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Phylogenomics of palearctic Formica species suggests a single origin of temporary parasitism and gives… Romiguier, Jonathan; Rolland, Jonathan; Morandin, Claire; Keller, Laurent Mar 28, 2018

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RESEARCH ARTICLE Open AccessPhylogenomics of palearctic Formicaspecies suggests a single origin oftemporary parasitism and gives insights tothe evolutionary pathway toward slave-making behaviourJonathan Romiguier1,2*, Jonathan Rolland1,3,4, Claire Morandin5 and Laurent Keller1AbstractBackground: The ants of the Formica genus are classical model species in evolutionary biology. In particular, Darwinused Formica as model species to better understand the evolution of slave-making, a parasitic behaviour whereworkers of another species are stolen to exploit their workforce. In his book “On the Origin of Species” (1859), Darwinfirst hypothesized that slave-making behaviour in Formica evolved in incremental steps from a free-living ancestor.Methods: The absence of a well-resolved phylogenetic tree of the genus prevent an assessment of whetherrelationships among Formica subgenera are compatible with this scenario. In this study, we resolve the relationshipsamong the 4 palearctic Formica subgenera (Formica str. s., Coptoformica, Raptiformica and Serviformica) using aphylogenomic dataset of 945 genes for 16 species.Results: We provide a reference tree resolving the relationships among the main Formica subgenera with highbootstrap supports.Discussion: The branching order of our tree suggests that the free-living lifestyle is ancestral in the Formica genus andthat parasitic colony founding could have evolved a single time, probably acting as a pre-adaptation to slave-makingbehaviour.Conclusion: This phylogenetic tree provides a solid backbone for future evolutionary studies in the Formica genus andslave-making behaviour.Keywords: Ants, Formica, Phylogenomics, Social parasitism, Slave-making, TranscriptomesBackgroundFrom birds to insects, many organisms can reduce thecosts of brood rearing by exploiting resources fromother species [1]. Certain ant species display an ad-vanced form of parasitim, social parasitism, whereby twospecies of social insects coexist in the same nest, one ofwhich is parasitically dependent on the other [2]. Slave-making in ants is a spectacular case of social parasitism.For example, the slave-making ant Formica sanguineainfiltrates nests of its slaves (e.g. the ant Formica fusca)to capture brood that are then reared inside the nest ofthe slave-making ant. After eclosion, the slave will per-form typical worker tasks such as foraging and defend-ing the colony [3]. In ants, slave-making behaviour isbelieved to have evolved nine times within two of the 21known subfamilies, the Formicinae and the Myrmicinae[2, 4, 5]. In fact, only 0.5% of the known ant species areactive slave makers [6], and the origins of slave-makingin ants are still not well understood. The Formica genusis historically renowned as a classical model for studying* Correspondence: jonathan.romiguier@umontpellier.fr1Department of Ecology and Evolution, Biophore, University of Lausanne,1015 Lausanne, Switzerland2CNRS UMR-5554, Institut des Sciences de l’Evolution de Montpellier,Université de Montpellier, 34095 Montpellier, FranceFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Romiguier et al. BMC Evolutionary Biology  (2018) 18:40 https://doi.org/10.1186/s12862-018-1159-4the evolution of social parasitism [6–8]. Reflecting theimportance of social parasitism in the genus, the classictaxonomic division of Formica in four subgenera ispartly based on host/parasite status.Palearctic Formica species are classically divided infour subgenera. The first subgenus, Serviformica (de-rived from the latin servire: “be a servant, be enslaved”),comprises many free-living species that are used as hostsby the three other subgenera. Contrary to the other sub-genera, a single Serviformica queen can found a new col-ony independently. The second and third subgenera,Coptoformica and Formica s. str, are often referred to as“wood ants” and have a similar ecology. They build largemounds from plant material and can start new coloniesby budding or temporary parasitism. Budding is aprocess whereby new queens and workers leave themother to initiate a new colony nearby. This strategy isparticularly common in species forming supercoloniesconsisting of many inter-connected nests [9–11]. In thecase of temporary parasitism, newly-mated queens enterthe nest of a Serviformica host species