Patterns of positive selection in seven ant genomes
Julien Roux, Eyal Privman, Sebastien Moretti, Josephine T. Daub, Marc Robinson-Rechavi, Laurent Keller
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For!latest!version,!please!visit:!http://mbe.oxfordjournals.org/content/early/2014/04/20/molbev.msu141.abstract! ! rticle Patterns of positive selection in seven ant gen omes
Julien' Roux ,' Eyal' Privman ,' Sébastien' Moretti ,' Josephine' T.' Daub ,' Marc'RobinsonERechavi ,'Laurent'Keller ' 'Department'of'Ecology'and'Evolution,'University'of'Lausanne,'1015'Lausanne,'Switzerland' 'SIB'Swiss'Institute'of'Bioinformatics,'1015'Lausanne,'Switzerland' VitalEIT'group,'SIB'Swiss'Institute'of'Bioinformatics,'1015'Lausanne,'Switzerland'' CMPG,'Institute'of'Ecology'and'Evolution,'University'of'Bern,'Baltzerstrasse'6,'3012'Bern,'Switzerland' Present' address:' Department' of' Human' Genetics,' University' of' Chicago,' Chicago,' IL'60637,'USA' 'Present'address:'Department'of'Evolutionary'and'Environmental'Biology,'University'of'Haifa,'Israel' ' Corresponding'author:'Julien'Roux'([email protected])'
Short title
Positive'selection'in'ant'genomes ! Keywords
Comparative'genomics,'sociality,' d N / d S ,'aging,'ageing,'lifespan,'immunity,'neurogenesis,'olfactory'receptors,'metabolism,'Hymenoptera,'bees,'Drosophila' Authors contributions
JR,' EP' and' LK' designed' the' study;' JR' and' EP' analyzed' data;' JTD,' SM' and' MRR'contributed'code'or'programs;'JR'and'LK'wrote'the'manuscript'with'input'from'MRR.' bstract
The' evolution' of' ants' is' marked' by' remarkable' adaptations' that' allowed' the'development' of' very' complex' social' systems.' To' identify' how' antEspecific' adaptations'are' associated' with' patterns' of' molecular' evolution,' we' searched' for' signs' of' positive'selection' on' aminoEacid' changes' in' proteins.' We' identified' 24' functional' categories' of'genes' which' were' enriched' for' positively' selected' genes' in' the' ant' lineage.' We' also'reanalyzed'genomeEwide'datasets'in'bees'and'flies'with'the'same'methodology,'to'check'whether'positive'selection'was'specific'to'ants'or'also'present'in'other'insects.'Notably,'genes' implicated' in' immunity' were' enriched' for' positively' selected' genes' in' the' three'lineages,' ruling' out' the' hypothesis' that' the' evolution' of' hygienic' behaviors' in' social'insects'caused'a'major'relaxation'of'selective'pressure'on'immune'genes.'Our'scan'also'indicated' that' genes' implicated' in' neurogenesis' and' olfaction' started' to' undergo'increased' positive' selection' before' the' evolution' of' sociality' in' Hymenoptera.' Finally,'the' comparison' between' these' three' lineages' allowed' us' to' pinpoint' molecular'evolution' patterns' that' were' specific' to' the' ant' lineage.' In' particular,' there' was' antEspecific' recurrent' positive' selection' on' genes' with' mitochondrial' functions,' suggesting'that'mitochondrial'activity'was'improved'during'the'evolution'of'this'lineage.'This'might'have'been'an'important'step'toward'the'evolution'of'extreme'lifespan'that'is'a'hallmark'of'ants.' ntroduction
Ants' constitute' an' extremely' successful' lineage' of' animals' which' has' colonized'virtually' all' ecosystems' on' Earth' (Hölldobler,' Wilson' 1990).' The' pivotal' feature' at' the'basis'of'this'ecological'success'is'their'highly'social'system'with'a'reproductive'division'of'labor,'where'one'or'a'few'queens'specialize'in'reproduction,'while'workers'conduct'all' the' colony' tasks' such' as' brood' care,' nest' maintenance' and' food' collection.' In' this'paper' we' take' advantage' of' the' recent' availability' of' seven' sequenced' ant' genomes'(Bonasio'et'al.'2010;'Nygaard'et'al.'2011;'Smith'et'al.'2011a;'Smith'et'al.'2011b;'Suen'et'al.' 2011;' Wurm' et' al.' 2011)' to' perform' a' genomeEwide' scan' for' positive' selection' on'aminoEacid'changes'in'protein'coding'genes'during'the'evolution'of'the'ant'lineage.'We'addressed'three'main'questions.'First,' we' compared' the' amount' of' positive' selection' in' functional' categories' of'genes.'Previous'largeEscale'scans'for'positive'selection'in'animals'indicated'that'positive'selection' predominantly' affects' certain' types' of' genes,' such' as' those' involved' in'evolutionary' arms' races,' sexual' selection' or' conflicts' with' pathogens' (Bakewell,' Shi,'Zhang'2007;'Drosophila'12'Genomes'Consortium'2007;'Kosiol'et'al.'2008;'Vamathevan'et' al.' 2008;' Oliver' et' al.' 2010;' George' et' al.' 2011;' Woodard' et' al.' 2011).' Such' genes'experienced'positive'selection'events'recurrently'on'broad'evolutionary'time'scales,'and'it'is'likely'that'they'contribute'to'a'fraction'of'the'positive'selection'events'that'occurred'in'the'ant'lineage.'To'identify'these'genes,'we'reasoned'that'they'likely'also'were'under'positive' selection' in' other' insect' lineages.' A' systematic' comparison' of' the' targets' of'positive' selection' from' published' studies' in' insects' is' not' straightforward' because'genomeEwide'scans'for'positive'selection'were'often'performed'with'different'methods'in'different'lineages.'For'example,'a'positive'selection'scan'on'12'Drosophila'species'(all'solitary)(Drosophila'12'Genomes'Consortium'2007)'used'the'site'test'of'Codeml'(Yang'et' al.' 2000),' which' is' aimed' at' detecting' recurrent' positive' selection' events' affecting'particular' sites' of' a' protein,' while' a' scan' on' 10' bee' species' (including' solitary,'primitively' social' and' highly' social' species)(Woodard' et' al.' 2011)' used' the' branch' test'(Yang'1998),'which'tends'to'detect'positive'selection'events'affecting'a'large'number'of'sites'of'a'protein'but'during'a'limited'period'of'time.'To'perform'a'robust'comparison'of'the' genes' that' were' under' positive' selection' in' ants' and' other' insects,' we' conducted'similar'scans'for'positive'selection'in'ants'and'the'flies'and'bees'outgroups.'An'example'of'genes'expected'to'be'repeatedly'under'positive'selection'in'insects'are'genes'involved'n'defense'and'immunity'(Drosophila'12'Genomes'Consortium'2007;'Bulmer'2010).'On'the'basis'of'the'observed'smaller'set'of'immunity'genes'in'the'honeybee'compared'to'
D.&melanogaster, ' it' has' been' suggested' that' selective' pressure' on' these' genes' might' have'been' relaxed' in' social' insects,' perhaps' because' they' have' social' hygienic' behaviors'(Honeybee' Genome' Sequencing' Consortium.' 2006;' Smith' et' al.' 2008;' Viljakainen' et' al.'2009;'Smith'et'al.'2011a;'Suen'et'al.'2011;'Harpur,'Zayed'2013).'However,'the'addition'of' several' newly' sequenced' insect' genomes' revealed' that' the' important' gene'complement'in'fruit'fly'is'a'derived'character'(Werren'et'al.'2010;'Fischman,'Woodard,'Robinson' 2011;' Smith' et' al.' 2011b).' We' used' our' datasets' to' test' whether' there' was'evidence'for'weaker'positive'selection'on'immunity'genes'in'ants'and'bees'compared'to'flies.' Next,' we' aimed' at' detecting' sets' of' genes' involved' in' functions' likely' to' reflect'antEspecific'adaptations.'We'focused'on'three'main'adaptations.'The'first'relates'to'the'wide' range' of' coordinated' collective' behaviors' associated' with' division' of' labor' in' ant'societies.' Complex' cooperative' behaviors' occur' among' nestmates' for' tasks' such' as'communal' nest' construction' and' defense,' brood' rearing,' social' hygienic' behavior' and'collective'foraging'(Hölldobler,'Wilson'1990).'It'has'been'suggested'that'the'evolution'of'social'interactions'may'be'tracked'down'to'molecular'changes'affecting'nervous'system'development'and'function.'In'particular'it'may'translate'into'increased'rates'of'positive'selection'on'nervous'systemErelated'genes,'as'documented'in'primitively'social'lineages'of' bees,' which' evolved' social' behaviors' independently' from' ants' (Fischman,' Woodard,'Robinson' 2011;' Woodard' et' al.' 2011).' Complex' collective' behaviors' also' require'efficient'communication'systems,'that'are'essentially'mediated'by'chemical'signaling'in'social' insects.' Ants' identify' nestmates' from' nonEnestmates,' as' well' as' ants' from' other'species,'through'their'scent.'Individuals'also'use'various'types'of'pheromones'as'alarm'signals' and' to' mark' their' trails' and' territories.' It' has' therefore' been' suggested' that'genes' involved' in' chemical' signaling,' notably' pheromone' production' and' perception,'should' experience' increased' positive' selection' in' ants' compared' to' solitary' insects'(Ingram,' Oefner,' Gordon' 2005;' Robertson,' Wanner' 2006;' Bonasio' et' al.' 2010;' Smith' et'al.' 2011a;' Wurm' et' al.' 2011;' Kulmuni,' Wurm,' Pamilo' 2013;' Leboeuf,' Benton,' Keller'2013).'A'manually'curated'dataset'of'873'olfactory'receptor'genes'allowed'us'to'conduct'a'test'for'increased'positive'selection'on'these'genes'in'ants.'he'second'type'of'potential'molecular'adaptation'relates'to'phenotypic'plasticity'among' castes.' Although' queens' and' workers' usually' develop' from' totipotent' eggs'(Schwander' et' al.' 2010),' they' display' dramatic' morphological' and' physiological'differences.'Queens'are'often'larger,'have'wings'and'have'much'more'highly'developed'ovaries'than'workers'which'often'are'sterile'and'lack'a'sperm'storage'organ'(Hölldobler,'Wilson'1990).'In'most'species'the'differences'between'castes'result'from'developmental'differences'induced'by'environmental'factors'rather'than'genetic'differences'(Abouheif,'Wray'2002;'Schwander'et'al.'2010;'Penick,'Prager,'Liebig'2012;'Rajakumar'et'al.'2012).'We' therefore' investigated' whether' there' was' evidence' for' increased' positive' selection'in' genes' and' pathways' potentially' involved' in' developmental' plasticity' (Smith' et' al.'2008;'Fischman,'Woodard,'Robinson'2011).'A' third' and' interesting' type' of' antEspecific' adaptation' relates' to' the' extremely'long'lifespan'of'ant'queens,'which'can'live'more'than'20'years'in'some'species'(Keller,'Genoud'1997;'Jemielity'et'al.'2005).'This'corresponds'to'a'100Efold'increase'in'lifespan'compared'to'solitary'insects.'The'variation'in'lifespan'among'castes'is'also'remarkable,'with'queens'living'up'to'10'times'longer'than'workers'and'500'times'longer'than'males.
