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Supplementary material
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Colony fusion and worker reproduction after queen loss in army ants
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Daniel J. C. Kronauer1,*, Caspar Schöning2, Patrizia d’Ettorre, Jacobus J. Boomsma
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Centre for Social Evolution, Department of Biology, University of Copenhagen,
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Universitetsparken 15, 2100 Copenhagen, Denmark
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Cambridge, MA 02138, USA
Present address: Museum of Comparative Zoology, Harvard University, 26 Oxford Street,
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Neuendorf, Germany
Present address: Länderinstitut für Bienenkunde, Friedrich-Engels-Strasse 32, 16540 Hohen
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*Correspondence: dkron@fas.harvard.edu
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Details of the queen-deprived Dorylus (Anomma) molestus colonies:
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In addition to the samples listed below, 78 individuals were genotyped for mitochondrial
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DNA and nuclear microsatellites to estimate background allele frequencies.
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1. Colony Q12:
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The queen was removed and a brood/worker sample collected on January 9th 2007. The
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colony started to emigrate into the nest of colony JC02 on January 14th. A worker sample of
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the mixed colony was taken on January 16th. A worker sample of JC02 had been collected
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before the fusion on December 19th 2006. The queen of Q12, ten workers of JC02 collected
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before the fusion, and 30 workers of the mixed colony were genotyped for microsatellites.
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The queen of Q12, one worker of JC02 from before the fusion, and four workers of the mixed
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colony were sequenced for mitochondrial DNA. Of the mixed workers, nine were assigned to
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the queen of Q12, and 21 to the queen of JC02. The two colonies did not share recent
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ancestry, as they had different mitochondrial haplotypes. No workers were analyzed for
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cuticular hydrocarbons.
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2. Colony Q13:
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The queen was removed and a brood/worker sample collected on January 10th 2007. The
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colony subsequently emigrated on January 11th and January 22nd. After the second emigration
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the colony continued to forage daily. The nest was opened on April 4th 2007 and workers and
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31 male larvae were collected. The colony did not contain any more brood at this point. The
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queen, 30 workers, and the 31 male larvae were genotyped for microsatellites, and the queen
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was sequenced for mitochondrial DNA. The following samples were analyzed for cuticular
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hydrocarbons: the queen of colony Q13; samples of five workers each, collected on January
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10th (before queen removal), on January 11th (one day after queen removal), and on February
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21st (six weeks after queen removal).
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3. Colony Q17
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The queen was removed and a brood/worker sample collected on January 23rd 2007. The
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colony subsequently emigrated on January 24th, February 1st, February 20th, March 5th, and
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March 14th. Because the colony continued to forage normally and contained brood we
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suspected that it might have merged with a queenright colony. A sample of workers and brood
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was collected from the emigration trail on March 14th. The queen and ten workers collected
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on January 23rd, as well as 34 workers collected on March 14th were genotyped for
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microsatellites, and the queen and one worker collected on March 14th were sequenced for
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mitochondrial DNA. None of the workers collected on March 14th was assigned to the queen
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of Q17 and we conclude that we lost track of the focal colony at some point during the study
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period. Another possibility would be that the great majority of workers from colony Q17 had
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died in the mixed colony in the meantime, so that we did not detect any Q17 workers in the
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sample taken on March 14th, 50 days after queen removal. The ultimate fate of the queenless
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colony remains uncertain. No workers were analyzed for cuticular hydrocarbons.
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4. Colony JC26
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The queen was removed and a brood/worker sample collected on January 28th 2007. The
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colony emigrated into the nest of colony JC33 on February 2nd 2007. On February 3rd, another
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colony seemed to emigrate into the same nest and we surmised that this might have been
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colony Q15 from which we had unsuccessfully tried to remove the queen on January 17th. The
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queen of JC26 and ten workers collected on January 28th, ten workers collected from JC33
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before the fusion, ten workers collected from Q15 on January 17th, and 30 workers collected
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from the inferred mixed colony on February 7th were genotyped for microsatellites, and the
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queen of JC26 and one worker each from JC33 and Q15 were sequenced for mitochondrial
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DNA. All workers of JC26 and JC33 were offspring of the queen of JC26. The queen
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genotypes reconstructed from the workers of JC26 and JC33 were identical to the observed
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genotype of the sampled JC26 queen, and 40 % of the repeatedly sampled patrilines (workers
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with genetically identical fathers) in the entire sample after the suspected fusion were shared
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between colonies (due to the very high mating frequencies, not all patrilines were detected in
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both colonies). This means that we either sampled from two subsequent nests of the orphaned
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colony without a fusion having occurred, or that fusion occurred between an orphaned mother
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colony and its daughter colony after recent colony fission. During colony fission, the old D.