where they expeland replace the original queen to use the host workersas helpers. Host workers are then gradually replaced bythe daughters of the temporary parasite queen. Finally,the fourth subgenus (Raptiformica, derived from thelatin raptus: “to seize”) contains the only Formica speciesthat practice slave-making, which is the most spectacularform of social parasitism in the genus. During a processcalled slave-raiding, Raptiformica workers capture broodof Serviformica species to increase the worker force oftheir own colony. After emerging in the slave-makernest, the Serviformica workers behave as if they were intheir own colony. Seasonal slave-raiding allows a con-tinuous replenishing of slaves from neighboring hostnests. In addition to slave-raiding, all species of the sub-genus Raptiformica also initiate new colonies by tempor-ary parasitism, similarly to Coptoformica and Formica s.str. Only one species of Raptiformica lives in thepalearctic region (F. sanguinea, which is the type-speciesof the subgenus), while all other species (11) are foundin the nearctic region [12].The evolutionary pathway toward slavery has been ex-tensively discussed [5, 8, 13–17]. In his book “On theOrigin of Species” [8], Darwin first suggested that slave-raiding in the genus Formica might evolve progressivelythrough an intermediary step of brood predationwhereby some individuals would not be eaten and thuslead to accidental “slave-making”. Building on the ideaof gradual evolution from free-living lifestyle to slave-making, Santschi [18] suggested that temporary parasiticcolony founding is an intermediary step towards slave-making. During parasitic colony founding, the queenuses workers of other species as helpers, which may fa-cilitate the use of slaves acquired after raids. By contrast,Wheeler [17] proposed that parasitic colony foundingevolved several times independently in Formica and isnot an intermediary step toward slavery. Finally,Buschinger [14] proposed that brood transport amongnests of a multi-nest colony (i.e., polydomy) acted as anearly step towards brood robbing, as seen in slave-raiding. Alloway [16] extended this theory by suggestingthat brood exchange among nests of a multi-nest colonyevolved toward selfish brood robbing during territorialbattles. Such intra-specific brood robbing could have ul-timately led to the inter-specific slave-raiding observedin Raptiformica species.Discriminating between these hypotheses requires a ro-bust phylogeny of the genus Formica. Several molecularphylogenetic studies have tried to resolve the phylogeny ofpalearctic Formica [19–21], but the relationship amongsubgenera is still unclear, probably because of the lownumber of loci used for these studies (e.g. allozymes andthe cytb mitochondrial gene). A resolved phylogenetic treeof the subgenera is necessary to answer two key questionsregarding the evolutionary pathway toward slavery in theFormica genus. The first is whether the ancestral lifestyleof the Formica genus is similar to the free-living Servifor-mica species. This question could not be answered tillnow, because the exact position of Serviformica in the treewas unknown and the monophyly of this subgenus hasalso never been clearly supported by molecular data [20].The second question is whether parasitic colony foundingdid evolve once or repeatedly. A monophyletic cladegrouping all social parasites (subgenera Raptiformica,Coptoformica and Formica s. str) would suggest a singleorigin of temporary parasitism in Formica, supporting theidea that parasitic colony founding has been a prerequisitefor slave-making to emerge in Raptiformica [18]. Alterna-tively, if these three subgenera of social parasites do notform a monophyletic clade, this would instead support theview that temporary parasitism and slave-making are notevolutionarily tied and evolved several times independ-ently [17]. Because Raptiformica slave-makers and woodants Coptoformica and Formica s. str. Often build multi-nest colonies (i.e polydomy) [9], a clade grouping thesethree subgenera would also provide support to the theorythat brood raiding of Raptiformica slave-makers is derivedfrom brood transport among nests of a polydomous col-ony, as suggested by several authors [5, 14, 16].To reconstruct a robust phylogeny of the Formicagenus, we generated a large transcriptomic dataset in-cluding 10 different species from the four Formica sub-genera (Formica s. str., Coptoformica, Raptiformica andServiformica). We completed our phylogenomic datasetwith six Formica transcriptomes available from the lit-erature [22], and resolved