonian
supercolony was excluded from the analysis,
only 26% (P < 0.001) of the variance in the
chemical cues could be attributed to genetic
distances between pairs of nesting sites. In
insects, cuticular hydrocarbon synthesis is
regulated by enzymes, which are themselves
under the control of specific genes.
Modification at these genes is sufficient to
prevent the biosynthesis of a hydrocarbon
(Wicker-Thomas C, 2008). This has been
clearly shown for *Drosophila melanogaster*,
where a modification at the gene desat1
coding for a desaturase that is involved in
sex pheromone synthesis disrupts individual
sexual behavior (Ueyama et al., 2005).

Thus, there could be genetic differences
at one or several loci between the Main and
Corsican supercolonies that may account for
odour and behavioural differences despite
no clear genetic differentiation at neutral
markers between supercolonies. Such a
situation has been demonstrated in the fire
ant *Solenopsis invicta* where a single genetic
Mendelian factor results in strong differences
in odour and behaviour between the
monogyne and polygyne social forms, which
show no differentiation at neutral nuclear
markers (Keller & Ross, 1998; Ross & Keller,
1998; Wang et al., 2008). In line with this
finding, high levels of variation in aggression
remained unexplained by the overall genetic
distance at the microsatellite marker loci
between the Main and Corsican
supercolonies (r² = 0.24, P < 0.001). High
aggression was present between the Main
and Corsican supercolonies even when
nesting sites shared relatively high
proportions of alleles, and inversely no
aggression was observed between some
nesting sites sharing low proportions of
alleles (Tableau 14 & Tableau 15). Consistent with
aggression being sometimes— but not
always— decoupled from genetic
differentiation at neutral markers,
BUCKOWSKI et al. (Buczkowski et al., 2004)
found no clear relationship between
intraspecific aggression and genetic similarity
at microsatellite markers between nest pairs
from California while Tsutsui et al. (Tsutsui
et al., 2000) reported higher aggression
between nests being genetically more
divergent in the Argentine ant. Likewise, no
relationship was detected between neutral
markers and aggression across Australia (Suhr
et al., 2011). Similar results were recently
found in the invasive pharaoh ant (Schmidt
et al., 2010) and in both the native
polygynous ants Formica fusca (Helantera et
al., 2011) and Formica exsecta (Martin et al.,
2012) where an increase in genetic diversity
at neutral makers did not result in an

increase of recognition cue diversity or
changes in agonistic behaviour. In the
invasive pharaoh ant, no qualitative
difference was detected in chemical cues
among colonies despite a very high genetic
differentiation (FST = 0.72) at neutral
markers (Schmidt et al., 2010). At the
opposite, the Main and Corsican
supercolonies differed qualitatively at 16
CHCs despite the weak genetic
differentiation (FST = 0.06). Most of the
studies suggesting the genetic basis of
recognition cues were usually based on the
correlation using neutral markers. However,
as correlation does not imply causation, it
may offer limited insights into relationships
between behaviour, chemistry and genetics
and may have limited power in unravelling
the pattern of nestmate discrimination.
Indeed, it is possible that only a few genes
could impact chemical diversity, irrespective
of relatedness.

Two hypotheses have been proposed
for the origin of the Corsican supercolony.
Either the Corsican supercolony originated
from the Main supercolony by geographic
isolation (split hypothesis), or it originated
from an independent introduction event
from the ants' native range (Blight et al.,
2010b). A slightly alternative explanation is
that one or both supercolonies did not
originate from native populations but from
introduced populations. The finding of a
high genetic similarity between the Main and
Corsican supercolonies both at the
microsatellite loci investigated and the sharing of the same mitochondrial haplotype (H1) supports the view that they may have a common origin, either from the same supercolony (in the native range or another introduced population) or that one originates from the other. In that respect, our finding of the Corsican supercolony being genetically more diverse and having more private alleles than the Main supercolony indicates that, if one of the two supercolonies derives from the other, it is the Main supercolony deriving from the Corsican supercolony rather than the reverse as had been previously proposed. It would be interesting to collect more nesting sites (in particular, for the Main supercolony), to investigate more thoroughly whether the Main supercolony really derives from the Corsican supercolony.

Historical records suggest that Argentine ants were first introduced into Madeira, which served as a source of propagules that were dispersed and invaded the Canary Islands and continental Europe (Wetterer & Wetterer, 2006). In south-eastern France, Argentine ants were first recorded in Cannes (~10 km from THE) and near Toulon (between sites EMB and DAN) at the beginning of the twentieth century (Marchal, 1917) (Carte 4).

Argentine ants were apparently transported from South America to Cannes with the importation of Orchids (Chopard, 1921), whereas the propagule introduced near

Toulon originated from the Canary Islands via the introduction of palm trees (Veyret, 1961). These historical data support the view that Argentine ants in southeastern France may originate from at least two introduction events. Interestingly, there is a good match between the historical data and current distribution of the two supercolonies. Continental nesting sites assigned to the Main European supercolony are located near Toulon, whereas those assigned to the Corsican supercolony are located near Cannes.

The results obtained in this study not only enhance our understanding of the origin of Argentine ant supercolonies in Southern Europe but also shed some light on questions regarding the general origin and evolution of invasive unicolonial ant populations (Helantera et al., 2009). It has been suggested that splitting of supercolonies can occur either through geographical isolation or behavioural processes (Drescher et al., 2010; Sunamura et al., 2011), both mechanisms contributing to reduced gene flow between supercolonies. Reduced gene flow may, in turn, lead to genetic and chemical differentiation between supercolonies. Previous work showed a complete absence of ongoing gene flow between the Main and Catalonian supercolonies (Jaquiy et al., 2005), which could lead to a process of speciation (Drescher et al., 2010; Helantera et al., 2009). For example, workers from the Main and the Catalonian supercolonies share only 18 of 123 CHCs in their profiles. This is a greater
difference than that reported among six
Formica species in which only 14 of 41
cuticular compounds differed qualitatively
between species (Martin *et al.*, 2008b). Such
difference in chemical profiles is remarkable as
in most ant species, qualitative differences is
observed between species(Hefetz, 2007).
Qualitative differences in cuticular hydro-
carbon profiles were previously detected
between the native and introduced ranges of
*Wasmannia auropunctata* (Errard *et al.*, 2005)
and *Linepithema humile* (Brandt *et al.*, 2009),
and between introduced supercolonies in both
the invasive yellow crazy ant *Anoplolepis
gracilipes* (Drescher *et al.*, 2010) and *L. humile*
(Blight *et al.*, 2010b; Brandt *et al.*, 2009).

It has been suggested that when
Argentine ant supercolonies overlap, this can
lead to their fusion (Vasquez & Silverman,
2008). It is currently unknown whether gene
flow still occurs between the Corsican and
Main supercolonies or whether they are
completely reproductively isolated as is the
case of the Main and Catalonian
supercolonies. Interestingly, our results show
that the behavioural differentiation between
nesting sites of the Main and Corsican
supercolonies is greater than the genetic and
chemical distance. This may suggest that the
behavioural response to chemical and
genetic differentiation is not linear but rather
depends on some threshold differentiation at
some specific genes and chemical
compounds. In line with this view, the
genetic differentiation between the Main and

Corsican supercolonies was lower than the
minimal genetic differentiation among
aggressive supercolonies in the native range
(Vogel et al., 2009). A nonlinear function
was recently found in the ant *F. exsecta*
where a small change in the Z9-alkene
profile resulted in a rapid shift from
nonaggressive to aggressive behaviours
(Martin et al., 2012). In that respect, it
would be interesting to conduct transects
with the aim of determining how the
genetic, chemical and behavioural clines vary
across the borders of the Main and Corsican
supercolonies and see how these variables
vary over time.

This study provides new insights into
the complex nestmate recognition system of
invasive ants which is a main factor driving
their evolution. To the best of our
knowledge, it constitutes the first report of
aggression between supercolonies that are
not clearly discriminated by genetics in
introduced populations of the Argentine ant.
Contrary to previous opinion, we
demonstrate that genetically similar nests at
microsatellite markers do not necessarily
belong to the same supercolony, highlighting
the importance of conducting more
qualitative and quantitative studies on levels
of aggression between supercolonies and
combines such data with genetic and
chemical data.
## ACKNOWLEDGEMENTS

Comments made by four anonymous reviewers have greatly improved this manuscript. This work was supported by a grant from the Office de l'Environnement de la Corse, the Direction Régionale de l'Environnement de Corse and the Provence-Alpes-Côtes d'Azur region, and the Swiss NSF.

## DATA ACCESSIBILITY

DNA sequences: *Linepithema humile* haplotype H1 cytochrome b (Cytb) gene, partial cds; mitochondrial, GenBank: FJ466647.1. *Linepithema humile* haplotype H9 cytochrome b (Cytb) gene, partial cds; mitochondrial. GenBank: FJ466655.1.

Microsatellite genotypes for each individual, DNA sequence alignment, cuticular hydrocarbons and results of behavioural tests: DRYAD entry doi:10.5061/dryad.1kc982n5.

Three video sequences of confrontations between the Main (EMB) and Corsican (OL) supercolonies, between the Corsican (BIG) and Catalonian supercolonies, and within the Main supercolony (CEY) are available as accessible data on Dryad. Examples of confrontations between the Main (CAN) and Corsican (OL) super-colonies, between the Corsican (BIG) and Catalonian supercolonies, and within the

Main supercolony (ŒY). DRYAD entry doi:10.5061/dryad.1kc982n5.