So' far,' a' limited' number' of' molecular' candidates' have' been' identified' to' explain' this'pattern,'mainly'inspired'from'work'in'Drosophila & (Jemielity'et'al.'2005;'Keller,'Jemielity'2006) . ' We' therefore' investigated' if' there' was' evidence' of' positive' selection' on' genes'that'have'previously'been'associated'with'aging'in'model'organisms.'It'is'possible'that'positive'selection'acted'on'the'same'sets'of'genes'in'the'bee'lineage,'where'queens'also'live' longer' than' other' castes' and' than' solitary' insects,' but' such' a' signal' should' not' be'observed' in' shortElived' species' of' the' Drosophila' lineage.' To' further' assess' the' link'between' positive' selection' and' aging,' we' investigated' whether' genes' that' experienced'positive' selection' in' ants' were' genes' shown' in' Drosophila& melanogaster ' to' be'differentially' expressed & between' old' and' young' individuals,' and' between' oxygenEstressed'and'control'individuals'(Landis'et'al.'2004).'Finally,' we' investigated' whether' there' was' a' difference' in' the' level' of' positive'selection'between'genes'showing'biased'expression'in'queens,'workers'and'males.'The'efficiency' of' natural' selection' acting' on' an' advantageous' mutation—and' thus' the'probability' of' its' longEterm' fixation—is' proportional' to' its' effect' on' fitness' (Duret'2008).' The' fitness' effects' of' mutations' in' genes' that' are' expressed' only' in' nonEreproductive' workers' are' indirect,' so' everything' else' being' equal,' selection' should' be'ess' efficient' at' fixing' them' than' mutations' on' genes' expressed' in' queens' and' males.'This' could' translate' into' lower' levels' of' positive' selection' on' genes' expressed'specifically' in' workers' compared' to' males' and' queens' (Linksvayer,' Wade' 2009;' Hall,'Goodisman' 2012).' We' therefore' analyzed' previously' published' microarray' data' from'the' red' fire' ant' Solenopsis& invicta& (Ometto' et' al.' 2011),' and' compared' the' amount' of'positive' selection' between' groups' of' genes' varying' in' the' level' of' casteEbiased'expression.'
Results
Pervasive positive selection detected in ants
To' detect' positive' selection' episodes' that' acted' on' protein' coding' genes' during'the'evolution'of'the'ant'lineage,'the'branchEsite'test'of'Codeml'was'run'on'4,261'protein'alignments' of' singleEcopy' orthologs' composed' of' four' to' seven' ant' and' three' to' five'outgroup' species' (see' Materials' and' Methods).' All' branches' that' led' to' ant' species' in'each' gene' family' tree' (including' 2' hymenopteran' and' 13' ant' branches,' Figure' 1)' were'successively' tested' for' the' presence' of' episodic' positive' selection.' As' many' as' 1,832'singleEcopy' orthologs' families' (43%)' displayed' a' signal' of' positive' selection' (at' 10%'FDR)' in' at' least' one' of' the' branches' tested' (Table' S1).' In' 91%' of' the' significant'alignments,'at'least'one'residue'targeted'by'positive'selection'could'be'identified'with'a'posterior' probability' greater' than' 0.9' (Bayes' Empirical' Bayes' test,' Figure' 2)(Yang,'Wong,'Nielsen'2005).'There'was'evidence'for'positive'selection'in'at'least'one'branch'of'the'ant'lineage'for'830'(20%)'of'the'genes'analyzed.'For'74%'of'them'positive'selection'was' specific' to' ants' and' not' observed' in' the' basal' hymenopteran' branches' & tested' ( χ ' test' p<10 E15 ;' Table' 2),' similarly' to'previous' analyses' with'experimental' and' simulated' datasets.' This' likely' results' at' least' in' part' from' lower'power' of' the' branchEsite' test' in' shorter' branches' (Anisimova,' Yang' 2007;' Kosiol' et' al.'2008;' Studer' et' al.' 2008;' Fletcher,' Yang' 2010;' George' et' al.' 2011;' Gharib,' RobinsonERechavi' 2013).' Consistent' with' this' view,' there' was' a' significant' correlation' in' our'dataset' between' the' length' of' tested' branches' and' the' test' score' (logElikelihood' ratio;'pearman' correlation' ρ =0.41,' p<10 E15 ).' Additional' analyses' ruled' out' the' hypothesis'that' false' positives' caused' by' either' convergence' problems' of' the' test,' selective'constraints' acting' on' synonymous' sites,' saturation' of' synonymous' substitution' rate' d S 'or'sequencing'errors'could'be'responsible'for'this'pattern'(Supplementary'text).'Taken'together,'these'results'demonstrate'that'positive'selection'was'common'in'the' evolution' of' the' ant' genes.' The' proportion' of' significant' genes' was' similar' in'magnitude' in' the' outgroup' dataset' of' 10' bees' analyzed' with' the' same' methodology'(20%;' Table' S2),' but' even' higher' in' the' outgroup' dataset' of' 12' flies' (36%;' Table' S3).'This' difference' might' reflect' biological' differences' between' the' lineages,' such' as'effective' population' size' N E ,' but' also' differences' in' the' topology' and' branch' lengths' of'the' species' trees,' which' influence' the' power' to' detect' positive' selection' events' in'protein'alignments'(see'Supplementary'text).'To'compare'the'amount'of'positive'selection'experienced'by'different'functional'categories'of'genes,'we'classified'genes'based'on'their'Gene'Ontology'(GO)'annotation'in' D.& melanogaster ' orthologs,' and' performed' a' gene' set' enrichment' test' using' for' each'gene' family' a' score' reflecting' the' overall' occurrence' of' positive' selection' in' the' ant'lineage' (Materials' and' Methods;' Supplementary' text).' Such' an' approach' of' grouping'genes'enables'a'more'sensitive'search'for'positive'selection,'while'buffering'the'impact'of' potential' false' positives' (e.g.,' from' remaining' alignment' errors' or' GCEbiased' gene'conversion'events'which'are'difficult'to'distinguish'from'real'positive'selection'signals;'see' Supplementary' text' and' Tables' S20' to' S24).' TwentyEfour' functional' categories' of'genes'were'significantly'enriched'for'positively'selected'genes'in'the'ant'lineage'(at'20%'FDR;'Table'3).'A'large'number'of'them'(11'out'of'24)'were'related'to'mitochondria'and'mitochondrial'activity.'The'other'significant'categories'were'related'to'nervous'system'development,'behavior,'immunity,'protein'translation'and'degradation,'cell'membrane,'and'receptor'activity.'Thus'positive'selection'apparently'targeted'a'diverse'array'of'gene'functions'during'the'evolution'of'the'ant'lineage.''