the deepest nodes of the For-mica tree, giving insight to the evolutionary pathwaytoward slavery in the Formica genus.Romiguier et al. BMC Evolutionary Biology  (2018) 18:40 Page 2 of 8MethodsSampling and RNA extractionWe sampled a total of 10 species (F. gagates, F. fusca, F.selysi, F. rufibarbis, F. cunicularia, F. sanguinea, F. pra-tensis, F. paralugubris, F. polyctena and F. bruni) distrib-uted among the 4 palearctic subgenera Formica s. str.,Coptoformica, Raptiformica and Serviformica and usedone Polyergus species (P. rufescens) as an outgroup toroot our phylogeny. The whole body of one individual ofeach species was flash-frozen in liquid nitrogen thenstored at − 80 °C before RNA-extraction. Total RNA wasextracted using specific protocols for ants [23]. MainRNA-extraction steps of this protocol were tissue dis-ruption, lysate homogenization, isolation and purifica-tion of RNA. Prior to precipitation of the RNA withisopropanol, 10 μg of RNAase-free glycogen was addedto the aqueous phase to increase the RNA yield. Weused a NanoDrop spectrophotometer and an Agilent2100 Bioanalyzer to check the quantity and the integrityof RNA extractions.Transcriptome sequencing and assemblyComplementary libraries were prepared using IlluminaTrueSeq preparation kit. These libraries were sequencedon a HiSeq 2000 (Illumina) to produce 100-base-pairs(bp) paired-end reads. We used Trimmomatic to removeadapters and reads with length less than 60 bp and aver-age quality less than 30 [24]. De novo transcriptome as-semblies were performed using a combination of ABySS(Assembly By Short Sequences) and Cap3, following thestrategy of Romiguier et al. [25]. The contigs generatedby ABySS were used in two consecutive Cap3 runs. Illu-mina reads of all individuals were mapped to the denovo transcriptome assembly of its corresponding spe-cies using the BWA program [26]. The contigs with aper-individual average coverage below X2.5 werediscarded.Ortholog genes and alignmentsWe used the Trinity package [27] to predict Open Read-ing Frames (ORFs) and discarded ORFs shorter than200 bp. In contigs with ORFs longer than 200 bp, 5′ and3′ flanking non-coding sequences were deleted, thusproducing predicted coding sequences that are hereafterreferred to as genes. We performed this coding sequencedetection on our 11 (10 Formica + 1 Polyergus) speciesand repeated the same procedure on 5 supplementaryspecies (namely F. exsecta, F. pressilabris, F. truncatulus,F. aquilonia and F. cinerea) with transcriptomes avail-able from a recent article [22]. We used OrthoMCL [28]to retrieve 945 one-to-one ortholog genes among these16 species. We then aligned all these ortholog genesusing MACSE, a multiple sequence alignment softwarethat aligns nucleotide sequences with respect to theiramino-acid translation [29]. We set the options with acost of 10 for frameshift and 60 for stop codons, as ad-vised by the user manual for transcriptomic data [29].Phylogenetic analysesWe performed phylogenetic analyses using three differentmethods: Maximum likelihood methods (RAxML) [30],Bayesian methods (PhyloBayes) [31] and coalescencemethods (MP-EST) [32]. Maximum likelihood and Bayes-ian inferences are the two most common probabilistic treereconstruction methods, and were used on large alignmentsof concatenated genes (supermatrix approach). Coalescencemethods have a different but complementary philosophyand infer a species tree from multiple gene trees (supertreeapproach). All computations were performed at the Vital-IT (http://www.vital-it.ch) Center for high-performancecomputing of the SIB Swiss Institute of Bioinformatics.Maximum likelihood (RAxML)We concatenated all the ortholog genes in a singlesupermatrix alignment of 1270,080 bp (referred later asthe ALLPOSITIONS supermatrix), then refined thissupermatrix using the automated method implementedin trimal [33] to obtain a supermatrix of 970,619 bp (re-ferred later as the CLEAN supermatrix). We also used astricter cleaning procedure by eliminating all nucleotidepositions containing a gap in at least one of the 16 spe-cies, reducing the size of the alignments to 621,307 bp(referred later as the GAPLESS supermatrix). As geneswith high GC-content may dramatically bias tree recon-struction [34, 35], we also used an alignment concatenat-ing only the 50% most GC-poor genes of the dataset(472 genes, total of 647,706 bp, referred later as theGCPOOR supermatrix). The CLEAN, GAPLESS andGCPOOR supermatrices were analyzed with RAxML[30] using a GTR + GAMMA model with 500 bootstrapreplications. We compute a supplementary tree by parti-tioning the