O.B.'s main research focus is the ecology of invasive ants. This work is part of his PhD project on the social structure, invasion dynamics and ecological impacts of Argentine ants in Corsica, and of L.B.'s Master thesis project. The main research interest of V.V. is the population and evolutionary genetics of many organisms (e.g. ants and bacteria). A.H., M.R., and E.P. (OB PhD co-supervisor) are engaged in research on several aspects in the chemical ecology of social insects. The main research interest of J.O. (OB PhD co-supervisor) is the population and community structure of many insects. L.K. works on various aspects of evolutionary ecology and social behavior in ants.

## APPENDIX

Tableau 16 : List of the identified cuticular hydrocarbons of workers from Caldès d'Estrac (Catalonian supercolony), Ceyreste (Main supercolony) and Fréjus (Corsican supercolony)
<table><thead><tr><th>Peak no</th><th>Compound</th><th>Caldès d'Estrac</th><th>Ceyreste</th><th>Fréjus</th></tr></thead><tbody><tr><td>1</td><td>n-Pentacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>2</td><td>3-Methylpentacosane</td><td></td><td>+</td><td>+</td></tr><tr><td>3</td><td>n-Hexacosene</td><td>+</td><td></td><td></td></tr><tr><td>4</td><td>n-Hexacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>5</td><td>2-Methylhexacosane</td><td></td><td>+</td><td>+</td></tr><tr><td>6</td><td>n-Heptacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>7</td><td>13-Methylheptacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>8</td><td>7-Methylheptacosane</td><td>+</td><td></td><td>+</td></tr><tr><td>9</td><td>5-Methylheptacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>10</td><td>3-Methylheptacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>11a</td><td>5, 15-Dimethylheptacosane</td><td></td><td>+</td><td></td></tr><tr><td>11b</td><td>5, 11-Dimethylheptacosane</td><td></td><td></td><td>+</td></tr><tr><td>12</td><td>n-Octacosene</td><td>+</td><td></td><td></td></tr><tr><td>13</td><td>n-Octacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>14</td><td>4-Methyloctacosane</td><td>+</td><td></td><td>+</td></tr><tr><td>15</td><td>2-Methyloctacosane</td><td>+</td><td></td><td></td></tr><tr><td>16</td><td>n-Nonacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>17</td><td>11- + 13- + 15-Methylnonacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>18</td><td>7-Methylnonacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>19</td><td>5-Methylnonacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>20</td><td>3-Methylnonacosane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>21a</td><td>5, 9-Dimethylnonacosane</td><td>+</td><td></td><td></td></tr><tr><td>21b</td><td>5, 11-Dimethylnonacosane</td><td></td><td>+</td><td>+</td></tr><tr><td>22</td><td>n-Triacontane</td><td>+</td><td></td><td></td></tr><tr><td>23</td><td>14- + 16-Methyltriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>24</td><td>12-Methyltriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>25</td><td>8-Methyltriacontane</td><td></td><td></td><td></td></tr><tr><td>26</td><td>4-Methyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>27</td><td>2-Methyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>28</td><td>n-Hentriacontane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>29a</td><td>13- + 15-Methylhentriacontane</td><td>+</td><td></td><td></td></tr><tr><td>29b</td><td>11- + 13- + 15-Methylhentriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>30</td><td>8, 10, xy-Tetramethyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>31</td><td>5-Methylhentriacontane</td><td></td><td></td><td>+</td></tr><tr><td>32</td><td>3-Methylhentriacontane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>33</td><td>5, 13- + 5, 15-Dimethylhentriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>34</td><td>5, 11-Dimethylhentriacontane</td><td>+</td><td></td><td></td></tr><tr><td>35</td><td>5, 13, 15- + 5, 13, 17-Trimethylhentriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>36</td><td>10- + 12-Methyldotriacontane</td><td>+</td><td></td><td></td></tr><tr><td>37</td><td>5, 7, 11-Trimethylhentriacontane</td><td>+</td><td></td><td></td></tr><tr><td>38</td><td>14, 18- + 14, 16-Dimethyldotriacontane</td><td></td><td></td><td>+</td></tr><tr><td>39</td><td>8, 12- + 8, 14-Dimethyldotriacontane</td><td>+</td><td></td><td></td></tr><tr><td>40</td><td>4, 14- + 4, 16-Dimethyldotriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>41</td><td>8, 10, 14-Trimethyldotriacontane</td><td>+</td><td></td><td></td></tr><tr><td>42</td><td>6, 10, 14-Trimethyldotriacontane</td><td>+</td><td></td><td></td></tr><tr><td>43</td><td>6, 14, 16-Trimethyldotriacontane</td><td></td><td>+</td><td></td></tr><tr><td>44</td><td>4, 14, 16-Trimethyldotriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>45</td><td>n-Tritriacontane</td><td>+</td><td></td><td></td></tr><tr><td>46</td><td>9, 13, 17, 19-Tetramethylhentriacontane</td><td>+</td><td></td><td></td></tr><tr><td>47</td><td>10, 16, 22, 24-Tetramethyldotriacontane</td><td>+</td><td></td><td></td></tr><tr><td>48</td><td>13- + 15- + 17-Methyltriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>49</td><td>11- + 13- + 15-Methyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>50</td><td>8, 10, xy-Tetramethyldotriacontane</td><td>+</td><td></td><td></td></tr><tr><td>51</td><td>11, 19- + 11, 13-Dimethyltriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>52</td><td>11, 15- + 13, 15-Dimethyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>53</td><td>9, 13-Dimethyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>54</td><td>7, 13- + 7, 11-Dimethyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>55</td><td>5, 15- + 5, 17-Dimethyltriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>56</td><td>5, 11-Dimethyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>57</td><td>7, 11, 15-Trimethyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>58</td><td>7, 11, 13-Trimethyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>59a</td><td>5, 13, 15-Trimethyltriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>59b</td><td>5, 9, 13-Trimethyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>60</td><td>7, 11, 13, 17-Tetramethyltriacontane</td><td>+</td><td></td><td></td></tr><tr><td>61</td><td>14, 16- + 16, 18-Dimethyltetracontane</td><td></td><td>+</td><td>+</td></tr><tr><td>62</td><td>12, 14- + 12, 16-Dimethyltetracontane</td><td></td><td>+</td><td>+</td></tr><tr><td>63</td><td>8, 12- + 8, 14-Dimethyltetracontane</td><td>+</td><td></td><td></td></tr></tbody></table>
Continued