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molestus queen and accompanying workers leave parts of the worker force and the developing
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reproductive brood behind in the daughter nest (Raignier 1972). Therefore, shortly after
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fission, before the young queen in the daughter colony starts reproduction, workers from both
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fission products have the same mother and fathers. In such a case it would obviously not be
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possible to genetically assign workers of the mixed colony to one of the two original colonies.
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In this case, however, we regard the former scenario (no fusion) as more likely, mainly
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because there was no evidence for a reproductive brood being present in colony JC33. The
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workers collected on February 7th were all offspring of a different queen, which means that
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we had lost track of JC26 after the first suspected fusion and were not able to reconstruct the
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subsequent events. No workers were analyzed for cuticular hydrocarbons.
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5. Colony JC35
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The queen was removed and a brood/worker sample collected on February 10th 2007. On
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February 11th, the colony emigrated into a previous nest, and on February 15th it emigrated
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into a new nest via the original nest that had been dug up. At the new nest site, the workers
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did not simply enter but formed many trails on top of the nest and around it, covering an area
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of two times six meters. Some of the brood was deposited on the surface in two piles covered
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by workers, which is never the case during emigrations of normal colonies. Apparently the
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colony was fusing with a resident colony whose nest was initially not large enough to
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accommodate all newcomers. The new nest was opened and a brood/worker sample collected
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on February 16th. The queen of JC35 and 50 workers from the inferred mixed colony collected
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on February 16th were genotyped for microsatellites, and the queen of JC35 and three workers
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of the inferred mixed colony were sequenced for mitochondrial DNA. Of the mixed workers,
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17 were assigned to the queen of JC35, and 33 were assigned to a second queen, which was
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clearly not related to JC35 because mitochondrial haplotypes were different. No workers were
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analyzed for cuticular hydrocarbons.
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6. Colony JC18
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The queen was removed and a brood/worker sample collected on February 25th 2007. The
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colony emigrated on February 26th and then moved into the nest of colony JC36 on March 1st.
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The mixed colony emigrated to a new nest on the same day of the fusion. The queen of JC18,
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26 mixed workers collected on March 1st, ten worker larvae from JC36 collected on March
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1st, five mixed workers collected on March 7th, nine mixed workers collected on March 27th,
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and ten workers collected from JC36 on February 8th before the fusion, were genotyped for
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microsatellites. The queen of JC18, one worker from JC36 collected on February 8th, and one
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worker from the supposedly mixed colony were sequenced for mitochondrial DNA. Of the
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mixed workers collected on March 1st, 7 were assigned to the queen of JC18, and 19 to the
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queen of JC36 based on genetic data. All workers collected on March 7th and March 27th were
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assigned to the queen of JC36. The two queens had the same mitochondrial haplotype and
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shared an allele at each microsatellite locus. This means that they could have been mother and
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daughter. However, the evidence remains inconclusive because in neither colony did we
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detect a patriline that could have produced the respective mother queen of the other colony.
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Samples analyzed for cuticular hydrocarbons were as follows: the queen of colony JC18; four
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or five workers each from colony JC36 collected before the merger on February 7th, 14th, 21st,
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and 28th (19 total); five workers from colony JC18 collected on February 25th before queen
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removal, five workers collected on February 26th, one day after queen removal, and three
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workers collected on March 1st from the emigration trail into the JC36 nest. From the mixed
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colony after fusion we collected and analyzed seven workers on March 1st, five workers on
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March 7th, and nine workers on March 27th.
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The following colonies were studied in 2005. For these we did not carry out any analyses of
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cuticular hydrocarbons:
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7. Colony Q4
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The queen was removed and a brood/worker sample taken on October 23rd 2005. Colony Q4
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merged with another colony on October 30th. A mixed brood/worker sample was collected on
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December 24th, 55 days after the merger. The queen of Q4, 15 workers from Q4 before the
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fusion, and 20 workers and 15 larvae after the inferred fusion were genotyped for
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microsatellites, and the queen of Q4, one larva, and one worker after the fusion were
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sequenced for mitochondrial DNA. Seven workers of the mixed sample were assigned to the
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queen of Q4, while all larvae and 13 workers sampled after the fusion were assigned to a
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second queen, which was clearly not related to Q4 because mitochondrial haplotypes were
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different.