Usual targets of positive selection in insects
To'identify'GO'categories'that'experienced'positive'selection'not'only'in'ants'but'also'in'other'insects,'we'reanalyzed'the'fly'and'bee'datasets'with'the'same'methodology'used' for' the' ant' dataset.' These' analyses' revealed' 106' GO' categories' significantly'enriched' for' flies' and' 38' for' bees' (Tables' 4' and' 5;' Tables' S4' and' S5).' We' investigated'hich' categories' were' enriched' for' positively' selected' genes' in' the' three' lineages.' The'first'group'of'genes'commonly'enriched'in'ants,'flies'and'bees'was'related'to'proteolysis.'This' group' included' four' of' the' 24' significantly' enriched' GO' categories' in' ants'(“proteolysis”,' “metallopeptidase' activity”,' “peptidase' regulator' activity”' and' “hydroElyase'activity”),'8'of'the'106'GO'categories'enriched'in'flies'(“serineEtype'endopeptidase'activity”,' “endopeptidase' activity”,' “proteolysis”,' “metalloendopeptidase' activity”,'“peptidase' activity”,' “peptidase' activity,' acting' on' LEamino' acid' peptides”,'“metallopeptidase' activity”,' “exopeptidase' activity”),' and' 6' of' the' 38' GO' categories'enriched' bees' (“amine' metabolic' process”,' “metalloendopeptidase' activity”,'“metallopeptidase' activity”,' “signal' transduction”,' “cellular' amine' metabolic' process”'and'“cellular'amino'acid'metabolic'process”).'The'second'group'of'genes'enriched'for'positive'selection'signal'in'ants,'flies'and'bees' was' involved' in' response' to' stimuli.' There' was' an' enrichment' of' the' GO' category'“receptor' activity”' in' the' three' lineages' as' well' as' the' GO' categories' “transmembrane'receptor' activity”' and' “olfactory' receptor' activity”' in' flies.' This' class' of' genes' plays' a'pivotal'role'in'the'interactions'between'individuals'and'their'environment.'In'addition,'the'GO'categories'“response'to'biotic'stimulus”'and'“response'to'other'organism”'were'enriched'in'flies,'and'the'GO'category'“response'to'hormone'stimulus”'was'enriched'in'bees.'In'ants,'“response'to'ecdysone”'and'“response'to'steroid'hormone'stimulus”'were'marginally'significant'(FDR'='21%;'Table'S6).''Some' functions' were' enriched' for' positively' selected' genes' in' only' two' of' the'three'lineages.'These'included'GO'categories'related'to'immunity'that'were'enriched'in'ants' and' flies,' and' some' categories' related' to' metabolism' which' were' enriched' in' flies'and' bees.' Evidence' for' positive' selection' on' immunityErelated' functions' in' ants' came'from' a' significant' enrichment' of' the' GO' categories' “immune' system' development”' and'“hemopoietic' or' lymphoid' organ' development”,' the' organ' that' produces' during' larval development the'cells'mediating'the'immune'response'in'insects (Corley,'Lavine'2006).'Seven'GO'categories'related'to'immunity'were'also'enriched'in'flies'(“defense'response”,'“immune' system' process”,' “regulation' of' antimicrobial' humoral' response”,' “regulation'of' immune' effector' process”,' “regulation' of' defense' response”,' “immune' response”' and'“antimicrobial' humoral' response”).' The' absence' of' significant' enrichment' for' related'categories'in'bees'might'reflect'a'lack'of'power'of'the'gene'set'enrichment'test,'because'the' set' of' immunity' genes' is' small' in' the' honeybee' (Honeybee' Genome' Sequencing'onsortium.' 2006)' and' the' dataset' analyzed' was' further' depleted' in' genes' with'immunityErelated' functions' (Supplementary' text' and' Tables' S7).' Consistent' with' this'interpretation,' there' was' a' trend' in' the' direction' of' an' enrichment,' although' non'significant,' for' 5' of' the' 7' tested' GO' categories' related' to' immunity' in' bees' (data' not'shown),' suggesting' that' immunity' might' be' a' common' target' of' positive' selection' in'insects.''The' second' set' of' GO' categories' enriched' in' two' of' the' three' insect' lineages'included' various' metabolic' processes' and' their' regulators,' with' metabolism' of' chitin,'aminoglycan,' carbohydrate,' polysaccharide,' glucose,' hexose,' glycerolipid,' and'phosphatidylinositol' being' enriched' in' flies' and' metabolism' of' lipid,' aminoEacid,'nucleotide,'and'phosphorus'being'enriched'in'bees.'There'was'no'significant'enrichment'for' GO' categories' related' to' metabolism' in' ants,' but' some' categories' were' close' to'significance' (e.g.,' “chitin' metabolic' process”' and' “rRNA' metabolic' process”,' with'FDR=21%' and' 24%' respectively;' Table' S6).' Metabolic' functions,' such' as' aminoEacid,'fatty' acid,' lipid' or' RNA' metabolism,' were' significantly' enriched' in' ants' when' we' used'KEGG' pathways' annotation' instead' of' the' Gene' Ontology' to' perform' the' gene' set'enrichment'test,'as'well'as'when'the'singleEcopy'orthologs'dataset'was'reanalyzed'with'another'multiple'alignment'method'and'a'different'quality'filtering'method'(Table'S25'and' S26;' Supplementary' text).' It' thus' seems' that' metabolism' is' a' common' target' of'positive'selection'in'insects.'
Social behaviors
Two' of' the' GO' categories' enriched' in' ants' (“neuron' recognition”' and' “adult'locomotory' behavior”)' might' potentially' be' linked' to' the' evolution' of' neural' systems'and' behavior' (Table' 3).' The' first' category' was' “neuron' recognition”.' However,' GO'categories' related' to' neural' systems' were' also' enriched' in' a' nonEsocial' hymenoptera'lineage' (“regulation' of' synaptogenesis”,' “mushroom' body' development”' and' “memory”'on' branch'
DCX9EMAP,& turtle& and & beethoven .' Mutational' analyses' of' these' genes' in'
Drosophila ' suggest'that' they' play' an' important' role' in' sensory' perception' functions.' Adult' flies' carrying' a'piggyBac' insertion' in'
DCX9EMAP ' are' uncoordinated' and' deaf' and' display' loss' of'mechanosensory'transduction'and'amplification'(Bechstedt'et'al.'2010).'
Turtle 'plays'an'essential'role'in'the'execution'of'coordinated'motor'output'in'complex'behaviors'in'flies,'notably' regarding' the' response' to' tactile' stimulation' (Bodily' et' al.' 2001).' Finally,' beethoven ' is' involved' in' male' courtship' behavior,' adult' walking' behavior' and' sensory'perception' of' sound' in' flies' (Tauber,' Eberl' 2001).' This' suggests' that' positive' selection'might'have'been'important'for'the'evolution'of'sensory'perception'functions'in'ants.'A'specific'analysis'of'olfactory'receptor'genes'(ORs)'did'not'provide'support'for'the'evolution'of'sociality'being'associated'with'increased'levels'of'positive'selection'on'ORs.' A' scan' for' positive' selection' across' branches' of' a' tree' gathering' 873' manually'annotated'ORs'from'two'ants'(
Pogonomyrmex&barbatus 'and'
Linepithema&humile )'and'the'solitary' wasp'
Nasonia& vitripennis ' (see' Materials' and' Methods),' revealed' that' positive'selection' was' pervasive,' with' 277' branches' (23%)' displaying' significant' signals' for'positive'selection'(Figure'3,'Figure'S1).'However,'positive'selection'was'detected'in'only'19%' of' the' 929' branches' leading' to' ant' species,' whereas' as' many' as' 40%' of' the' 156'branches'leading'to'wasps'were'under'positive'selection'(Fisher'exact'test'p=7.6eE9).'