<table><thead><tr><th>Peak no</th><th>Compound</th><th>Caldès d'Estrac</th><th>Ceyreste</th><th>Fréjus</th></tr></thead><tbody><tr><td>64</td><td>4,14-Dimethyltetracontane</td><td></td><td>+</td><td>+</td></tr><tr><td>65</td><td>8,12,20- +8,12, 18-Trimethyltetracontane</td><td></td><td></td><td>+</td></tr><tr><td>66</td><td>8,12,16-Trimethyltetracontane</td><td>+</td><td></td><td></td></tr><tr><td>67</td><td>8,10,14-Trimethyltetracontane</td><td>+</td><td></td><td></td></tr><tr><td>68</td><td>11,x,y,z-TetraMeC33</td><td>+</td><td></td><td></td></tr><tr><td>69</td><td>x,y,z-Trimethyltetracontane</td><td>+</td><td></td><td></td></tr><tr><td>70</td><td>6,12,20-Trimethyltetracontane</td><td>+</td><td></td><td></td></tr><tr><td>71</td><td>6,14,18-Trimethyltetracontane</td><td></td><td>+</td><td>+</td></tr><tr><td>72</td><td>n-Pentatriacontane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>73</td><td>4,14,18-Trimethyltetracontane</td><td></td><td>+</td><td>+</td></tr><tr><td>74</td><td>13- + 15- + 17-Methylpentatriacontane</td><td>+</td><td>+</td><td>+</td></tr><tr><td>75</td><td>13,17- + 15,19-Dimethylpentatriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>76</td><td>11,19- + 13,19- + 15,19-Dimethylpentatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>77</td><td>5,15-Dimethylpentatriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>78</td><td>13,17,21-Trimethylpentatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>79</td><td>9,13,15-Trimethylpentatriacontane</td><td>+</td><td>+</td><td></td></tr><tr><td>80</td><td>7,11,15-Trimethylpentatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>81</td><td>n-Hexatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>82</td><td>5,13,17- +5,13,15-Trimethylpentatriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>83</td><td>7,11,15,17-Tetramethylpentatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>84</td><td>14,16-Dimethylhexatriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>85</td><td>12,14-Dimethylhexatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>86</td><td>6,14- +6,18-Dimethylhexatriacontane</td><td></td><td></td><td>+</td></tr><tr><td>87</td><td>x,y,z-Trimethylhexatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>88</td><td>8,12,20-Trimethylhexatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>89</td><td>6,14,18-Trimethylhexatriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>90</td><td>4,14,18-Trimethylhexatriacontane</td><td></td><td></td><td>+</td></tr><tr><td>91</td><td>13- + 15- + 17- + 19- Methylheptatriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>92</td><td>7-Methylheptatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>93</td><td>13,15- + 15,19- + 17,19-Dimethylheptatriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>94</td><td>13,17- + 15,21- + 13,19-Dimethylheptatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>95</td><td>5,15-Dimethylheptatriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>96</td><td>9,13,15-Trimethylheptatriacontane</td><td></td><td>+</td><td></td></tr><tr><td>97</td><td>11,19,23-Trimethylheptatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>98</td><td>9,13,15-Trimethylheptatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>99</td><td>7,13,15-Trimethylheptatriacontane</td><td>+</td><td>+</td><td></td></tr><tr><td>100</td><td>5,15,17-Trimethylheptatriacontane</td><td></td><td>+</td><td>+</td></tr><tr><td>101</td><td>9,13,15,18-Tetramethylheptatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>102</td><td>7,11,15,19-Tetramethylheptatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>103</td><td>14,16- + 14,18-Dimethyloctatriacontae</td><td></td><td>+</td><td>+</td></tr><tr><td>104</td><td>12,18- + 14,18-Dimethyloctatriacontae</td><td>+</td><td></td><td></td></tr><tr><td>105</td><td>12,14,20- + 12,16,20-Trimethyloctatriacontae</td><td>+</td><td></td><td></td></tr><tr><td>106</td><td>8,12,20-Trimethyloctatriacontae</td><td>+</td><td></td><td></td></tr><tr><td>107</td><td>8,12,16,20-Tetramethyloctatriacontae</td><td>+</td><td></td><td></td></tr><tr><td>108</td><td>13 + 15 + 17 + 19-Methylnonatriacontane</td><td>+</td><td></td><td>+</td></tr><tr><td>109</td><td>13,17- + 17,19-Dimethylnonatriacontane</td><td></td><td></td><td>+</td></tr><tr><td>110</td><td>11,17- + 13,17- + 13,19-Methylnonatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>111</td><td>11,17,19- + 13,17,19-Trimethylnonatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>112</td><td>9,13,17-Trimethylnonatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>113</td><td>7,13,17- + 7,11,17-Trimethylnonatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>114</td><td>9,13,17,19-Tetramethylnonatriacontane</td><td>+</td><td></td><td></td></tr><tr><td>115</td><td>12,16,22-Trimethyltetracontane</td><td>+</td><td></td><td></td></tr><tr><td>116</td><td>13,19- + 13,21-Dimethylhentetracontane</td><td>+</td><td></td><td></td></tr><tr><td>117</td><td>13,19,23-Trimethylhentetracontane</td><td>+</td><td></td><td></td></tr><tr><td>118</td><td>9,13,19-Trimethylhentetracontane</td><td>+</td><td></td><td></td></tr><tr><td>119</td><td>x,y-Dimethyltritricacontane</td><td>+</td><td></td><td></td></tr><tr><td>120</td><td>13,19,23-Trimethyltritricacontane</td><td>+</td><td></td><td></td></tr><tr><td>121</td><td>9,13,21-Trimethyltritricacontane</td><td>+</td><td></td><td></td></tr><tr><td>122</td><td>x,y,z-Trimethylpentatetracontane?</td><td>+</td><td></td><td></td></tr><tr><td>123</td><td>x,y,z-Trimethylpentatetracontane?</td><td>+</td><td></td><td></td></tr><tr><td>124</td><td>x,y,z-Trimethylpentatetracontane?</td><td>+</td><td></td><td></td></tr><tr><td>125</td><td>x,y,z-Trimethylheptatetracontane?</td><td>+</td><td></td><td></td></tr><tr><td>126</td><td>x,y,z-Trimethylheptatetracontane?</td><td>+</td><td></td><td></td></tr><tr><td>127</td><td>x,y,z-Trimethylheptatetracontane?</td><td>+</td><td></td><td></td></tr><tr><td>128</td><td>x,y,z-Trimethylnonatetracontane?</td><td>+</td><td></td><td></td></tr><tr><td>129</td><td>x,y,z-Trimethylnonatetracontane?</td><td>+</td><td></td><td></td></tr><tr><td>130</td><td>x,y,z-Trimethylhenpentacontane?</td><td>+</td><td></td><td></td></tr><tr><td>131</td><td>x,y,z-Trimethylhenpentacontane?</td><td>+</td><td></td><td></td></tr><tr><td></td><td>total</td><td>97</td><td>49</td><td>55</td></tr></tbody></table>
# A peaceful zone bordering two Argentine ant (*Linepithema humile*) supercolonies

Laurence Berville • Olivier Blight • Marielle

Renucci • Abraham Hefetz • Erick Provost

Received: 28 January 2013/Accepted: 14 May 2013

© Springer Basel 2013

## Abstract :

In invasion areas, the Argentine ant (*Linepithema humile*) forms huge supercolonies with free exchange of individuals among nests. Two continental supercolonies on the French Mediterranean coast, the Main European supercolony and the Corsican supercolony, are known to contain workers showing moderate to high levels of inter-supercolony aggression. We performed chemical and behavioral assays using workers from eight coastal sites in southeastern France covering a geographical zone that includes nests of the two supercolonies, to determine the nature of worker interactions in a supercolony boundary zone. We examined how the chemical and behavioral clines vary across the borders of the Main and Corsican supercolonies. Our results on Giens peninsula and Porquerolles Island populations bordering the Main supercolony reveal the existence of a peaceful border zone with no aggression between workers of the Main supercolony and the Corsican supercolony. The chemical results, however, exhibited qualitative similarity to those observed in the Corsican supercolony and revealed both quantitative and qualitative chemical differences from those observed in the Main supercolony. These peaceful populations may result from fusion and/or hybridization of the two supercolonies, and we hypothesize that such peaceful border zones are instrumental in the evolution of supercolonies.

## Key words

*Linepithema humile*; cuticular hydrocarbons; fusion; territory border; aggressive behavior; unicoloniality.
<figure><img src="image_71.png" /><figcaption>Carte 5 : Geographical location of the eight sites of Linepithema humile. Sampling sites include the Corsican supercolony sites [Anthéor, Fréjus and Théoule] the Main supercolony sites [Ceyreste, Danio, and Les Embiez Island], and the two new sites: Giens Peninsula, and Porquerolles Island.</figcaption></figure>

## Introduction

The Argentine ant, *Linepithema humile*, invaded the Mediterranean coasts one hundred and fifty years ago (Wetterer & Wetterer, 2006). As with other invasive ants, the loss of territorial aggressiveness within a supercolony contributes to its invasion success (Suarez *et al.*, 1999; Suarez *et al.*, 2001). *L. humile* was first introduced into Madeira, this island subs-equently becoming a source of propagules for the invasion of the Canary Islands and later, continental Europe (Wetterer & Wetterer, 2006). Propagules introduced at the beginning of the twentieth century near Toulon (Marchal, 1917) via palm-trees (Veyret, 1961) are those assigned to the Main European supercolony, most likely the Madeira population. A second invasion of southern France was recorded in Cannes (Marchal, 1917), with the

importation of orchids and tropical ferns by horticulturalists (Chopard, 1921). In southern Europe, GIRAUD et al. (Giraud et al., 2002) identified two supercolonies, the Catalonian supercolony and the Main supercolony, ranging over 6,000 km from the Spanish Atlantic coast to northern Italy and characterized both by a general lack of aggression and by genetic homogeneity. Recently, BLIGHT et al. (Blight et al., 2012; Blight et al., 2010b) identified a third supercolony situated in southern France and Corsica, which was termed the Corsican supercolony, which borders the Main supercolony. These two supercolonies were found to be chemically and behaviorally, but not genetically, distinct (Blight et al., 2012). In the light of this discovery, we conducted behavioral and chemical experiments in geographical border zones between these
two supercolonies in order to gain insight
into what happens at the boundaries
between two supercolonies. We deter-mined
1.) Whether this zone is a peaceful or a
confrontation zone, and 2.) How the
chemical and behavioral clines vary across
the border of the Main and Corsican
supercolonies ?

**Materials & methods**

To compare patterns of intraspecific
aggression and cuticular hydrocarbon
profiles of Argentine ants in the border zone
between the two supercolonies, we selected
eight sites over a distance of 110 Km: three
sites contain the Main supercolony [Ceyreste
(Cey), Les Embiez Island (Emb), and Danio
(Dan) near Toulon], three sites contain the
Corsican supercolony [Anthéor (Ant), Fréjus
(Fre), and Théoule (The)(Blight et al., 2012)]
and two sites [Giens peninsula (Gie):
43°02'41.39"N / 6°08'02.56"E and
Porquerolles Island (Por): 43°00'00.28"N /
6°12'18.03"E] constitute the border between
the two supercolonies (Carte 5). Ants were
maintained in the laboratory at 23 ± 2°C on
suitably moist soil. In addition to the
arthropods collected along with the original
substrate and which served as a protein
source, the colonies were supplied with
honey and water *ad libitum*.