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8. Colony Q2
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The queen was removed and a brood/worker sample taken on October 20th 2005. The colony
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subsequently emigrated four times: two, three, 36, and 43 days after queen removal. During
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the fourth emigration, the colony merged with another colony. A mixed brood/worker sample
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was collected on December 29th, 28 days after the merger. The queen of Q2, 15 workers from
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Q2 before the fusion, and 20 workers and 15 larvae after the fusion were genotyped for
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microsatellites, and the queen of Q2 and one larva sampled after the fusion were sequenced
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for mitochondrial DNA. One worker of the mixed sample was assigned to the queen of Q2,
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while 19 workers and all larvae sampled after the fusion were assigned to a second queen,
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which was clearly not related to Q2 because mitochondrial haplotypes were different.
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9. Colony Q1
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The queen was removed and a brood/worker sample taken on October 20th 2005. Ten days
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later, the colony merged with a neighbouring colony, which turned out to contain a sexual
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brood. While this was a case where the fusion could be clearly observed due to epigaeic
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emigration trails, unfortunately only two male larvae from the adopting colony were
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preserved for genetic analyses, which is insufficient to confirm the merger genetically.
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However, microsatellite and mitochondrial genotypes of the queen of colony Q1 and both
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males from the adopting colony clearly showed that the two colonies were not related as they
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had different mitochondrial haplotypes.
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10. Colony Q5
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The queen was removed and a brood/worker sample taken on October 28th 2005. The colony
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emigrated four times before we lost track on December 20th. The last known nest was dug up
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but neither workers nor brood were found. The ultimate fate of this colony remains uncertain.
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A tentative model to compare fitness benefits after queen loss:
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Making a number of simplifying assumptions, the condition under which colony fusion with a
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random neighbouring colony would be preferred over male production by orphaned workers
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can be expressed as:
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x * rf * 6000 > rm * N
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where:
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x = the proportion of the original worker force and brood of an orphaned colony that is left at
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the moment of fusion, relative to the average colony at the time of fission. For simplicity we
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assume that there is no differential worker or brood mortality after fusion. Based on the field
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observations that we have, we will use 0.3 as a crude approximation of this term.
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rf = the average relatedness of the orphaned workers to the reproductive offspring of the
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adopting colony
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6000 = a crude estimate of the approximate fitness gain from a colony fission (assuming that
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3000 males are produced and that fitness returns from male and female function are equal, i.e.
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that the daughter colony headed by a young queen is worth as much in terms of fitness as the
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3000 males). The latter assumption directly follows from assuming that the population sex
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allocation ratio is at equilibrium and that this equilibrium is equal for both queens and
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workers because of the extreme degree of multiple mating.
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rm = the average relatedness of workers to male offspring produced by an orphaned colony,
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i.e. approximately 0.125 because of the highly polyandrous mating system
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N = the average number of males produced by an orphaned colony. The only estimate we
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have would be ca. 30. We cannot exclude that some orphaned colonies could raise a larger
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male brood, but there are also serious doubts that the single male brood that we sampled
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would have ever made it to the adult stage (see text). We therefore use the value of 30 as a
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crude approximation.
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If we substitute these values in the equation above, we find that any value of rf that exceeds
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0.002 would be sufficient to make inclusive fitness gains from colony fusion larger than those
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obtained from male production. Even if we use more conservative values for some parameters
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(N = 50; x = 0.1), the threshold relatedness value remains low (rf = 0.01). Although a more
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fully developed model parameterized with better field data might further adjust these
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threshold values, there seems little doubt that very small values of rf are sufficient to make
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colony fusions with random neighbours adaptive. The typical army ant population viscosity
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caused by queens dispersing on foot is likely to create these relatedness incentives, but this
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remains an inference of common sense (i.e. relatedness cannot be negative and rf = 0 requires
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random mating and dispersal of both sexes throughout the population, which we know is not
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true in army ants). As we have explained in the main text, much larger sample sizes and/or
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higher numbers of genetic markers would be needed to prove that an average of, say, rf = 0.01
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is statistically different from zero. We therefore suffice noting that both values obtained above
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(rf = 0.002 and 0.01) are covered by all the confidence intervals for our nuclear relatedness
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estimates.
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Supplementary note:
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The study of six orphaned D. wilverthi colonies by Raignier (1972) is worth mentioning
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because none of these colonies apparently merged with an adjacent colony. Similarly, Leroux
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(1982) monitored colony fissions and colony deaths in D. nigricans over a period of four
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years, but did not witness a single colony fusion. He did, however, observe more than twice as
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many colony fissions as colony deaths. One possible explanation for this apparent
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discrepancy is that both authors assumed that, after a fusion had occurred, they had lost track
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of the queenless colony and did not report such cases. As has been mentioned above, the
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partly subterranean emigrations of these army ants can lead to uncertainty about colony
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identity when colonies are followed over extended periods. Another possibility is that
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inherent differences between army ant species or even populations make colony fusions
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adaptive in one case, but not in the other. Such differences could, for example, pertain to
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population structure and density, or the feasibility of worker reproduction. Similar studies on
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additional species and populations therefore have great potential to elucidate the relative
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contributions of these factors.