Phenotypic plasticity among castes
None'of'the'GO'categories'enriched'for'positively'selected'genes'in'ants'could'be'linked' to' phenotypic' plasticity' (i.e.,' caste' differences).' In' particular,' there' was' no'evidence' of' a' significant' enrichment' for' GO' categories' related' to' morphology' or'morphogenesis' in' the' ant' lineage.' Another' enrichment' test' using' annotations' obtained'from' mutant' phenotypes' in'
D.& melanogaster ' (which' are' more' relevant' than' GO'annotations'to'analyze'genes'involved'in'morphogenesis'since'genes'sets'mostly'refer'to'anatomical' structures)' also' provided' no' clear' support' for' positive' selection' on' genes'associated'with'phenotypic'plasticity'in'ants'(Table'S12).'However,'among'the'genes'with'the'highest'support'for'positive'selection'in'ants'(Table' 1),' two' genes' had' a' role' in' wing' development' (
Guanine& nucleotide& exchange&actor& in& mesoderm ' and'
Methylthioribose919phosphate& isomerase )' and' one' in' larval'development'(
Dis3 ) ,& suggesting'that'even'if'positive'selection'did'not'act'consistently'on'large' sets' of' genes' related' to' morphogenesis,' it' could' have' acted' specifically' on' a' few'individual'genes.' Mitochondrial genes
Eleven'GO'categories'enriched'for'positively'selected'genes'in'ants'were'related'to' mitochondrial' activity' (e.g.,' “mitochondrial' electron' transport”,' “mitochondrial'matrix”,' “mitochondrial' respiratory' chain”,' “NADH' dehydrogenase' ubiquinone' activity”'and' “oxidoreductase' activity”;' Table' 3).' The' mitochondrial' processes' under' positive'selection' were' not' restricted' to' respiration' and' energy' production,' but' also' included'translation'(“organellar'ribosome”,'“mitochondrial'small/large'ribosomal'subunit”).'GO'categories' related' to' mitochondria' were' also' enriched' for' positively' selected' genes' on'many' individual' branches' of' the' ant' lineage' analyzed' separately' (Table' S9),' and' in' a'larger'dataset'including'duplicated'genes'analyzed'with'the'site'test'(see'Materials'and'Methods;' Table' 1,' Table' S13' and' S19).' This' indicates' that' recurrent' events' of' positive'selection'occurred'on'genes'with'mitochondrial'functions'during'the'evolution'of'the'ant'lineage.'In'contrast,'mitochondriaErelated'GO'categories'did'not'display'any'enrichment'for'positively'selected'genes'in'flies'and'bees'(Tables'4'and'5),'despite'a'high'power'to'detect' it' on' the' respective' datasets' (Supplementary' text).' Similarly,' no' mitochondrial'function' was' significantly' enriched' in' the' branches' ifespan genes
There' was' a' significant' enrichment' for' positively' selected' genes' in' the' ant'orthologs' of'
D.& melanogaster ' genes' that' were' downEregulated' in' 61' daysEold' flies'compared' to' 10' daysEold' flies,' based' on' a' published' microarray' analysis' (p=0.011;'below'Bonferroni'threshold'α=0.05/4=0.0125;'Table'S16)(Landis'et'al.'2004).'Two' other' genes' known' to' be' involved' in' aging' were' among' the' topEscoring'genes'for'positive'selection'in'our'dataset.'The'first'was'
Tequila ,'which'has'been'shown'to' be' associated' with' aging' in' an' experimental' evolution' study' in'
D.& melanogaster '(Remolina' et' al.' 2012).' The' other' was'
Mitochondrial& trifunctional& protein& α& subunit,& whose'knockEout'also'reduces'lifespan'in'
D.&melanogaster '(Kishita,'Tsuda,'Aigaki'2012).'Although' not' in' the' list' of' top' hits,'
Sod2 ' (
Superoxide& dismutase& [Mn],& mitochondrial),& a'gene & known'to'have'antioxidant'activity'and'whose'overexpression' has'been'shown'to'be'associated'with'increased'lifespan'in'some'strains'of' D.&melanogaster& (Mockett'et'al.'1999;'Curtis'et'al.'2007),'underwent'positive'selection'at'the'base'of'the'Hymenoptera'lineage'(FDR=0.0073)'and'in'the'
Accromyrmex&echinatior 'branch'(FDR=9.6eE8).'
Selective pressure on genes with caste-biased expression
There' was' a' marginally' significant' enrichment' for' positively' selected' genes'among' genes' with' biased' expression' in' adult' workers' in'
S.& invicta& (effect' size=1.2,'p=0.025;'not'significant'after'Bonferroni'correction'α=0.05/6=0.0083;'Table'S17)'and'a'stronger' enrichment' for' genes' with' queenEbiased' expression' in' adults' (effect' size=1.8,'p=0.0028).' Surprisingly,' however,' there' was' a' pattern' of' weaker' enrichment' for' genes'with' maleEbiased' expression' in' adults' (effect' size=1.04,' p=0.2381).' At' the' pupal' stage,'we' did' not' detect' a' significant' enrichment' for' positively' selected' genes' among' any'group' of' genes' showing' casteEbiased' expression.' But' similarly' to' the' adult' stage,' the'enrichment' effect' size' was' higher' for' genes' with' queenEbiased' expression' (effect'size=1.2)'than'for'genes'with'workerEbiased'expression'(effect'size=1.1),'and'it'was'the'lowest'for'genes'showing'maleEbiased'expression'(effect'size=1.06).'
Discussion
In'this'paper,'we'report'results'from'a'genomeEwide'scan'for'positive'selection'in'protein'coding'sequences'of'seven'ant'genomes,'using'the'rigorous'branchEsite'model'of'Codeml' (Zhang,' Nielsen,' Yang' 2005)' with' stringent' data' quality' control.' Positive'election' was' detected' in' the' ant' lineage' for' 20%' of' the' gene' families' analyzed.' This'proportion' is' similar' in' magnitude' to' the' values' observed' in' the' other' two' insect'lineages'that'we'reanalyzed'in'this'study:'20%'in'the'10'bee'species,'and'36%'in'the'12'flies'species.'Our' analysis' identified' similarities' in' patterns' of' positive' selection' between' the'ants' and' other' insect' lineages.' Notably,' at' the' broadest' phylogenetic' scale' that' our'datasets' allowed' us' to' study,' functional' categories' related' to' proteolysis,' metabolism,'response' to' stimuli,' and' immunity,' were' enriched' for' positively' selected' genes' in' ants,'bees' and' flies.' Interestingly,' studies' in' mammals,' fishes' and' urchins' also' provided'evidence' for' positive' selection' on' similar' functional' categories' (Kosiol' et' al.' 2008;'Studer' et' al.' 2008;' Oliver' et' al.' 2010;' MontoyaEBurgos' 2011).' Recurrent' positive'selection' on' such' long' evolutionary' time' scales' is' typical' of' genes' involved' in' the'interaction' with' changing' environments' or' in' conflict' and' competition,' such' as'evolutionary' arms' races' between' sexes' or' between' different' species,' which' cause' the'perpetuation' of' adaptations' and' counterEadaptations' in' competing' sets' of' coEevolving'genes' (Dawkins,' Krebs' 1979).' It' is' notable' that' positive' selection' patterns' on' these'categories'of'genes'do'not'seem'to'reflect'or'be'strongly'affected'by'the'large'lifeEhistory'differences' between' lineages' analyzed' here,' for' example' the' evolution' of' eusociality' in'the' hymenopteran' lineages.' In' particular,' our' results' on' immunityErelated' genes'challenge'the'hypothesis'that'hygienic'behaviors'in'social'insects'could'have'relaxed'the'selective'pressure'on'immune'genes,'since'this'should'be'reflected'in'reduced'levels'of'positive' selection' on' these' genes' (Honeybee' Genome' Sequencing' Consortium.' 2006;'Smith' et' al.' 2008;' Viljakainen' et' al.' 2009;' Werren' et' al.' 2010;' Fischman,' Woodard,'Robinson'2011;'Smith'et'al.'2011a;'Smith'et'al.'2011b;'Suen'et'al.'2011;'Harpur,'Zayed'2013).'Our' analysis' indicated' that' genes' involved' in' neurogenesis' were' under' positive'selection' in' ants' and' the' primitively' social' lineages' of' bees.' It' was' previously'hypothesized' that' stronger' selection' on' genes' related' to' brain' function' and'development'should'be'observed'in'eusocial'Hymenoptera'species'due'to'high'cognitive'demands'associated'with'social'life'(Fischman,'Woodard,'Robinson'2011).'However,'our'results' are' not' consistent' with' this' prediction' because' we' also' uncovered' signs' of'positive'selection'at'the'base'of'the'Hymenoptera'lineage,'that'is'before'the'evolution'of'sociality.' Interestingly,' a' similar' pattern' had' previously' been' reported' with' brain' orphological+data.