Worker aggression between all possible
combinations of colony fragment pairing,
each comprising 100 individuals, was
assessed as described in BLIGHT *et al.* (Blight

et al., 2012). Assays were conducted in 3
replicates (n = 28 x 3 = 84), and
confrontations within a nesting site were
recorded as controls (n = 8). Behavioral
interactions between workers were observed
and recorded for 20 minutes immediately
after group exposure and discriminated using
a five-step scale [1: touch; 2: avoidance; 3:
aggression (<2s, lunging, biting); 4:
asymmetrical intense aggression and 5:
symmetrical intense fighting]. Scores of 3, 4
and 5 indicated aggressive interactions.
Confrontations were stopped after 24h, and
then the numbers of both live and dead
workers were counted and recorded as a
percentage. For cuticular hydrocarbon
(CHC) analyses, eight workers were collected
from each site and maintained at -18°C. Each
worker was immersed in 10µl of hexane for
15 minutes to extract CHCs. Quantification
was achieved by injecting 3µl of each extract
into a gas chromatograph equipped with a
FID detector (GC/FID). For the running
conditions see (Blight *et al.*, 2012).
Comparison of CHC profiles was based on
integrating 50 peaks and calculating their
proportions. For CHC identification we ran
samples from Giens by gas chromatography
/mass spectrometry (GC/MS) and compared
the mass spectra of eluting compounds with
standard data and published spectra. A more
detailed description of both behavioral tests
and chemical analyses is presented in BLIGHT
*et al*. (Blight *et al.*, 2012).
<table><caption>Tableau 17 : List of cuticular hydrocarbons identified from Giens workers. Peak numbers after Blight et al. (Blight et al., 2012).</caption><thead><tr><th>Peak numbers</th><th>Compounds</th><th>Giens</th><th>Fréjus</th></tr></thead><tbody><tr><td>1</td><td>n-C25</td><td>+</td><td>++</td></tr><tr><td>2</td><td>3-MeC25</td><td>Trace</td><td>Trace</td></tr><tr><td>4</td><td>n-C26</td><td>+</td><td>+</td></tr><tr><td>5</td><td>2-MeC26</td><td>Trace</td><td>Trace</td></tr><tr><td>6</td><td>n-C27</td><td>++</td><td>+++</td></tr><tr><td>7</td><td>13-MeC27</td><td>Trace</td><td>Trace</td></tr><tr><td>8</td><td>7-MeC27</td><td>Trace</td><td>Trace</td></tr><tr><td>9</td><td>5-MeC27</td><td>Trace</td><td>Trace</td></tr><tr><td>10</td><td>3-MeC27</td><td>+</td><td>++</td></tr><tr><td>11b</td><td>5,11-MeC27</td><td>Trace</td><td>Trace</td></tr><tr><td>13</td><td>n-C28</td><td>++</td><td>+</td></tr><tr><td>14</td><td>4-MeC28</td><td>Trace</td><td>Trace</td></tr><tr><td>16</td><td>n-C29</td><td>+</td><td>+</td></tr><tr><td>17</td><td>11- + 13+ 15-MeC29</td><td>Trace</td><td>Trace</td></tr><tr><td>18</td><td>7-MeC29</td><td>Trace</td><td>Trace</td></tr><tr><td>19</td><td>5-MeC29</td><td>Trace</td><td>Trace</td></tr><tr><td>20</td><td>3-MeC29</td><td>+</td><td>+</td></tr><tr><td>21b</td><td>5,11-diMeC29</td><td>Trace</td><td>+</td></tr><tr><td>23</td><td>14- +16-MeC30</td><td>Trace</td><td>+</td></tr><tr><td>24</td><td>12-MeC30</td><td>Trace</td><td>+</td></tr><tr><td>28</td><td>n-C31</td><td>Trace</td><td>+</td></tr><tr><td>29b</td><td>11-+ 13- + 15-MeC31</td><td>+</td><td>+</td></tr><tr><td>31</td><td>5-MeC31</td><td>Trace</td><td>+</td></tr><tr><td>32</td><td>3-MeC31</td><td>+</td><td>+</td></tr><tr><td>33</td><td>5,13 + 5,15 -diMeC31</td><td>+</td><td>+</td></tr><tr><td>35</td><td>5,13,15- + 5,13,17-triMeC31</td><td>++</td><td>++</td></tr><tr><td>38</td><td>14,18- + 14,16-diMeC32</td><td>+</td><td>+</td></tr><tr><td>40</td><td>4,14- +4,16-diMeC32</td><td>Trace</td><td>+</td></tr><tr><td>44</td><td>4,14,16-triMeC32</td><td>+</td><td>+</td></tr><tr><td>48</td><td>13- +15- +17-MeC33</td><td>++</td><td>+</td></tr><tr><td>51</td><td>11,19- + 11,13-diMeC33</td><td>+</td><td>+</td></tr><tr><td>55</td><td>5,15- + 5,17-diMeC33</td><td>++</td><td>+++</td></tr><tr><td>59a</td><td>5,13,15-triMeC33</td><td>+++</td><td>++++</td></tr><tr><td>61</td><td>14,16- + 16,18-diMeC34</td><td>++</td><td>++</td></tr><tr><td>62</td><td>12,14- +12,16-diMeC34</td><td>+</td><td>+</td></tr><tr><td>64</td><td>4,14-diMeC34</td><td>Trace</td><td>+</td></tr><tr><td>65</td><td>8,12,20- +8,12,18-triMeC34</td><td>+</td><td>+</td></tr><tr><td>71</td><td>6,14,18-triMeC34</td><td>++</td><td>+</td></tr><tr><td>72</td><td>n-C35</td><td>++</td><td>++</td></tr><tr><td>73</td><td>4,14,18-triMeC34</td><td>+++</td><td>++</td></tr><tr><td>74</td><td>13- +15- +17-MeC35</td><td>++</td><td>++</td></tr><tr><td>75</td><td>13,17- +15,19-diMeC35</td><td>+++</td><td>++</td></tr><tr><td>77</td><td>5,15-diMeC35</td><td>+++</td><td>+++</td></tr><tr><td>82</td><td>5,13,17- +5,13,15-triMeC35</td><td>++++</td><td>++++</td></tr><tr><td>84</td><td>14,16-diMeC36</td><td>+++</td><td>+++</td></tr><tr><td>86</td><td>6,14- +6,18-diMeC36</td><td>+</td><td>+</td></tr><tr><td>89</td><td>6,14,18-triMeC36</td><td>++</td><td>+</td></tr><tr><td>90</td><td>4,14,18-triMeC36</td><td>++</td><td>++</td></tr><tr><td>91</td><td>13- + 15- + 17- +19-MeC37</td><td>+++</td><td>+</td></tr><tr><td>93</td><td>13,15- + 15,19- + 17,19-diMeC37</td><td>+++</td><td>++</td></tr><tr><td>95</td><td>5,15-diMeC37</td><td>+++</td><td>++</td></tr><tr><td>100</td><td>5,15,17-triMeC37</td><td>++++</td><td>++++</td></tr><tr><td>103</td><td>14,16- +14,18-diMeC38</td><td>++</td><td>++</td></tr><tr><td>108</td><td>13 + 15 + 17 +19-MeC39</td><td>+</td><td>+</td></tr><tr><td>109</td><td>13,17- +17,19-diMeC39</td><td>+</td><td>+</td></tr><tr><td>Total :</td><td></td><td>55</td><td></td></tr></tbody></table>
The *Kruskal-Wallis* tests for comparing aggression levels for all possible combinations of site pairing were performed using the maximum score per trial. Aggressiveness during each encounter was compared by ANOVA (*Bartlett test*) and the percentage of ants killed was compared using the non-parametric *Mann-Whitney U* test (Sigmastat 3.1).

We used multivariate exploratory techniques based on the classification of the principal chemical components (PCA) to distinguish important factors and to estimate correlation among variables (Statistica v.6 software). To discriminate all the CHC profiles, we performed a projection of the variables based on twenty-one factors which accounted for more than 95.5 % of the total variance of the 50 peaks. We also conducted a separate *K-means* cluster analysis to group together data points (factor coordinates of the variables) showing similar cuticular hydrocarbon profiles. A *Mantel test* based on the *Pearson test* (200 000 random permutations) was used to explore the possibility of an association

between levels of chemical distance (Euclidian distance based on CHCs), levels of aggression and distance in kilometers between all nesting sites (XLSTAT 7.5.2 software). Results & discussion

Aggression assays are commonly used to study nestmate recognition in ants (Chen & Nonacs, 2000; Tsutsui *et al.*, 2000), and so far, workers from Argentine ant supercolonies have always proved to be behaviorally distinguishable (Blight *et al.*, 2012; Blight *et al.*, 2010b; Giraud *et al.*, 2002). This is confirmed here (*Tableau 18*). Indeed, no aggression was recorded between workers from the same nesting site [Controls, n=8, *Tableau 18*]: mean aggressivity (1±0 SE); mean mortality after 24hrs (in percent: 1.8 ± 1.1 SE)] and from sites of the same supercolony [Main supercolony, n=9: mean aggressivity (1 ± 0 SE); mean mortality (2.2 ± 1.2 SE); Corsican supercolony, n=9: mean aggressivity (1.11 ± 0.14SE); mean mortality (4 ± 2.3 SE)]. In contrast, the level of aggression between workers from the two supercolonies varied from moderate to high

## Errur ! Source du renvoi introuvable. : Percentages of mortality after 24h (top) and mean indices of aggression (bottom) for each nest pair across replicates. Values are mean ± SE.