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Table S1. GenBank accession numbers of mitochondrial haplotypes and allele frequencies from the eastern slope population of Mt. Kenya
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(overall; n = 30) and the study plot (local; n = 47). Variable positions among a total of 781 base-pairs are shown.
GenBank acc. no. Haplotype Variable sites in DNA alignment
Allele frequencies
132 177 227 280 357 358 376 436 472 559 610 611 628 649 733 overall
local
GU065698
1
C
A
C
G
T
C
C
C
G
T
C
C
T
G
T
0.133
0.043
GU065699
2
C
G
C
G
T
C
C
C
T
T
C
C
T
A
C
0.200
0.745
GU065700
3
C
G
C
G
T
C
C
C
T
T
C
C
T
G
C
0.267
0.213
GU065701
4
T
A
C
G
T
C
C
C
T
T
C
C
C
G
T
0.067
0.000
GU065702
5
C
G
C
G
T
C
C
C
T
T
T
C
T
G
C
0.067
0.000
GU065704
6
C
G
C
G
T
C
T
C
T
T
C
C
T
G
C
0.033
0.000
GU065703
7
C
A
C
G
T
C
C
C
T
T
C
C
T
G
T
0.200
0.000
EF413797
8
?
?
T
A
C
T
C
T
T
C
C
G
T
A
T
0.033
0.000
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Table S2. Identification and relative proportion of the 50 compounds consistently found in
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the cuticular hydrocarbon profile of D. molestus. Numbers correspond to those in figure 2a,
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which depicts representative gas-chromatograms. The relative proportions were calculated
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based on the cuticular hydrocarbon profiles of workers of the three colonies shown in figure 3
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before any experimental manipulations.
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Peak No. Retention time Identification
Mean (%) SD (%)
1
13.79
n-C21
0.164
0.052
2
15.98
n-C22
0.346
0.128
3
17.67
C23:1
0.374
0.123
4
18.36
n-C23
8.496
1.374
5
19.12
11-meC23
0.821
0.096
6
19.75
3-meC23
0.287
0.216
7
20.63
n-C24
0.535
0.282
8
22.29
C25:1
0.408
0.162
9
22.40
C25:1
2.800
1.351
10
22.56
C25:1
0.717
0.188
11
23.02
n-C25
2.466
0.656
12
23.82
13-meC25
4.172
0.572
13
24.19
5-meC25
0.545
0.278
14
24.48
5,13-dimeC25
0.384
0.166
15
24.59
C26:1
0.273
0.099
16
24.72
3-meC25
0.687
0.129
17
26.11
13-meC25
0.344
0.096
18
26.61
C26:1
0.617
0.146
12
19
26.95
C27:1
3.166
1.079
20
27.07
C27:1
5.268
2.623
21
27.17
C27:1
8.725
3.869
22
27.32
C27:1
3.291
1.539
23
27.66
n-C27
0.549
0.169
24
28.42
13-meC27
3.289
0.584
25
28.61
7-meC27
0.517
0.094
26
28.82
5-meC27
0.511
0.260
27
28.98
11,15-dimeC27 0.990
0.142
28
29.18
C28:1
0.636
0.313
29
29.32
dimeC27
0.923
0.252
30
29.38
C28:1
0.579
0.232
31
29.48
C28:1
0.749
0.347
32
30.58
unknown
1.446
0.977
33
30.76
unknown
0.378
0.318
34
30.95
C29:1
1.014
0.401
35
31.10
C29:1
1.812
0.505
36
31.20
C29:1
1.097
0.345
37
31.45
C29:1
3.295
0.869
38
31.80
C29:1
25.233
4.288
39
31.88
C29:1
2.701
0.600
40
32.15
n-C29
0.796
0.153
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32.82
13-meC29
1.004
0.161
42
33.30
13,17-dimeC29 0.524
0.099
43
33.56
dimeC29
0.087
0.220
13
44
34.94
unknown
0.269
0.226
45
35.27
C30:1
1.023
0.357
46
35.40
C30:1
1.367
0.247
47
35.68
C31:1
0.938
0.224
48
35.95
C31:1
2.534
0.348
49
36.37
n-C31
0.222
0.099
50
37.62
dimeC31
0.500
0.142
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Figure S1. Schematic map of the main study plot and the altitudinal and horizontal transect
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on the eastern slope of Mt. Kenya.
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