ALLPOSITIONS supermatrix by codon posi-tions (i.e. different parameter estimation for the sitesbelonging to the 1st, 2nd or 3rd codon position) usingRAxML and a GTR +GAMMA model (500 bootstrapreplications).Bayesian method (PhyloBayes)For Bayesian inference we used PhyloBayes MPI [31]with a CAT-GTR model. This model takes into accountsite-specific nucleotide preferences, which better modelsthe level of heterogeneity seen in real data and is wellsuited to large multigene alignments [36, 37]. Becausethis method is computationally more costly than a max-imum likelihood approach (RAxML), it was only runusing the GAPLESS supermatrix (621,307 bp). We runtwo independent Markov chains and convergence wasassessed by comparing the two independent MarkovRomiguier et al. BMC Evolutionary Biology  (2018) 18:40 Page 3 of 8chains with bpcomp and tracecomp tools from Phylo-Bayes. We stopped the inferences after 15,000 genera-tions, with a maximum discrepancy in clade support of0 (maxdiff metrics from bpcomp), a minimal effectivesample size of 50 (effsize metrics from tracecomp) and amaximal relative difference in posterior mean estimatesof 0.3 (red_diff metrics from tracecomp). The appropri-ate number of generations to discard as “burn-in” (1000)was assessed visually using Tracer 1.6.Coalescence based method (MP-EST)Recently developed coalescence-based methods use mul-tiple gene trees to reconstruct phylogenies. Contrary tothe other phylogenetic methods used in this article, thismethod does not use a concatenated sequence of all thegenes but builds a species tree based on every individualgene tree. The main advantage of this approach is to bet-ter take into account incomplete lineage sorting [32, 38], aphenomenon whereby different gene trees differ from thespecies tree [39]. We used MP-EST (Maximum Pseudoli-kelihood Estimation of the Species Tree), a coalescence-based method that estimates a species tree from a set ofgene trees by maximizing a pseudo likelihood function[32]. We built individual gene trees with RAxML (GTR +GAMMA model, 500 bootstrap replicates) and used theresulting 500 bootstrap replicates of each gene tree (avail-able as supplementary material) to compute a species treewithMP-EST through the STRAW web server [40].SH tests of monophylyTo test for the monophyly of the Serviformica subgen-era, we performed Shimodaira-Hasegawa tests [41] asimplemented in RAxML. We used the CLEAN superma-trix to compare the maximum likelihood value of a treethat constrains the monophyly of Serviformica species tothe maximum likelihood value of the best unconstrainedtree.ResultsPhylogenetic analysesWe generated a phylogenomic dataset of 965 orthologgenes in 16 species that we concatenated in a singlemulti-gene alignment cleaned using three different pro-cedures (CLEAN, GAPLESS and GCPOOR, see Materialand Methods for details) and analysed these data (super-matrices or individual gene trees) with three differentphylogenetic methods (maximum likelihood withRAxML, bayesian inference with PhyloBayes and asupertree coalescence-based method with MP-EST, seeMaterial and Methods for details). All analyses retrievedessentially the same phylogenetic relationships with onlyfew discrepancies. These discrepancies concerned rela-tionships among highly related species, in particular inthe Formica str. s. subgenus (Fig. 1). This result is notsurprising given that there are many cases of hybrids inthis taxonomic group and even colonies may compriseseveral species of this subgenus [42–48]. It is likely thathybridization is associated with significant gene flowamong species, which, in turn, will cause discrepanciesamong gene trees and thus hamper species tree recon-structions, regardless of the method used [49]. Bayesianinference (PhyloBayes) recovered the highest supportvalues while the coalescence-based approach (MP-EST)retrieved globally slightly lower support values (Fig. 1).Fig. 1 Molecular phylogeny of Formica. Branch lengths and topology are based on the Maximum-likelihood analysis (CLEAN supermatrix). Supportof the three methods (Maximum-likelihood, Bayesian and Coalescence-based method) is indicated for each node. When a method does not re-trieve a node, the support value is replaced by a “-”Romiguier et al. BMC Evolutionary Biology  (2018) 18:40 Page 4 of 8Phylogenetic trees retrieved for each analysis are avail-able in the Supplementary Material section (Add-itional file 1: Fig. S1, Additional file 2: Fig. S2,Additional file 3: Fig. S3, Additional file 4: Fig. S4, Add-itional file 5: Fig. S5 and Additional file 6: Fig. S6). Thetree of the RAxML + CLEAN