<table><thead><tr><th rowspan="2"></th><th colspan="3">Main Supercolony</th><th colspan="2">Border zone</th><th colspan="3">Corsican supercolony</th></tr><tr><th>Ceyreste</th><th>Les Embiez</th><th>Danio</th><th>Giens</th><th>Porquerolles</th><th>Fréjus</th><th>Anthéor</th><th>Théoule</th></tr></thead><tbody><tr><td>Ceyreste</td><td></td><td>1.5 ± =0.4</td><td>1.9 ± =1</td><td>4.1 ± =1</td><td>5.1 ± =2.1</td><td>3.6 ± =1.2</td><td>8.4 ± =5.7</td><td>4.2 ± =2.3</td></tr><tr><td>Les Embiez</td><td>1 ± =0</td><td></td><td>3.2 ± =2.6</td><td>5 ± =0.7</td><td>6.3 ± =0.9</td><td>7.6 ± =4.2</td><td><strong>18.6 ± =1.2</strong></td><td><strong>17.8 ± =11.7</strong></td></tr><tr><td>Danio</td><td>1 ± =0</td><td>1 ± =0</td><td></td><td>3.5 ± =0.7</td><td>4.6 ± =1.3</td><td>4.7 ± =1.3</td><td><strong>29.2 ± =16.9</strong></td><td><strong>18.6 ± =15.8</strong></td></tr><tr><td>Giens</td><td>1 ± =0</td><td>2 ± =0.6</td><td>1 ± =0</td><td></td><td>6.8 ± =3.1</td><td>7 ± =2.9</td><td>0.6 ± =7.7</td><td>2.6 ± =1.2</td></tr><tr><td>Porquerolles</td><td>1.6 ± =0.8</td><td>2.2 ± =0.7</td><td>2.6 ± =0.8</td><td>1 ± =0</td><td></td><td>6.8 ± =4.5</td><td>4.6 ± =1.4</td><td>4.1 ± =3.2</td></tr><tr><td>Fréjus</td><td><strong>3.6 ± =0.4</strong></td><td><strong>4 ± =0.6</strong></td><td><strong>3.6 ± =0.7</strong></td><td>1 ± =0</td><td>1 ± =0</td><td></td><td>6.3 ± =0.7</td><td>2.1 ± =1.1</td></tr><tr><td>Anthéor</td><td><strong>3.6 ± =0.4</strong></td><td><strong>4.3 ± =0.4</strong></td><td><strong>4 ± =0</strong></td><td>1.3 ± =0.4</td><td>1.3 ± =0.4</td><td>1 ± =0</td><td></td><td>3.7 ± =2.6</td></tr><tr><td>Théoule</td><td><strong>3.5 ± =0.6</strong></td><td><strong>3.8 ± =0.2</strong></td><td><strong>3.3 ± =0.8</strong></td><td>1.3 ± =0.4</td><td>1 ± =0</td><td>1 ± =0</td><td>1.3 ± =0.4</td><td></td></tr></tbody><tfoot><tr><td colspan="9" style="text-align: center;">Mean indices of aggression for each nest pair</td></tr></tfoot></table>

Withing-nest confrontations :

Mean aggressivity : 1 ± SE =0

Mean of dead workers : 1.8 ± SE =1.1
[n=27, mean aggressivity (3.77 ± 0.13SE); mean mortality (12.5 ± 10 SE)] and was significantly higher than the level of aggression within the Main and Corsican supercolonies (t-test, t= 44.915 P <0.0001). Against this relatively clear pattern, what stood out was a complete lack of aggression when workers from two sites, Giens and Porquerolles, were involved in confrontations with workers from both the Main [n=18, mean aggressivity (1.7 ±0.5SE); mean mortality (4.7 ± 1.3SE)] and the Corsican supercolonies [n=18, mean aggressivity (1.2 ±0.2SE); mean mortality (5.4 ± 3SE)]. These levels of aggression weren't significantly different from those shown in assays intra-supercolony, in controls or within Giens and Porquerolles (ANOVA, Bartlett test, p<0.0001; n=65) (Annexe 1). To the best of our knowledge, this is the first investigation of a buffer zone between two L. humile supercolonies. Surprisingly, although it was impossible to behaviorally assign the Giens and Porquerolles populations to one or the other supercolony, there was a clear chemical demarcation.

A PCA based on the proportions of the
major CHCs of the eight sites analyzed
separated the nesting sites of the Corsican
supercolony from those of the Main
supercolony (Factor 1: 17.22%) (Figure 23). The
K-means analysis distinguished two chemical
groups, with all individuals from a given
location assigned to the same group. The first
chemical group comprised all nests from the
Corsican supercolony, including Giens and
Porquerolles Island. The second chemical group
comprised all the Main supercolony nests. This

was confirmed by the identification of cuticular
hydrocarbons. The profile of the workers from
Giens (Figure 21) was qualitatively identical to
the profile of the workers from the Corsican
supercolony (Blight et al., 2012), dominated by
methyl-branched alkanes [mono-, di-, tri-
(Tableau 17)]. The profiles of the workers from
Porquerolles and Giens did not differ
qualitatively, while the profiles of the workers
from the Corsican and Main supercolonies
differed both quantitatively and qualitatively.

While the Giens and Porquerolles
populations could not be clearly assigned to
one or the other supercolony on the basis of
the behavioral data, the chemical analyses
clearly placed both populations within the
Corsican supercolony. Assuming that CHCs are
the chemicals that mediate nestmate
recognition, the absence of aggression from
Giens and Porquerolles populations towards
workers of the Main supercolony remains
unexplained. What is particularly interesting in
these findings is that the same order of
magnitude of qualitative differences in CHC
profiles leads in one case from moderate to
high levels of aggression (Main versus Corsican
supercolonies), while in the other case we
observed no aggression (Giens or Proquerolles
versus Main supercolony). This suggests that the
aggressiveness observed here and in BLIGHT et
al. (Blight et al., 2012) between workers from
the two supercolonies may be not the direct
expression of qualitative differences in CHC
profiles. Instead, it may result from quantitative
differences in specific hydrocarbons involved in
nestmate recognition.
In 2007, GREEN and GORDON (Greene & Gordon, 2007) showed that when nestspecific Argentine ant hydrocarbon proportions were

supercolonies. For further clarification, we performed correlation analyses between chemical distance and either geographical distance or aggression level (Figure 22 a & b).

Figure 21: Gas chromatogram of total body wash of a L. humile worker from Giens. Peak numbers after Blight et al. (Blight et al., 2012), corresponding to the list of CHCs identified in Tableau 16, p138.

<figure><img src="image_72.png" /></figure>

Figure 22 abc: Scatterplot of pairwise chemical, geographical (Km) and behavioral distances between all nesting site of L. humile. Gray circles represent inter-supercolony comparison (n=9), black circles represent Giens or Porquerolles comparison with both Main and Corsican supercolonies (n=12), black lozenges represent intra-Main supercolony comparison (n=3), gray lozenges represent intra-Corsican supercolony comparison (n=3) and black triangles represent comparison between Giens and Porquerolles.

a

<figure><img src="image_73.png" /></figure>

b

<figure><img src="image_74.png" /></figure>

c

<figure><img src="image_75.png" /></figure>

modified, a significantly greater proportion of ants behaved aggressively towards conspecifics.

Our results suggest that the Giens and Porquerolles nesting sites constitute a peaceful border zone at the boundaries of the two

There was no correlation between the chemical distance and either geographical distance (Figure 22a; r²=0.019, P=0.417) or aggression level (Figure 22b; r²=0.029, P=0.415). There was, however, a nonlinear correlation between
Figure 23 : PCA analysis based on 50 major cuticular hydrocarbons from the 8 sites of Linepithema humile. 7 or 8 workers per site (n = 61). Sampling sites include Anthéor (A), Ceyreste (C), Danio (D) (Toulon), Les Embiez Island (E), Fréjus (F), Giens Peninsula (G), Porquerolles Island (P)

error ! Source du renvoi introuvable.

and Théoule (T). The projection was performed on
factorial plane 1-2. Axis 1 accounts for 17.22% of the total
variance and axis 2 for 11.13%. Confidence limit showed a
range of 95% of the distribution of each supercolony.

aggression level and geographical distance
(Figure 22c; r²=0.494, P=0.009). To explain this
peaceful border zone, at least two hypotheses
can be proposed; between-colony hybridization
and colony fusion. Between-colony
hybridization could occur when males from
two different, but neighboring, supercolonies
are accepted by the respective alien workers
and copulate with alien females, thus creating a
hybrid population (=supercolony) with a blend
of both supercolonies' alleles. SUNAMURA et al.
(Sunamura et al., 2011) found that the level of
aggression against alien males was similar to
that against alien workers. Here, there was

considerable variation in levels of aggression
among workers during encounters between sites
of the Corsican and Main supercolonies, with
some workers eliciting fights, whereas others
moved freely from one nest to the other. This
variability of aggressive behaviors may increase
the likelihood of some alien males being
accepted by workers, thus allowing a gene flow
between the two supercolonies. Conversely,
when aggression is high, it may prevent any
potential gene flow by rejecting alien males
(Drescher et al., 2010; Sunamura et al., 2011).
For instance, no gene flow was recorded
between the Main and the Catalonian
supercolonies even at local scale (Jaquery et al., 2005). Aggression can even increase at the border of two highly different supercolonies. Several studies indicated that the intensity of aggression toward non-nestmates varies under specific circumstances, such as repeated contacts with a neighboring colony (Gordon, 1989a; Thomas et al., 1999) THOMAS et al. (Thomas et al., 2006) showed that neighboring workers (less than 500m apart) from two Argentine ant supercolonies exhibited higher levels of aggression than did non-neighboring workers. In these cases, both aggression and genetic distance were high. Here the low level of genetic differentiation recorded between the two supercolonies (Fst=0.06)(Blight et al., 2012) may explain the moderate level of aggression, which may permit hybridization between the Main and the Corsican supercolonies. It may have resulted in hybrids not aggressive towards workers of the two "mother" supercolonies, regardless of how far apart their nests are (from 5 to 85 km).