+A+comparative+analysis+of+insects+showed+that+the+size+of+mushroom+body+ started+ to+ increase+ at+ the+ base+ of+ the+ Euhymenopteran+ (Orussioidea+Apocrita)+lineage,+approximately+90+Myr+before+the+evolution+of+sociality+in+the+Aculeata,+and+that+there+ was+ no+ clear+ correlation+ between+ the+ size+ of+ brain+ components+ and+ the+ levels+ of+sociality+ or+ cognition+ capabilities+ (Farris,+ Schulmeister+ 2011;+ Lihoreau,+ Latty,+ Chittka+2012).+To+account+for+this+observation,+Fischman+and+colleagues+tried+to+identify+factors,+other+than+sociality,+that+may+have+placed+unique+selective+pressure+on+brain+evolution+in+species+of+the+Hymenoptera+lineage+(Fischman,+Woodard,+Robinson+2011).+Based+on+the+ observation+ that+ there+ was+ less+ positive+ selection+ on+ neurogenesis+ genes+ in+ highly+social+bees+than+in+primitively+social+bees,+they+proposed+that+cognitive+challenges+might+be+ associated+ with+ the+ mode+ of+ colony+ founding+ in+ social+ Hymenoptera.+ In+ particular,+primitively+social+bees,+which+transit+from+a+solitary+phase+during+the+process+of+colony+founding,+ to+ a+ social+ phase+ could+ experience+ higher+ cognitive+ needs+ than+ highly+ social+bees,+ which+ never+ go+ through+ a+ solitary+ phase.+ However,+ our+ results+ are+ also+inconsistent+with+this+model+since+increased+positive+selection+was+observed+before+the+evolution+of+sociality+in+Hymenoptera.+A+comprehensive+survey+of+positive+selection+on+neurogenesis+ genes+ in+ Hymenoptera+ species,+ including+ species+ basal+ to+ the+ lineage,+ is+required+ to+ identify+ precisely+ when+ the+ selective+ regime+ of+ these+ genes+ started+ to+change,+and+in+which+hymenopteran+subSlineages+it+was+maintained.+Our+ results+ also+ challenge+ the+ hypothesis+ that+ genes+ involved+ in+ chemical+signaling+ experienced+ increased+ positive+ selection+ in+ social+ insects+ (Ingram,+ Oefner,+Gordon+2005;+Robertson,+Wanner+2006;+Bonasio+et+al.+2010;+Smith+et+al.+2011a;+Wurm+et+ al.+ 2011;+ Zhou+ et+ al.+ 2012;+ Leboeuf,+ Benton,+ Keller+ 2013).+ The+ analysis+ of+ olfactory+receptor+ repertoires+ in+ two+ ants+ and+ a+ nonSsocial+ wasp+ indicate+ that+ positive+ selection+on+aminoSacid+substitutions+was+surprisingly+less+frequent+in+ant+than+in+wasp+branches.+Given+ the+ limited+ number+ of+ species+ used+ in+ this+ analysis,+ future+ work+ should+concentrate+ on+ generating+ extensive+ annotation+ of+ olfactory+ receptors+ from+ more+Hymenoptera+ as+ well+ as+ outgroup+ species+ to+ identify+ characters+ or+ traits+ that+ could+ be+associated+with+the+pattern+of+positive+selection+on+olfactory+receptors.+Although+our+analyses+did+not+provide+support+for+previous+hypotheses+about+the+expected+ effect+ of+ social+ evolution+ on+ gene+ evolution,+ we+ identified+ several+ interesting+functional+ categories+ which+ were+ enriched+ for+ positively+ selected+ genes+ exclusively+ in+the+ ant+ lineage,+ possibly+ reflecting+ antSspecific+ adaptations.+ The+ most+ consistent+ and+ obust'result'was'that'genes'functioning'in'the'mitochondria'were'particularly'likely'to'be'under'positive'selection.'Mitochondrial'activity'plays'an'important'role'in'the'process'of' reproductive' isolation' and' speciation' (Lee' et' al.' 2008;' Burton,' Barreto' 2012),'interactions' with' endosymbionts' such' as'
Wolbachia ' (Werren' 1997),' diseases'(Cortopassi'2002;'Richly,'Chinnery,'Leister'2003;'Trifunovic'et'al.'2004;'Trifunovic'et'al.'2005),' and' in' the' process' of' aging' (Lenaz' 1998;' Cortopassi' 2002;' Kowald,' Kirkwood'2011).'In'that'respect'it'is'notable'that'the'evolution'of'sociality'has'been'accompanied'by'a'nearly'100Efold'increase'in'lifespan'of'queens'compared'to'their'solitary'ancestors'(Keller,' Genoud' 1997;' Jemielity' et' al.' 2005).' Three' lines' of' evidence' suggest' that'increased' lifespan' of' queens' might' be' related' to' increased' positive' selection' on'mitochondrial'genes'in'the'ant'lineage.'First,' lifespan' extension' in' insects,' but' also' in' other' lineages' such' as' birds' and'bats,'appears'to'be'associated'with'decreased'production'of'Reactive'Oxidative'Species'(ROS)(PerezECampo'et'al.'1998;'BrunetERossinni'2004;'Parker'et'al.'2004;'Corona'et'al.'2005;' Jemielity' et' al.' 2005).' ROS' are' a' normal' byEproduct' of' cellular' metabolism.' In'particular,' one' major' contributor' to' oxidative' damage' is' hydrogen' peroxide' (H O ),'which' is' produced' from' leaks' of' the' respiratory' chain' in' the' mitochondria' (Harman'1972;'Lenaz'1998;'Finkel,'Holbrook'2000;'Cui,'Kong,'Zhang'2012).'Positive'selection'in'ants' on' genes' functioning' in' the' mitochondria' may' thus' reflect' selection' to' increase'mitochondrial'efficiency'and'reduce'ROS'production.'Interestingly,'positive'selection'on'genes'with'mitochondrial'functions'was'previously'documented'in'the'bat'lineage'(Shen'et'al.'2010;'Zhang'et'al.'2013),'which'include'species'with'exceptional'longevity'(BrunetERossinni,' Austad' 2004).' In' the' bat' Myotis& lucifugus, ' ROS' production' was' also' shown' to'be'significantly'lower'than'in'two'similar'sized'mammal'species'(a'mouse'and'a'shrew)'although'the'metabolic'rates,'and'thus'mitochondrial'activity,'of'the'former'were'much'higher'because'of'flight'demands'(BrunetERossinni'2004).'Second,'on'the'basis'of'gene'expression'data'obtained'in'the'fire'ant'
S.&invicta ,'our'analyses' revealed' that' positive' selection' was' strongest' on' genes' with' queenEbiased'expression,' intermediate' on' genes' with' workerEbiased' expression,' and' weakest' on'genes'with'maleEbiased'expression.'This'association'between'levels'of'positive'selection'and' casteEbiased' differences' in' gene' expression' cannot' be' simply' accounted' by'differences' in' expression' levels' of' mitochondrial' genes' (which' are' enriched' for'positively'selected'genes'in'ants)'since'in'
S.&invicta 'mitochondrial'genes'are'significantly' ess' expressed' in' queens' than' in' workers' at' the' larval' stage,' and' not' differentially'expressed'at'the'adult'stage'(Figure'S2).'The'finding'of'higher'levels'of'positive'selection'for'genes'more'highly'expressed'in'the'castes'with'the'longer'lifespan'(queens'can'live'decades' in' some' species,' whereas' workers' have' lifespan' in' the' order' of' months,' and'males'in'the'order'of'days)'suggests'that'increased'positive'selection'on'queenEspecific'genes'could'be'related'to'longer'lifespan.'Third,' our' analyses' showed' that' the' levels' of' positive' selection' were' higher' on'orthologs'of'genes'which'are'downEregulated'during'aging'in'flies.'These'genes'include'numerous'energy'metabolism'genes,'and'their'downEregulation'in'old'flies'is'thought'to'reflect' a' decline' of' normal' and' functional' mitochondria' with' age' (Yui,' Ohno,' Matsuura'2003;'Landis'et'al.'2004).'The'finding'of'increased'levels'of'positive'selection'on'genes'whose'expression'declines'at'older'ages'suggests'that'the'function'of'these'genes'might'be' improved' in' ants,' potentially' delaying' the' loss' of' normal' activity' in' mitochondria'with' age.' It' would' be' interesting' to' test' if' parallel' mechanisms' also' evolved' in' the' ant'lineage'to'maintain'the'expression'of'these'genes'and'delay'the'decline'of'mitochondria'activity'through'lifespan'in'queens.'In'contrast'to'ants,'there'was'no'evidence'of'elevated'levels'of'positive'selection'on' mitochondrial' functions' in' bees.' As' most' social' species,' bees' also' evolved' longer'queen' lifespans' (more' than' 2' years)' compared' to' males' and' workers' (a' few'weeks)(Keller,' Genoud' 1997;' Munch,' Amdam,' Wolschin' 2008).' There' are' four' possible'explanations' for' the' difference' between' ants' and' bees' in' the' level' of' positive' selection'on' mitochondrial' genes.' First,' lifespan' differences' between' castes' are' less' pronounced'in'bees,'where'queens'live'up'to'2E5'years,'than'in'ants,'where'queens'can'live'up'to'30'years,'possibly'resulting'in'lower'selective'pressure'to'increase'lifespan'in'bees'than'in'ants.' Second,' because' eusociality' evolved' independently' in' ants' and' bees' it' is' possible'that'extended'queen'lifespans'evolved'by'different'molecular'mechanisms'(Jemielity'et'al.' 2005;' Jobson,' Nabholz,' Galtier' 2010).' For' example,' vitellogenin' may' play' a' more'central' role' for' aging' in' bees' than' ants' (Amdam,' Omholt' 2002;' Corona' et' al.' 2007;'Munch,' Amdam,' Wolschin' 2008).' Third,' the' evolution' of' mitochondriaErelated' genes'may' have' been' differently' constrained' in' ants' and' bees.' For' example,' metabolic' rates'differ'greatly'between'flying'bee'workers'and'nonEflying'ant'workers'because'flight'is'an'energetically' costly' behavior' requiring' highly' elevated' metabolic' rates' (Jensen,' HolmEJensen' 1980;' Suarez' 2000;' Niven,' Scharlemann' 2005).' Because' metabolism' and'itochondrial' activity' are' closely' connected,' lower' metabolic' rates' in' ants' might' have'alleviated' functional' constraints' on' mitochondriaErelated' genes,' allowing' selection' to'act'on'lifespan'extension.'Fourth,'the'GC'content'in'bee'genomes'was'shown'to'be'lower'than' in' ant' genomes' (Honeybee' Genome' Sequencing' Consortium.' 