analysis is used as the ref-erence for the topology and branch lengths in Fig. 1while the nodal support of the bayesian (PhyloBayes)and coalescent-based approach (MP-EST) are mappedon each node. Exactly the same topology is obtained bypartitioning the dataset by codon positions (RAxML +ALLPOSITIONS analysis, Additional file 6: Fig. S6).Non-monophyly of ServiformicaOur results do not support the monophyly of the sub-genus Serviformica. Phylogenetic analyses of the six spe-cies of this subgenus indicate with high support valuesthat these species are clustered in three different mono-phyletic clades (Fig. 1), namely (F. fusca + F. cinerea + F.selysi), (F. cunicularia + F. rufescens) and (F. gagates).To further validate the non-monophyly of the Servifor-mica subgenus, we performed a Shimodaira-Hasegawatest [41] by comparing the likelihood of a tree constrain-ing the monophyly of the six Serviformica species withthe likelihood of the unconstrained tree retrieved in theRAxML + CLEAN analysis. The likelihood of the uncon-strained tree was significantly higher than the likelihoodof the tree constraining the monophyly of Serviformica(respectively − 1,865,962 and − 1,867,685, SH test p-value< 0.01), confirming the non-monophyly of Serviformica.Monophyly of social parasitesAll the analyses support with maximal values the mono-phyly of the Coptoformica and Formica str. s. subgenera(Fig. 1). This result confirms previous phylogenetic stud-ies [19, 20]. More interestingly, we also retrieved amonophyletic clade grouping together the temporary so-cial parasite subgenera Coptoformica, Formica s. str andRaptiformica. The support for this grouping is unam-biguous and maximal in all the phylogenies constructedin our study (100 in the three RAxML Maximal likeli-hood analyses, 1.0 for the PhyloBayes Bayesian inferenceand 100 for the MP-EST shortcut coalescence approach).This result contrasts with previous studies that failed toretrieve a high bootstrap support for the monophyly ofthe temporary social parasite clade [19, 20]. Coptofor-mica, Formica s. str. and Raptiformica subgenera shareimportant ecological traits, such as the loss of the abilityto independently found new colonies and temporaryparasitic colony founding. A single clade grouping thesesubgenera suggests that they inherited the ability toparasite Serviformica nests from a common ancestor.This result suggests a common origin of socialparasitism in both wood ants (Formica str. s. and Copto-formica) and slave-makers (Raptiformica).Phylogenetic position of Nearctic Formica speciesAlthough our species sampling includes all describedpalearctic Formica subgenera, it lacks representatives ofnearctic species, particularly species of two described ne-arctic groups of slave species, namely the F. neogagatesgroup and the F. pallidefulva group [12]. To confirmthat these two nearctic groups of slave species do notbelong to the clades of social parasites (Formica str. s,Coptoformica and Raptiformica), which may affect ourconclusion of a single origin of slave-making, we built anadditional phylogeny based on the cox1 sequence of allFormica species available in GeneBank (i.e., 41 species,19 with a nearctic distribution). As expected by the shortlength of the alignment (1270 bp), the resulting phylo-genetic tree (Additional file 7: Fig. S7) has few well-resolved nodes (i.e. bootstrap support > 70), but there isgood support for the F. neogagates group (representedby F. neogagates, F. perpilosa and F. lasioides) and the F.pallidefulva group (represented by F. pallidefulva) beingnot part of the parasitic clades. Rather, these two groupsappear to be the two most basal clades of this Formicaphylogeny (supported by a bootstrap of 87). Among theother well-resolved phylogenetic relationships, this ana-lysis also retrieved three clades corresponding to thethree social parasites subgenera, namely Raptiformica(bootstrap of 94), Formica str. s. species (bootstrap of87) and Coptoformica species (bootstrap of 94). Import-antly, all the nearctic Raptiformica species (F. wheeleri,F. aserva and F. subintegra) cluster with the palearctic F.sanguinea. This well-supported monophyly of themorphologically-defined Raptiformica subgenus thus in-dicates that slave-raiding did not evolve independentlyin the palearctic and nearctic regions, supporting theview of a single origin of slave-making in the Formicagenus.DiscussionThe six species of the subgenus Serviformica clusteredin three different monophyletic clades (Fig. 1). Previousstudies already questioned the monophyly of Servifor-mica, but the low number of molecular