According to the hypothesis of colony fusion, some workers with broods from one supercolony could be accepted by the other supercolony. This may be the result of supercolony boundary expansion, followed by colony members mixing and thereby creating a blend of recognition cues across the newly merged colony.

The colony fusion hypothesis is supported by the works of VÁSQUEZ and SILVERMAN (Vasquez & Silverman, 2008) and VÁSQUEZ et al. (Vasquez et al., 2009). These

authors carried out experiments in the laboratory showing colony fusion between aggressive colonies. They suggest that fusion between different colonies may be one route towards unicoloniality for *L. humile* populations, even if, at the start, workers show strong levels of aggression. In a recent study, VÁSQUEZ et al. (Vasquez et al., 2012) demonstrated that fusion of aggressive Argentine ant colonies can lead to larger broods and worker populations with greater numbers of queens. All these queens take part in the production of workers, leading to greater genetic diversity, which may increase their acceptance of foreign individuals.

This surprising result raises questions about the origin and evolution at regional scale of two distinct supercolonies. The peaceful colonies identified at their borders may constitute a “buffer” zone facilitating the progressive union of the workers of the two groups. We can hypothesize the existence of several buffer zones facilitated by human exchange along the Mediterranean coast leading to the emergence of a supercolony. Future studies could usefully seek to confirm the existence of such zones at other sites.

## ACKNOWLEDGEMENTS

This project was supported by the Provence-Alpes-Côte d'Azur Region. We thank STÉPHANE GREFF for their help with the GC-MS. Our thanks to M. SWEETKO for English language revision.
<figure><img src="image_76.png" /></figure>

Annexe 1 : Indice d'agressivité et pourcentage de mortalité des ouvrières L. humile pour chaque confrontation.
# IV. Suivi d'une invasion : résistance locale et dynamique d'expansion

## *Article D:*

*Tapinoma nigerrimum: a natural barrier to Argentine ant expansion?*

L. BERVILLE* M. RENUCCI§ AND E. PROVOST§
(In preparation)

*Aix-Marseille Université, §CNRS
Institut Méditerranéen de Biodiversité et d'Ecologie marine et continentale (UMR CNRS-IRD), Université Paul Cézanne, Europôle Méditerranéen de l'Arbois BP 80, 13545 Aix-en-Provence Cedex 4, France
« Les plus grands ennemis des fourmis sont les autres fourmis tout comme les plus grands ennemis des Hommes sont d'autres Hommes »

AUGUSTE FOREL
Avant-propos :

Lorsque des espèces sont transportées dans une nouvelle région, elles peuvent s'établir puis persister, proliférer et s'étendre (Mack et al., 2000). Les fourmis d'Argentine ont commencé ce processus il y a plus d'un siècle en France. Comme nous venons de le voir dans les chapitres précédents, l'invasion s'étend le long du littoral de Provence. Quand elles s'établissent dans un milieu qui leurs est optimal, cette espèce exclut, la plupart du temps, la majorité des fourmis locales (Cole et al., 1992) et les autres arthropodes (Liebherr & Krushelnycky, 2007). Cependant, la résistance d'une ou plusieurs espèces indigènes face à l'invasion de la fourmi d'Argentine a été observée dans certaines régions. C'est le cas en Corse (Blight et al., 2009; Blight et al., 2010a).

En Corse, malgré une forte expansion depuis dix ans, la fourmi d'Argentine n'est présente que de façon discontinue le long des côtes. Cette distribution hétérogène peut-être due à des facteurs biotiques ou abiotiques, comme des résistances des communautés locales (Casevitz-Weulersse & Brun, 1999). Dans leurs travaux, BLIGHT et al. (Blight et al., 2009) ont noté l'absence de co-occurrence entre la fourmi d'Argentine et une espèce locale du genre *Tapinoma*, due probablement à des exigences écologiques proches. Au Portugal, *Tapinoma nigerrimum* est considérée comme une espèce dominante lors de la compétition pour la nourriture avec la fourmi d'Argentine (Way et al., 1997). Pour tester l'hypothèse d'une résistance biotique des interactions comportementales entre les deux espèces ont été menées au laboratoire (Blight et al., 2010a). Cette étude a démontré les capacités de *T. nigerrimum* à résister face aux fourmis d'Argentine.

Récemment, une co-occurrence entre des *Tapinoma sp.* et des *L. humile* a été observée sur l'archipel du Frioul [Marseille – Bouches du Rhône (Berville, 2011b)]. Dans le second chapitre nous avons démontré que les *Tapinoma sp.* présentent sur l'île du Frioul étaient des *T. nigerrimum*. Dans ce chapitre, nous nous sommes intéressés à cette co-occurrence qui pourrait nous permettre d'étudier les mécanismes de compétition entre ces deux espèces soulevés par les récents travaux de BLIGHT et al. (Blight et al., 2009; Blight et al., 2010a). La connaissance des aptitudes de résistance des *T. nigerrimum* face aux fourmis d'Argentine *in natura* pourrait nous permettre d'identifier les possibilités de réussite d'un moyen de bio-contrôle original tel que le transfert de *T. nigerrimum*.
Problématiques :

- Au laboratoire, quelles sont les aptitudes de résistance de *T. nigerrimum* à défendre leurs territoires face à l'intrusion de fourmis d'Argentine ? Jusqu'à quel désavantage numérique les *T. nigerrimum* arrivent-elles à éradiquer les fourmis d'Argentine ?

- *In natura*, quelles sont les distributions des deux espèces sur l'Archipel ? Quelles sont les capacités de compétition de *L. humile* et *T. nigerrimum* ? Sur le long terme, observe-t-on une résistance des fourmis *T. nigerrimum* face à l'invasion des fourmis d'Argentine ? Observe-t-on une différence dans la vitesse de l'invasion de la fourmi d'Argentine dans les zones avec *T. nigerrimum* comparées aux zones naturelles sans *T. nigerrimum* ? Quelles sont les aptitudes de *T. nigerrimum* à défendre leurs territoires face à l'intrusion de fourmis d'Argentine ?
**Article D: *Tapinoma nigerrimum*: a natural barrier to Argentine ant expansion?

**Abstract**

The establishment and spread of invasive species may be affected by abiotic factors, various characteristics of the invader, resistance of the native community and interactions among all these variables. This complexity makes the study of the success of biological invasions a major challenge for ecology. In southern France, the invasive Argentine ant (*Linepithema humile*) is well established, increasing its distribution range, and actively displacing native ant species. Here we employ a range of laboratory and field approaches to test the extent to which the presence of native ants limits post-establishment invasion success in Argentine ants. The rate of invasion was measured on both natural and urban areas and on an invasion front between *Tapinoma nigerrimum*, a native ant, and *L. humile*. The expansion rate of *L. humile* was not constant. This species, able to spread on average at a rate of 6 m/year in natural areas, retreated when faced with *T. nigerrimum*. Prior to the field study, two laboratory assays were conducted on *L. humile* and *T. nigerrimum*: asymmetrical group interactions and competition interactions involving resident colony status. They confirm the ability of *T. nigerrimum* to compete with *L. humile* by invading Argentine ant nests, while the reverse was never observed, as well as by controlling food. Our study also demonstrates that *T. nigerrimum* can offer strong competition even in a situation of numerical inferiority. Together, these findings suggest that *T. nigerrimum* may be of paramount importance in limiting the rate of spread of the Argentine ant.

**Key words**

Argentine ant. *Tapinoma nigerrimum*. Biological invasions. Biotic resistance. Rate of invasion. Resident effect.
Introduction

Global biodiversity is currently
threatened by invasive species, most of which
have been redistributed through expansion of
international trade (Mack *et al.*, 2000).
Invasions occur when species are transported
and become established in a new range in
which they proliferate, persist and spread
(Mack *et al.*, 2000). Because of their success
in establishment and ecological dominance,
ant are considered severe economic and
ecological pests (Holway *et al.*, 2002a; Vega
& Rust, 2001). Invasion by ants can cause
ecological disruption affecting both
continental and island ecosystems throughout
the world (Holway *et al.*, 2002a), being
particularly harmful on islands [see examples
in (Kenis *et al.*, 2009)].