2006;' Simola' et' al.'2013).' Some' parts' of' the' bee' genomes,' in' particular' their' mitochondrial' genomes'(Crozier,' Crozier' 1993;' Gotzek,' Clarke,' Shoemaker' 2010;' Tan' et' al.' 2011),' display'extreme' bias' in' nucleotide' composition,' which' leads' to' significant' effect' on' both' the'codon'usage'patterns'and'amino'acid'composition'of'proteins'and'may'have'interfered'with'the'action'of'positive'selection.'If' positive' selection' acted' to' optimize' the' functioning' of' mitochondria' in' ants,' it'could' be' expected' that' the' mitochondrial' genome' itself' should' be' targeted' by' positive'selection.' However,' mitochondrial' genes' generally' exhibit' very' low' d N / d S ' ratios'(Montooth,' Rand' 2008)' and' there' was' no' clear' evidence' in' our' results' for' positive'selection' on' the' 13' genes' of' the' mitochondrial' genome' itself.' This' suggests' that'innovations' related' to' mitochondrial' activity' could' arise' more' easily' on' nuclear' genes,'whereas' mitochondrial' genes' seem' more' likely' to' maintain' conserved' core'functionalities.'In' conclusion,' this' study' provides' a' detailed' analysis' of' the' extent' of' positive'selection' events' on' proteinEcoding' genes' in' seven' ant' species.' Because' false' positives'are' a' major' concern' for' wholeEgenome' scans' for' positive' selection,' we' used' a'conservative' methodology.' We' also' reanalyzed' data' in' bees' and' flies' with' the' same'methods' to' permit' an' unbiased' and' robust' comparison' of' positive' selection' between'lineages.' The' comparison' between' these' three' lineages' provided' interesting'perspectives' on' the' evolution' of' genes' implicated' in' immunity,' neurogenesis' and'olfaction,' and' allowed' us' to' pinpoint' positive' selection' events' that' were' specific' to' the'ant' lineage.' In' particular,' we' found' that' the' evolution' of' extreme' lifespan' in' ants' was'associated' with' positive' selection' on' genes' with' mitochondrial' functions,' suggesting'that'a'more'efficient'functioning'of'mitochondrial'genes'might'have'been'an'important'step'towards'the'extreme'lifespan'extension'that'characterizes'this'lineage.'It'would'be'interesting'to'complement'this'study'by'scans'for'genes'under'lineageEspecific'strong'or'relaxed'purifying'selection,'to'get'a'more'global'picture'of'natural'selection'patterns'in'ant' genomes,' and' uncover' additional' genes' that' could' have' played' a' significant' role'during'the'evolution'of'the'ant'lineage.' aterials and M ethods Single-copy orthologs gene families dataset
Protein' coding' gene' sequences' of' the' 7' ant' genomes' were' downloaded' from' the'Hymenoptera' Genome' Database'(http://hymenopteragenome.org/ant_genomes/)(MunozETorres'et'al.'2011).''The'complete'annotated'gene'sets'were'OGS_1.0'for'
Acromyrmex&echinatior& (Nygaard'et'al.'2011),'OGS_1.2'for'
Atta&cephalotes '(Suen'et'al.'2011),'OGS_2.2.3'for'
Solenopsis&invicta '(Wurm' et' al.' 2011),' OGS_1.2' for'
Pogonomyrmex& barbatus ' (Smith' et' al.' 2011b),' OGS_3.3'for'
Camponotus&floridanus& (Bonasio'et'al.'2010),'OSG_1.2'for'
Linepithema&humile& (Smith'et' al.' 2011a)' and' OGS_3.3' for'
Harpegnathos& saltator ' (Bonasio' et' al.' 2010).' Coding'sequences' of' 5' outgroup' species' were' downloaded' from' the' Hymenoptera' Genome'Database' for' the' honey' bee' (
Apis& mellifera&
Amel_pre_release2)(Honeybee' Genome'Sequencing' Consortium.' 2006)' and' the' jewel' wasp' (
Nasonia& Vitripenis&
OGS_v1.2)(Werren' et' al.' 2010),' from' Flybase' (Tweedie' et' al.' 2009)' for' the' fruit' fly'(
Drosophila& melanogaster&
FB5.29)(Adams' et' al.' 2000),' from' BeetleBase' (Kim' et' al.'2010b)' for' the' flour' beetle' (
Tribolium& castaneum&
Tcas_3.0)(
Tribolium ' Genome'Sequencing'Consortium.'2008),'and'from'vectorBase'(Lawson'et'al.'2009)'for'the'body'louse'(
Pediculus&humanus&
PhumU1.2)(Kirkness'et'al.'2010).'Gene' families' were' obtained' from' a' custom' run' of' the' OrthoDB' pipeline' for' the'Ant' Genomic' Consortium' (http://cegg.unige.ch/orthodbants' and'http://bioinfo.unil.ch/supdata/Roux_positive_selection_ants/orthoDB_run.zip;' pipeline'of' OrthoDB' release' 4)(Waterhouse' et' al.' 2011;' Simola' et' al.' 2013).' Briefly,' OrthoDB'implements' a' Best' Reciprocal' Hit' clustering' algorithm' based' on' allEagainstEall' SmithEWaterman' protein' sequence' comparisons.' The' longest' alternatively' spliced' form' of'genes' is' used.' The' orthologous' groups' are' built' at' different' taxonomic' levels' and' it' is'possible' to' query' for' specific' phyletic' profiles' by' combining' the' criteria' of' absent,'present,' singleEcopy,' multiEcopy' or' no' restriction,' for' each' species' within' the' studied'clade.' Gene' families' including' strictly' one' ortholog' in' each' of' the' 12' species' were'selected'(2,756'gene'families).'Because'annotations'of'the'studied'genomes'are'likely'to'be' incomplete' (Simola' et' al.' 2013),' families' with' a' few' missing' genes' –' gene' losses' or'nannotated'genes'–'were'included,'with'the'restriction'that'at'least'4'genes'out'of'the'7'ant' species,' and' 3' genes' out' of' the' 5' outgroup' species' should' be' present' in' the' gene'family.' Simola' et' al.' (2013)' have' shown' that' among' the' seven' ant' species,' there' were'generally'few'lost'or'missing'genes,'apart'from'
S.&invicta '(less'than'400'
S.&invicta 'genes'were' missing' in' singleEcopy' orthologs' families)' and'
A.& echinatior ' (less' than' 150'
A.&echinatior ' genes' were' missing' in' singleEcopy' orthologs' families).' Our' gene' family'selection'criteria'allow'handling'such'a'moderate'amount'of'missing'genes'in'families.'In'order'to'transfer'functional'annotations'from'
D.&melanogaster
Extensive gene families dataset
Another' dataset' gathered' all' gene' families' from' the' OrthoDB' database' that' could' pass'our' quality' filters,' and' notably' families' that' experienced' duplications.' The' CDS' were'filtered' as' described' above.' AminoEacid' sequences' were' aligned' using' PAGAN' version'0.47' (Loytynoja,' Vilella,' Goldman' 2012).' The' program' GUIDANCE' (v1.1)' was' used' to'assess' alignment' confidence' and' mask' unreliably' aligned' residues' (Penn' et' al.' 2010;'Privman,' Penn,' Pupko' 2012).' The' combination' of' a' phylogenyEaware' aligner' (PAGAN'replaces'PRANK'(Löytynoja,'Goldman'2008)'and'is'based'on'the'same'principle)'and'of'this'filtering'algorithm'was'shown'to'perform'the'best'in'recent'benchmark'studies'on'simulated' data' (Jordan,' Goldman' 2012;' Privman,' Penn,' Pupko' 2012).' Gene' family'phylogenies' were' built' using' RAxML' (v7.2.9)(Stamatakis' 2006)' from' the' amino' acid'sequences,'with'the'LG'matrix'and'the'CAT'model.'Amino'acid'alignments'were'reverseEtranslated' into' the' corresponding' codon' alignments.' This' resulted' in' 6,186' families'tested,'with'an'average'of'11'genes,'and'an'average'length'of'filtered'alignment'of'3,129'nt.'(median'of'2,385'nt.,'ranging'from'a'minimum'of'192'nt.'to'a'maximum'of'20,556'nt.).'