markers pre-vented sufficiently high support values (> 70) to give aclear answer [20, 21]. Our results, which are based on alarge phylogenomic dataset, demonstrate that Servifor-mica should not be considered as a subgenus anymore,but is a paraphyletic group of species occupying a basalposition in the Formica genus. Because all Serviformicaspecies are free living (i.e., able to start new colonies ontheir own), this indicates that a free living lifestyle is ashared ancestral state (i.e. plesiomorphy) of ServiformicaRomiguier et al. BMC Evolutionary Biology  (2018) 18:40 Page 5 of 8species, and then is the ancestral state of the Formicagenus.Our results are consistent with two previous theoriesproposed to explain the evolution towards slavery inFormica. The first is that parasitic colony founding is anintermediary step from independent colony founding toslave-making [15]. The second is that brood transportamong nests of polydomous colonies preceded broodrobbing observed in slave-raiding [14]. The branchingorder of our phylogeny suggests an evolutionary pathwaytoward slavery in several steps. The basal position of Ser-viformica species in the Formica phylogeny suggests afree-living ancestor with independent colony founding(white star in Fig. 1). From this ancestral state, ourphylogenetic trees support a single loss of independentcolony founding (grey star in Fig. 1) in both wood ants(Coptoformica and Formica s. str.) and slave-makers(Raptiformica). Dependent colony founding has beensuggested as an adaptation to unfavorable cold habitatwhere success of independent colony founding is limitedby high queen mortality [2, 6, 50]. This is supported bythe alpine/boreal distribution of Formica social parasitesand the fact that they all build mound nests from plantmaterials, which is known to increase thermal isolation[51]. To adapt to cold habitats, the ancestor of Formicasocial parasites may have avoided independent colonyfounding by allowing the return of mated queens in theparental colony, a hypothesis supported by the high oc-currence of polygyny in the social parasite clades Rapti-formica, Coptoformica and Formica s. str. As suggestedby Buschinger [14], parasitic colony founding is thenlikely to have evolved from a state where queensreturned to an established nest of their species to exploitthe workforce and the security of other species nests.The finding that the Raptiformica slave-maker subgenusis nested in the monophyletic clade grouping the twowood-ant subgenera (Formica s. str and Coptoformica)suggests that slave-raiding evolved at some point from awood-ant ancestor (black star in Fig. 1). As typically seenin both wood-ant and Raptiformica species, such an an-cestor of the Raptiformica slave-makers is likely to havefeatured polydomous (multi-nests) colonies, as suggestedby Buschinger’s hypothesis [14] whereby slave-raidingevolved from opportunistic brood transport among nestsof large polydomous colonies.While our phylogenomic dataset offers an unprece-dented amount of genetic information for the Formicagenus (up to 1270,080 bp), one of its limitations is theexclusively palearctic distribution of the species sampled.This sampling issue is unlikely to affect our conclusionsregarding the non-monophyly of Serviformica, but canaffect our conclusions regarding the monophyly of socialparasites (Formica s. str. + Coptoformica + Raptiformica).Based on our analysis of the cox1 sequence of 41 species(including a total of 19 nearctic species, Additional file7: Fig. S7), we can however reasonably exclude the possi-bility that nearctic groups of slave species (F. pallide-fulva group and F. neogagates group) cluster with socialparasites. Furthermore, most of the social parasite spe-cies (19 out of 22) are clustered in their expected socialparasite subgenus, namely Coptoformica, Formica s. str.or Raptiformica (Additional file 7: Fig. S7). However, thisgene analysis of a single gene does not allow one to givea clear position of F. uralensis, F. dakotensis and F. ulkei,three species that have been reported to practice tem-porary parasitism during colony founding [2]. These spe-cies are traditionally thought to be part of the Formica s.str. Subgenus (for F. uralensis and F. dakotensis) or theCoptoformica subgenus (F. ulkei), but their subgenus af-filiation is here not confirmed, an issue already knownfor F. uralensis that has a notoriously controversialphylogenetic position [19, 20]. Future phylogenomicsdataset analyses should include these controversial spe-cies in order to clarify their position in the Formicaphylogeny and confirm whether