Successful establishment and spread of
species depends on invasiveness (the
characteristics of the invasive species) and
invasibility (recipient environment: e.g.
abiotic factors, predation and competition).
In the case of invasion on islands, human
transport is the necessary vector. The
Argentine ant (*Linepithema humile*) is a
common invasive species worldwide in
subtropical and Mediterranean climates
(Wetterer *et al.*, 2009), often found on
islands like the Azores, Corsica (Blight *et al.*,
2009), Madeira, Malta, Port-Cros,
Porquerolles (Berville *et al.*, 2012b) and Sicily
[for a review see (Wetterer *et al.*, 2009)].
Once established due to lack of predators, L.

humile causes the decrease and even the
disappearance of many organisms, including
many native ant species (Fluker &
Beardsle.Jw, 1970; Rowles & O'Dowd, 2007;
Sanders et al., 2001; Suarez et al., 1998;
Ward, 1987), other arthropods (Cole et al.,
1992; Holway, 1998a; Human & Gordon,
1996; Zee & Holway, 2006), plants (Bond &
Slingsby, 1984; Carney et al., 2003; Christian,
2001; Gomez & Oliveras, 2003) and
vertebrates (Holway et al., 2002a). L. humile
competitively displaces (Cammell et al., 1996)
and drastically reduces populations of ant
species (Rowles & O'Dowd, 2007) by both
interference and exploitative competition
(Human & Gordon, 1996; Zee & Holway,
2006). While studies have investigated the
rate of spread of L. humile invasion, in the
USA (Erickson, 1971; Holway, 1995, 1998b;
Human & Gordon, 1996) and in Spain
(Casellas-Febrellas D, 2004; Roura-Pascual et
al., 2010) to our knowledge, no such study
has addressed the case of an island ecosystem.

L. humile shows characteristics typical
of tramp species (Holway *et al.*, 2002a;
Passera, 1994), such as high polygyny
(Holldobler & Wilson, 1977), unicoloniality
(Giraud *et al.*, 2002) or reproduction by
budding (Heller & Gordon, 2006; Passera &
Keller, 1994). Argentine ants typically form
supercolonies (Giraud *et al.*, 2002; Tsutsui *et
al.*, 2001). Among nests belonging to the
same supercolony there is no sign of
intraspecific aggression, which allows them to
reach a high density of workers (Holway,
1998b). Various factors may explain and
contribute to the advantage of *L. humile*
over native ants, among which the most
important are considered to be fight
strategies (Buczkowski & Bennett, 2008;
Human & Gordon, 1999), cooperation or
defensive technique (Buczkowski & Bennett,
2008), aggressiveness towards other species
(Carpintero & Reyes-Lopez, 2008; Holway,
1999) or supercolonies (Blight et al., 2012;
Blight et al., 2010b), numerical superiority
(Holway, 1999). Nevertheless, some native
ants resist the Argentine ant (Blight et al.,
2010a; Human & Gordon, 1996; Rice &
Silverman, 2013; Suarez et al., 1998). Local
resistance is ordinarily associated with local
dominant ant species (Carpintero & Reyes-
Lopez, 2008; Walters & Mackay, 2005), as
with *Lasius grandis* in Madeira (Wetterer &
Wetterer, 2006), *Tapinoma simrothi* in
Algeria (Bernard, 1950) or *Tapinoma
nigerrimum* in both Portugal (Way et al.,
1997) and Corsica (Blight et al., 2009). In
addition, some ants, like *Prenolepis imparis* in
northern California, persist in invaded areas
(Holway, 1998b; Suarez et al., 1998), using
chemical defense (Sorrells et al., 2011).

Our team previously demonstrated
the ability of *T. nigerrimum* to fight and kill
Argentine ants in the laboratory (Blight *et al*.,

2010a). *T. nigerrimum* was more efficient in
both exploitative and interference
competition. Interspecific competition can
have a major impact on ant community
distribution (Holway, 1999; Human &
Gordon, 1996). Strong competition often
occurs when the invader encounters an
ecologically dominant species with a large
population (Walters & Mackay, 2005) and
with whom it shares very similar biology.
Both *T. nigerrimum* and *L. humile* belong to
the subfamily Dolichoderinae, both form
large polygynous and polydomous colonies
and both are associated with
anthropogenically disturbed habitats (Acosta
Salmerón et al., 1983; Passera, 1977). Both
species have similar behaviors, like quick
moving and mass-recruitment foraging
strategy (Carpintero et al., 2007); they look
for the same type of habitat and share a
similar diet. The most common form of
aggressive behavior between *L. humile* and *T.
nigerrimum* is physical attack, but chemical
attack can also occur (Carpintero & Reyes-
Lopez, 2008). *T. nigerrimum* anal glands
 synthesize a mixture of methyl-heptone,
propyl-isobutyl-ketone and iridodial (Pavan
& Trave, 1958). Ketones give the secretion its
odor and its insecticidal properties.

Iridodial has no insecticidal properties
and is non-volatile.
<figure><img src="image_77.png" /><figcaption>Carte 6 : Map showing the location of Frioul archipelago with Pomègues and Ratonneau islands (MapInfo 7.8).</figcaption></figure>

<figure><img src="image_78.png" /><figcaption>Figure 24 : Artificial nest where workers of both species were tested for asymmetrical group interactions.</figcaption></figure>

However, in air, it "[...] polymerizes into a viscous yellow substance that increases the effectiveness of the [...] defensive [...] secretion by adhering to the predator" (Roth & Eisner, 1962) or assailant, as well as retarding the evaporative loss of the toxic volatile components mixed with it (Pavan & Trave, 1958; Roth & Eisner, 1962). The Argentine ant, for its part, synthesizes iridomyrmecin, a powerful insecticide (Roth & Eisner, 1962).

Because of the temporal and spatial scales involved, field experiments may lack the rigor of manipulative experiments. Nevertheless, they are the best way to quantify certain ecological phenomena (Diamond, 1986). On the Frioul archipelago off the Provence coast facing Marseille (southern France), both *T. nigerrimum* and *L. humile* are detected. This natural co-occurrence at the island scale is interesting, considering, the invasiveness of *L. humile*.
and the relative fragility of island populations (Rizali et al., 2010; Wetterer et al., 2006; Wetterer et al., 2004; Wetterer et al., 2001) notably in Mediterranean Island ecosystems (Espadaler, 2006; Espadaler & Gomez, 2004). Because of the relative nearness of the Frioul archipelago to one of the invasion sources (Marseille), extensive human transport (350 000 visitors/year), the small size of the territory (2 km²) and the presence of rare and/or endangered species [e.g. Geckonidae: *Euleptes europaea*, Plumbaginaceae: *Limonium pseudominutum Erben*, birds: *Calonectris diomedea diomedea* or *Puffinus yelkouan*], this island could be regarded as an “open sky laboratory” for studying the biological traits of these species.

The successful establishment and spread of the Argentine ant should thus depend both on its invasiveness against *T. nigerrimum* and its tolerance of abiotic conditions. Here we employ a range of laboratory and field approaches to test the extent to which the presence of *T. nigerrimum* limits post-establishment invasion success in Argentine ants. At colony level, we use laboratory experiments to examine competitive interactions between controlled numbers of Argentine ants and *T. nigerrimum*. In ant fights, the winner is likely to be numerically superior; however, numerical strength may be mitigated by individual ability to fight. We examine two questions in the laboratory: 1) how far the

ability of *T. nigerrimum* to resist *L. humile* depends on their relative numbers of workers and 2) whether the status of resident or of intruder plays an important role. Usually, when species become owners, their aggressiveness towards intruders increases (Carpintero & Reyes-Lopez, 2008). At community level, we examine in natura, on the Frioul Archipelago, where Argentine ants are established, the expansion of *L. humile* against the native *T. nigerrimum*. First, we estimate *L. humile* rate of invasion over two years at multiple invasion contact zones [with native ants but not *T. nigerrimum* and in a human-disturbed area]. Then, we determine the rate of invasion of the Argentine ant in the presence of *T. nigerrimum*, and explore the possible persistence of an invasion front between *T.nigerrimum* and *L. humile*. We examine in this field study whether the presence of *T. nigerrimum* impacts *L. humile's* ability to spread.

## Methods and materials

### Laboratory study

*T. nigerrimum* and *L. humile* colony fragments used for the laboratory experiments were collected in the Frioul archipelago (Ratonneau Island) off the coast of Provence, southern France, in August 2009 (Carte 6). Colony fragments were placed in a plastic nest (360 x 240 x 110 mm) container filled with original nest
substrate. Ants were fed with honey and water *ad libitum*, in addition to proteins provided by arthropods caught with their original substrate. Colonies were maintained at 20 ± 3°C under natural photoperiod.

*Assay on relative numbers of workers*

For each trial assay, workers of both species were placed in closed artificial nests (60 x 50 x 43mm) where they were kept for two hours to acclimatize. Artificial nests were connected with plastic tubes to a common foraging arena (88 x 60 x 50 mm) with honey in the center (Figure 24). *T. nigerrimum* were in a proportion of 3/2 (90 T.n versus 60 L.h), 1/1 (100 T.n versus 100 L.h), 3/4 (60 T.n versus 80 L.h), 2/3 (60 T.n versus 90 L.h), 1/2 (50 T.n versus 100 L.h), and 1/3 (30 T.n versus 90 L.h). Proportions were chosen so as to determine the balance of power between the two species. Assays on the highest and lowest *T. nigerrimum* proportions (3/2 and 1/3) were conducted five times and the rest were conducted ten times, for a total of 50 trials. The trial continued until the death of all individuals of one species. We counted the number of victories for each species in each trial, and results are given as the percentage of victories.