Mitochondrial gene families dataset
Contigs'corresponding'to'mitochondrial'genomes'could'be'downloaded'for'5'of'the'7'ant'genomes' (
Acromyrmex& echinatior,& Atta& cephalotes,& Solenopsis& invicta,& Pogonomyrmex&barbatus& and'
Linepithema&humile ).'They'were'submitted'to'MITOS,'a'web'server'for'the'annotation' of' metazoan' mitochondrial' genomes' (http://mitos.bioinf.uniEleipzig.de/index.py)(Bernt' et' al.' 2012).' This' gave' us' the' predicted' coordinates' of' 13'mitochondrial' protein' coding' genes' in' these' species.' Frameshift' errors' or' incomplete'gene' predictions' were' manually' corrected.' Mitochondrial' genes' from' the' outgroup'species'
Apis& mellifera ,' Nasonia& Vitripenis ' and'
Tribolium ' castaneum' were' downloaded'from' Genbank' (accessions' L06178;' EU746609.1' and' EU746613.1;' AJ312413.2' and'NC_003081.2' respectively).' Mitochondrial' genes' from'
Drosophila& melanogaster& were'ownloaded' from' Flybase' at'ftp://ftp.flybase.net/genomes/Drosophila_melanogaster/dmel_r5.43_FB2012_01/fasta/dmelEdmel_mitochondrion_genomeECDSEr5.43.fasta.gz.'The'alignment'and'filtering'steps'for' the' 13' mitochondrial' gene' families' were' identical' to' the' dataset' of' singleEcopy'orthologs'nuclear'gene'families'(see'above).'A'total'of'119'branches'were'tested'in'this'dataset'(average'of'9.2'and'median'of'9'branches'per'family;'average'length'of'filtered'alignment' of' 641' nt.' and' median' of' =' 621' nt.,' ranging' from' a' minimum' of' 39' nt.' to' a'maximum'of'1,413'nt.).'
Twelve Drosophila dataset
SingleEcopy' ortholog' gene' families' from' the' 12' sequenced' Drosophila' species' were'downloaded' from' ftp://ftp.flybase.net/12_species_analysis/clark_eisen/alignments/'(files' “all_species.guide_tree.longest.cds.tar.gz”' and'“all_species.guide_tree.longest.translation.tar.gz”)(Drosophila' 12' Genomes' Consortium'2007).' The' alignment' and' filtering' steps' for' these' gene' families' were' identical' to' the'dataset' of' singleEcopy' ortholog' gene' families' used' for' the' ant' analysis.' Out' of' 6,698'initially'downloaded'Drosophila'gene'families,'3,749'(56%)'passed'our'filters'and'could'be'tested'for'positive'selection,'resulting'in'77,495'branches'tested'(average'of'20.7'and'median' of' 21' branches' per' family;' average' length' of' filtered' alignment' of' 876' nt.' and'median'of'708'nt.,'ranging'from'a'minimum'of'15'nt.'to'a'maximum'of'14,535'nt.).'
Bee dataset
SingleEcopy' ortholog' gene' families' from' 10' bee' species' were' downloaded' from'http://insectsociogenomics.illinois.edu/.' This' set' of' gene' families' is' incomplete' as' it' is'derived'from'the'sequencing'of'expressed'sequence'tags'(using'454'Life'Science/Roche'GSEFLX'platform)'from'9'bee'species'(Woodard'et'al.'2011),'and'from'gene'models'of'the'honey' bee'
Apis& mellifera ' (Honeybee' Genome' Sequencing' Consortium.' 2006).' The'alignment' and' filtering' steps' for' these' gene' families' were' identical' to' the' dataset' of'singleEcopy' ortholog' gene' families' used' for' the' ant' analysis.' Out' of' 3,647' initially'downloaded' gene' families,' 2,256' (62%)' passed' our' filters' and' could' be' tested' for'positive' selection,' resulting' in' 20,169' branches' tested' (average' of' 8.9' and' median' of' 9'branches'per'family;'average'length'of'filtered'alignment'of'611'nt.'and'median'of'528'nt.,'ranging'from'a'minimum'of'27'nt.'to'a'maximum'of'3,945'nt.).' ranch-site test for positive selection
We' used' the' updated' branchEsite' test' (Zhang,' Nielsen,' Yang' 2005)' of' Codeml' from' the'package'PAML'v4.4c'(Yang'2007)'to'detect'Darwinian'positive'selection'experienced'by'a' gene' family' in' a' subset' of' sites' in' a' specific' branch' of' its' phylogenetic' tree.' This' test'was' previously' used' in' genomeEwide' scans' for' positive' selection' in' various' lineages'(Bakewell,' Shi,' Zhang' 2007;' Kosiol' et' al.' 2008;' Studer' et' al.' 2008;' Vamathevan' et' al.'2008;' Oliver' et' al.' 2010;' George' et' al.' 2011)' and' is' used' by' the' Selectome' project'(http://selectome.unil.ch)(Proux'et'al.'2009;'Moretti'et'al.'2014).'It'is'acknowledged'to'be'more'sensitive'for'the'detection'of'positive'selection'than'branch'tests'(Yang'1998)'or'site'tests'(Yang'et'al.'2000),'because'it'does'not'average'the'signal'over'all'codons'in'the'alignment'(branch'test)'nor'over'all'branches'of'the'phylogeny'(site'test)(Yang,'dos'Reis'2011).'It'is'also'robust'to'relaxation'of'purifying'selection'( ω 'close'to'1)'since'this'scenario' is' accounted' for' in' the' null' model' (Zhang' 2004;' Zhang,' Nielsen,' Yang' 2005).'The'alternative'model'is'contrasted'to'the'null'model'using'a'likelihoodEratio'test'(LRT),'where' logElikelihood' ratios' are' compared' to' a' chiEsquare' distribution' with' 1' degree' of'freedom' (Zhang,' Nielsen,' Yang' 2005).' Previous' studies' have' reported' the' branchEsite'test' to' be' conservative' in' this' setup' (Bakewell,' Shi,' Zhang' 2007;' Studer' et' al.' 2008;'Fletcher,'Yang'2010;'Yang,'dos'Reis'2011;'Gharib,'RobinsonERechavi'2013).'We'did'not'use'the' ω 'estimates'to'infer'the'strength'of'positive'selection'because'they'were'shown'to'be'unreliable'(Bakewell,'Shi,'Zhang'2007;'Yang,'dos'Reis'2011).'In'the'absence'of'a'specific' a&priori& hypothesis'regarding'which'branches'to'test'for' positive' selection,' our' implementation' runs' the' test' multiple' times' on' each' gene'family,' successively' changing' the' branch' selected' as' foreground.' The' branches'considered'as'foreground'are'highlighted'in'red'in'Figure'1.'This'approach'was'shown'to'be' legitimate' if' pEvalues' from' the' successive' tests' are' corrected' for' multiple' testing'(Anisimova,'Yang'2007;'Yang,'dos'Reis'2011).'We'applied'a'False'Discovery'Rate'(FDR)'correction' (Benjamini,' Hochberg' 1995)' over' all' the' pEvalues' treated' as' one' series'(number' of' branches' tested' ×' number' of' gene' families' tested).' In' the' ant' singleEcopy'orthologs' nuclear' dataset' we' analyzed' a' maximum' of' 15' branches' leading' to' the' 7' ant'species,' summing' to' 44,306' tests' performed.' In' the' ant' mitochondrial' dataset' we'analyzed' a' maximum' of' 11' branches' leading' to' 5' ant' species,' summing' to' 119' tests'(branches' in' red' in' Figure' S3).' In' the' Drosophila' singleEcopy' orthologs' dataset' we'analyzed' a' maximum' of' 21' branches,' leading' to' a' total' of' 77,495' tests' (Figure' S4).'inally'in'the'bee'dataset'we'analyzed'a'maximum'of'17'branches,'leading'to'a'total'of'20,169'tests'(Figure'S5).'All' computations' were' performed' using' Slimcodeml' (release' 4 th d ).'The'distribution'of'differences'was'bimodal,'with'a'first'major' mode' at' d =0,' gathering' most' data,' and' a' second' minor' mode' at' d ~1.' A' cutoff' at' d =0.004' clearly' separated' the' 2' peaks.' We' used' this' stringent' cutoff' ( d >0.004)' to'eliminate' all' tests' with' potential' convergence' problems' in' the' fixed' and' alternative'models'(see'Supplementary'text'and'Table'S23).'Values' of' d N ' and' d S ite test for positive selection The' site' test' (Yang' et' al.' 2000)' of' Codeml' from' the' package' PAML' v4.4e' (Yang' 2007),'allowing' the' d N /d S ' ratio' ( ω )' to' vary' among' sites,' was' run' on' the' extensive' dataset' of'6,186' families' (see' above).' We' contrasted' the' null' model' M8a' (beta' &' ω ' with' ω =1)' to'the' alternative' model' M8' (beta' &' ω ' with' ω ≥1)' with' 11' site' classes' (Swanson,' Nielsen,'Yang' 2003;' Wong' et' al.' 2004).' Examples' of' Codeml' control' files' used' are' provided' in'Supplementary'text.'Similar'to'the'branchEsite'test,'we'launched'3'independent'runs'for'both' the' alternative' and' null' hypotheses' for' each' gene' family' and' kept' the' best'likelihood' value' of' each' run' for' the' likelihood' ratio' test' (Table' S19).' The' likelihood'ratios' were' compared' to' a' ChiEsquare' distribution' with' 1' degree' of' freedom' as'recommended' in' PAML' user’s' guide'(http://abacus.gene.ucl.ac.uk/software/pamlDOC.pdf).' Reconstruction of ancestral G+C content
The' program' nhPhyml' (Galtier,' Gouy' 1998;' Guindon,' Gascuel' 2003;' Boussau,' Gouy'2006)'was'used'to'estimate'the'G+C'content'at'third'codon'positions'at'each'node'of'the'gene' family' trees' (topology' fixed,' transition/transversion' ratio' estimated,' alpha'parameter' estimated' with' eight' categories).' Following' Studer' et' al.' (2008),' we'calculated' the' shift' in' GC3' content' at' each' branch' as' the' difference' between' GC3'contents'at'the'nodes'delimitating'that'branch.'