parasitic colony found-ing appeared only once in the genus.ConclusionThis study resolves the phylogenetic relationships amongpalearctic Formica subgenera. Interestingly, our phylo-genetic tree reveals that the free-living Serviformica spe-cies do not form a monophyletic clade, and thatparasitic colony founding in wood ants and Raptiformicaslave-makers is likely to have a single origin. Slave-making behaviour is observed in nine different ant gen-era and has evolved several times repeatedly across theant phylogeny [6]. While slave-maker species and slavespecies tend to be closely related [52], the evolutionaryorigins of slave making itself remains obscure. Our re-sults suggest that parasitic colony founding is likely tobe an intermediary step between free-living hosts andslave-maker parasites in the Formica genus. Similarstudies in other genera containing slave-making species(e.g. Temnothorax, Harpagoxenus, Myrmoxenus, Proto-mognathus…) will be necessary to get a better global pic-ture of the evolution of slave-making in ants.Additional filesAdditional file 1 Figure S1 Phylogenetic tree of the CLEAN supermatrix(970,619 bp) built using RAxML (GTR + GAMMA model, 500 bootstrapreplications). (PDF 2 kb)Additional file 2 Figure S2 Phylogenetic tree of the GAPLESSsupermatrix (621,307 bp) built using RAxML (GTR + GAMMA model, 500bootstrap replications). (PDF 2 kb)Additional file 3 Figure S3 Phylogenetic tree of the GCPOORsupermatrix (647,706 bp) built using RAxML (GTR + GAMMA model, 500bootstrap replications). (PDF 2 kb)Romiguier et al. BMC Evolutionary Biology  (2018) 18:40 Page 6 of 8Additional file 4 Figure S4 Phylogenetic tree of the GAPLESSsupermatrix (621,307 bp) built using PhyloBayes (two independentMarkov chains, 15,000 generations). (PDF 2 kb)Additional file 5 Figure S5 Phylogenetic tree of the MP-EST analysisbased on 945 gene trees (500 bootstrap replications for each gene tree).(PDF 2 kb)Additional file 6 Figure S6 Phylogenetic tree of the ALLPOSITIONSsupermatrix (1270,080 bp) built using RAxML by partitioning thesupermatrix by codon positions (GTR + GAMMA model, 500 bootstrapreplications). (PDF 2 kb)Additional file 7 Figure S7 Phylogenetic tree based on the cox1mitochondrial gene of 41 Formica species borrowed from GeneBank(NCBI ID indicated between parentheses). The tree was built using RAxML(GTR + GAMMA, 500 bootstrap replications). Nodes supported by abootstrap inferior to 70 were removed. Nearctic species are highlightedin red. (PDF 35 kb)AknowledgementsWe thank Jessica Purcell, Timothée Brütsch, Michel Chapuisat, Nicolas Galtier,Marion Ballenghien, Rumsais Blatrix and Nicolas Faivre for their help duringsampling. We also thank Christophe Galkowski for help during speciesidentification. Some computations were performed at the Vital-IT (http://www.vital-it.ch) Center for high-performance computing (HPC) of the SIBSwiss Institute of Bioinformatics and the EPSRC-funded MidPlus HPC center.FundingThis work was supported by a Federation of European Biochemical Societies(FEBS) long-term fellowship to J. Romiguier, the Swiss NSF, and an ERC Ad-vanced Grant. C. Morandin was supported by the Academy of Finland (grandnumber 52411, 284666 to Centre of Excellence in Biological Interactions). J.Rolland received funding from the Swiss National Science Foundation(CRSIII3–147630, PI: Nicolas Salamin) at UNIL and from a Banting postdoctoralfellowship at UBC.Availability of data and materialsThe dataset generated during the current study (raw reads) are available inthe ENA (European Nucleotide Archive), repository (https://www.ebi.ac.uk/ena/data/view/PRJEB25332).Author’s contributionsJR and CM generated the data. JR analyzed the data. JR, JR, CM and LKwrote the manuscript. All authors read and approved the final manuscript.Ethics approval and consent to participateNot applicable.Consent for publicationNot applicable.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Department of Ecology and Evolution, Biophore, University of Lausanne,1015 Lausanne, Switzerland. 2CNRS UMR-5554, Institut des Sciences del’Evolution de Montpellier, Université de Montpellier, 34095 Montpellier,France. 3Department of Zoology, University of British Columbia, #4200-6270University Blvd, Vancouver, B.C., Canada. 4Swiss Institute of Bioinformatics,Quartier Sorge, 1015 Lausanne, Switzerland. 5Centre of Excellence inBiological Interactions, Department of Biosciences, University of Helsinki,Helsinki, Finland.Received: 29 March 2017 Accepted: 19 March 2018References1. Davies NB, Bourke AF, de L Brooke M. 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