*Assay on resource and territorial competition: resident status*

This bioassay focused on the defensive ability of a resident colony against intruding workers of the other species (n= 2x4 replicates). A colony fragment (450 workers and a queen) of one species was placed in an artificial nest with sand, called the resident colony. This resident colony was allowed to access a sand arena (360 x 240 x 135 mm) connected to a foraging arena (120x90 x 50 mm) with honey and water *ad libitum* (Figure 25).

Two days later, the same number of workers of the other species was placed in a second artificial nest (120x90x50mm). This second colony fragment was allowed to access both sand and foraging arenas after two hours of acclimatization, workers from both nests needing to cross the sand arena to access the foraging arena. We recorded both time and nature of the first aggressive behavioral interaction between workers of the two colony fragments. We used a five-step scale to discriminate behavioral interactions between ants [1: touch; 2: avoidance; 3: aggression (<2s, lunging, biting); 4: asymmetrical intense aggression and 5: symmetrical intense fighting] as described in BLIGHT et al. (2010b). Then we recorded 1) the presence of intruders in both artificial nests and 2) the species controlling the foraging area and the honey after 1h, 5h, 24h, 48h and 72 hours.
<figure><img src="image_79.png" /><figcaption>Figure 25: Artificial nest where workers of both species were tested for resource and territorial competition.</figcaption></figure>

Bioassays were stopped at the death of all workers from one species and workers of the surviving species were counted. As control experiments, so as to ascertain the mortality rate of each species, we placed 450 workers of one species at a time in another artificial nest and we counted the number of surviving workers after four days (two controls, one on *T. nigerrimum* and one on *L. humile*).

## Field study

Field observations were conducted in the Frioul archipelago (Calanques National Park, southern France on a 2 Km² site formed by Ratonneau and Pomègues islands (Carte 6) (687126 - 4794503, 31T: UTM). In 2009, both islands were the site of intensive investigation seeking the precise location of *L. humile* and *T. nigerrimum* (unpublished data). *T. nigerrimum* nests were found near the island's village, east of the area invaded by *L. humile*. Argentine ants were found only in a limited area within the village and the harbor, which was used for monitoring experiments between autumn 2009 and spring 2012.

## *Linepithema humile* nest monitoring

In order to calculate the area invaded by the Argentine ant, the limited area within the village and the harbor was thoroughly explored in autumn 2009 (T=0). Argentine ants were hunted every five meters, on the ground, in trees, under stones, inside buildings (e.g.: restaurants) and even in trash, then determined and geo-referenced. In this way, we obtained a map of the distribution of *L. humile* in the invaded area. In autumn 2011, the invaded area was examined again in the same way as in autumn 2009, to obtain the new Argentine ant distribution (T+2), and quantify the evolution of the invasion.

One hundred *L. humile* nests (Carte 7), well established with eggs and queens, were used as control of continuity of presence. These nests were identified in autumn 2009, and in autumns 2010 and 2011 we checked whether they were still there. The area of the Argentine ant invasion range was calculated with GIS software (Mapinfo© 7.5).
<figure><img src="image_80.png" /></figure>

Location of the study area on Ratonneau Island. The natural area is represented in dark grey and the urban area in light grey. Black triangles show the location of T. nigerrimum nests. Dots show the location of the hundred L. humile control nests found in 2009 and prospected in 2010 and 2011. The lines represent the eight transects (A to H).

<figure><img src="image_81.png" /><figcaption>Carte 8 : Left striped area represents the area invaded by the Argentine ant in 2011.</figcaption></figure>
*Rate of spread and abundance of Argentine ant*

To investigate the expansion of *L. humile*, we used a protocol similar to HOLWAY (1998b) in California and CASELLAS-FABRELLAS (2004) in Spain. We chose zones in contact with the invaded area [half the perimeter of the invaded area is coast (Carte 7)]. In each zone, we established a transect perpendicular to the invasion front of the Argentine ant invasion: one transect on the concrete dyke between the two islands [Transect A], two in the semi-urban area [Transects B, C], two in the urban area [Transects G and H] and three in natural areas [Transects D, E and F] (Carte 7). Transect A was laid on concrete dyke with only some large concrete pots of plants. During winter, parts of the dyke, only 50 cm above sea level can be washed over by waves. Transects B and C were laid on coastal vegetation. Transects D and E were laid on a natural area but at the edge of a highly irrigated zone. Transect F was laid on the same vegetation as transects D and E but far from any human disturbance. Transects G and H were laid near the main road.

The eight transects were placed twice a year (late spring and early autumn, n=7) and geo-referenced. Each contained ten baits set five meters apart and consisting of a plate of honey, ham and cat food to attract the ants. After two and an half hours, each plate was photographed and ant species and *L. humile* abundance recorded. We defined the

abundance of *L. humile* as the maximum number of Argentine ants found per transect. Abundance was categorized according to a numerical scale: "0" no ants, "1" one to five ants, "2" six to 25 ants, "3" 26 to 100, "4" more than one hundred ants. Transects were always placed in the exactly same location and were replicated in September 2009, early May and late September of both 2010 and 2011, and May 2012 (n=48). In September 2011, due to the construction of a building, we lost part of transect G.

We recorded presence/absence of *L. humile* on each bait of each transect for all replicates. Bait were graded from one (invaded area) to ten (non-invaded area). The rate of spread was represented by the distance (in meters) separating the last invaded bait at T*n*, and the last invaded bait at T*n+1*.

*Statistical analyses*

Prior to statistical analysis, all data normality distributions were tested using the *Shapiro-Wilk test*.

We tested for the effects of relative numbers of workers by using t-Test on the difference in percentage of victories of *T. nigerrimum* at the end of confrontations. Differences in mortality levels during the competitions for resources and space were compared using the non-parametric *Mann-Whitney U test* for independent data. The presence/absence of Argentine ants in the control nests was compared using the non-
parametric *Mann-Whitney U test*. Differences in rates of invasion by *L. humile* among transects did not follow a normal distribution; thus, a *Kruskall-Wallis test* was used and then a paired *Mann-Whitney U test*. Differences in *L. humile* abundance among transects were compared using an ANOVA and then a *Turkey test*.

All data were analyzed using XLstat and graphs were performed using Systat software: XLstatSigmaPlot 10.0.

## Results

Laboratory study

*Effects of number of T. nigerrimum or L. humile during confrontations.*

After 2 hours of acclimatization, as soon as the artificial nests were opened, aggressive asymmetrical interactions and fights between workers were observed ($T_{+6min} \pm 5min$). Whatever the initial ratio, confrontations were relatively short; 95% of the confrontations ended after 48 hours in the death of all the individuals of one species. We observed three significant outcomes during the asymmetrical group confrontations (Figure 26). First, when *T. nigerrimum* was vastly outnumbered by *L. humile* (*T. nigerrimum*: 1/3 *L. humile*, n=5), 100% of the confrontations were lost by *T. nigerrimum*. Second, when *T. nigerrimum* was less severely outnumbered by *L. humile*, 1/2 or 2/3, the outcome of fights was less radical. In 30% of the assays, *T. nigerrimum*

won despite being outnumbered. Third, *T. nigerrimum* were very successful, with a victory rate of over 80%, when the initial worker ratio was 1) to their advantage [*T. nigerrimum* proportions of 3/2], 2) equal in number [*T. nigerrimum* 1/1 *L. humile*] and 3) slightly to their disadvantage [*T. nigerrimum* proportions of 3/4]. *T. nigerrimum* were able to win fights up to a ratio of 3 *L.h* to 2 *T.n* (*t*-test: t= 2.776, P= 0.003).

*Competition for space and food in the large arena: resident status*

As soon as the second artificial nest was opened, intense asymmetrical fights were observed ($T_{+10min} \pm 4$ min). Aggression and mortality rate were always high when *L. humile* were defending their nest from the intruders (

Tableau 18). *T. nigerrimum* maximum mortality score was on average 60.31% ± 1.55 SE when *L. humile* were resident, whereas it was on average 34.11% ± 5.44 SE when *T. nigerrimum* were defending their territories from Argentine ants (P<0.0001, n=8, Mann-Whitney U test). In all eight confrontations, whatever the residency status, *L. humile* were exterminated by *T. nigerrimum* (Mortality rate = 100%), first in the foraging area and then in the Argentine ant nest. At the end of the experiment, *T. nigerrimum* were nesting in *L. humile* nests in 100% of the replicates. It took a mean of four days for *T. nigerrimum* to exterminate the Argentine ant.
<figure><img src="image_82.png" /><figcaption>Figure 26 : Percentage of victories of *T. nigerrimum* over *L. humile* workers according to initial ratio of both species (n=50), *< 0.003</figcaption></figure>

<table><caption>Tableau 18: Effect of residency status on the rate of death of *T. nigerrimum* and *L. humile* workers during the confrontations, n=10.</caption><thead><tr><th rowspan="2"></th><th colspan="4">Percentage of dead workers</th></tr><tr><th colspan="2">Residency status</th><th colspan="2">Intruder status</th></tr></thead><tbody><tr><td><em>L. humile</em></td><td>100%</td><td>SE ± 0</td><td>100%</td><td>SE ± 0</td></tr><tr><td><em>T. nigerrimum</em></td><td>34%</td><td>SE ± 5</td><td>60%</td><td>SE ± 1</td></tr><tr><td>Control (n=2)</td><td>8.40%</td><td>SE ± 2.3</td><td></td><td></td></tr></tbody></table>