Olfactory receptors family
Olfactory'receptors'are'difficult'to'process'in'automated'pipelines'since'they'are'characterized' by' dynamic' patterns' of' duplications' and' pseudogenization' during'evolution' (Nozawa,' Nei' 2007).' Furthermore,' the' sequences' of' ORs' are' highly' variable'and'notoriously'difficult'for'automatic'gene'annotation.'Accordingly,'our'main'dataset'of'singleEcopy'orthologs'was'depleted'in'genes'involved'in'olfaction'(Tables'S7,'S8,'S10'and'S11)' and' GO' categories' related' to' olfaction' could' not' be' tested' for' enrichment' of'positively'selected'genes'because'they'included'too'few'annotated'genes.'We'therefore'used' a' more' comprehensive' dataset' of' 873' manually' annotated' protein' coding'sequences'of'OR'genes'(excluding'suspected'pseudogenes)'provided'by'Hugh'Robertson'for'
P.& barbatus ' (291' genes)(Smith' et' al.' 2011b),'
L.& humile ' (320' genes)(Smith' et' al.'2011a)' and'
N.& vitripennis ' (262' genes)(Werren' et' al.' 2010).' Nucleotide' sequences' were'ranslated' and' amino' acid' sequences' were' aligned' using' MAFFT' (Katoh' et' al.' 2005).'Unreliably' aligned' residues' were' masked' using' GUIDANCE' based' on' 32' bootstrap'samples'and'a'cutoff'of'0.2'that'was'chosen'so'that'the'15%'lowest'scoring'residues'are'masked' (Penn' et' al.' 2010;' Privman,' Penn,' Pupko' 2012).' Phylogeny' was' reconstructed'using' RAxML' with' the' JTT' substitution' matrix,' the' CAT' approximation' and' 100'bootstrap'samples'(Stamatakis'2006).'Because'the'resulting'gene'tree'was'too'large'for'an'analysis'with'the'branchEsite'test'of'Codeml,'we'divided'it'into'16'smaller'subtrees,'each' containing' less' than' 100' leaves.' Branches' with' as' high' as' possible' bootstrap'support' were' chosen' as' splitting' points.' The' 16' subtrees' include' all' ant' sequences' but'only' 105'
N.& vitripennis ' sequences.' The' sequences' from' each' subtree' were' realigned'using' PRANK' version' 100701' (Löytynoja,' Goldman' 2008)' and' reverseEtranslated' into'corresponding' codon' alignments.' GUIDANCE' was' used' to' mask' unreliably' aligned'codons' (0.8' cutoff).' Phylogeny' was' reconstructed' using' RAxML' as' above.' Out' of' 1,744'branches' in' the' initial' tree,' 1,400' branches' from' the' subtrees' were' tested' using' the'branchEsite' test' of' Codeml' (see' above),' and' the' computation' was' successful' (both' null'and'alternative'hypotheses)'for'1,184'branches.'Significant'branches'are'highlighted'in'red'in'Figure'3'and'in'Figure'S1.'Full'results'of'the'branchEsite'test'on'all'16'clades'are'shown'in'Table'S18.'A'full'tree'with'branch'names'and'bootstrap'values'is'provided'as'figure' S1.' Newick' trees' of' the' 16' individual' subtrees' along' with' annotation' of' tested'branches'are'available'in'Supplementary'text.'
Tests of functional category enrichment
Gene' Ontology' (GO)(Ashburner' et' al.' 2000)' annotations' for' gene' families' were' taken'from' the' annotation' of' the'
D.& melanogaster ' gene' member' they' include' (downloaded'from'http://flybase.org/static_pages/downloads/FB2011_02/go/gene_association.fb.gz).' The'annotation' of' children' GO' categories' was' propagated' to' their' parent' categories'following'the'GO'graph'structure.'Gene'Ontology'categories'mapped'to'10'genes'or'less'were'discarded'for'the'enrichment'analysis.'To' identify' over' and' underErepresented' functional' categories' present' in' the'datasets' used' in' this' study,' the' package' topGO' version' 2.4' (Alexa,' Rahnenfuhrer,'Lengauer' 2006)' of' Bioconductor' (Gentleman' et' al.' 2004)' was' used.' A' Fisher' exact' test'was' used,' combined' with' the' “elim”' algorithm' of' topGO,' which' decorrelates' the' graph'tructure' of' the' Gene' Ontology' to' reduce' nonEindependence' problems' (Alexa,'Rahnenfuhrer,' Lengauer' 2006).' The' reference' set' was' constituted' of' all' OrthoDB'families'including'a'
D.&melanogaster 'gene'with'GO'annotation.'Gene'ontology'categories'with'a'FDR'<'20%'are'reported'(Benjamini,'Hochberg'1995).'Regarding' the' functional' enrichment' of' genes' targeted' by' positive' selection,' the'Fisher' exact' test' approach' has' been' criticized' because' it' imposes' the' choice' of' an'arbitrary'cutoff'to'dichotomize'genes'into'“significant”'and'“nonEsignificant”'categories.'This'leads'to'a'loss'of'information'and'limits'the'power'and'robustness'of'this'method'(Allison' et' al.' 2006;' Tintle' et' al.' 2009;' Daub' et' al.' 2013).' To' test' for' GO' functional'categories' for' enrichment' for' positively' selected' genes,' we' instead' used' a' gene' set'enrichment' approach,' which' tests' whether' the' distribution' of' scores' of' genes' from' a'gene' set' differs' from' the' whole' dataset' scores' distribution,' allowing' the' detection' of'gene'sets'that'contain'many'marginally'significant'genes.'Different'implementations'for'this' approach' have' been' proposed.' The' most' widely' used' is' the' Gene' Set' Enrichment'Analysis' (GSEA)(Subramanian' et' al.' 2005),' but' it' was' shown' to' perform' relatively'poorly' (Kim,' Volsky' 2005;' Efron,' Tibshirani' 2007;' Tintle' et' al.' 2009).' Here,' we' used' a'SUMSTAT'test:'for'a'given'gene'set' g 'including' n 'genes,'the'SUMSTAT'statistic'is'defined'as'the'sum'of'scores'of'the' n 'genes.'This'statistic'was'shown'to'be'more'sensitive'than'a'panel'of'other'methods,'while'controlling'well'for'the'rate'of'false'positives'(Ackermann,'Strimmer'2009;'Tintle'et'al.'2009;'Fehringer'et'al.'2012;'Daub'et'al.'2013).'To'be'able'to'use'the'distribution'of'logElikelihood'ratios'of'the'positive'selection'test'–'which'follows'a' chiEsquare' distribution' with' 1' degree' of' freedom' and' spans' several' orders' of'magnitude'–'as'scores'in'the'SUMSTAT'test,'we'applied'a'fourth'root'transformation'as'variance' stabilizing' method.' This' transformation' conserves' the' ranks' of' gene' families'(see' http://udel.edu/~mcdonald/stattransform.html)(Canal' 2005;' McDonald' 2009).'According' to' the' Central' Limit' Theorem' the' distribution' of' SUMSTAT' scores' from'random' gene' sets' approaches' a' normal' distribution' whose' mean' and' variance' derives'from'the'mean'and'variance'of'the'scores'of'the'complete'list'of'tested'genes' G :'' E ( SUMSTAT ) = n ⋅ E ( G ) 'and' Var ( SUMSTAT ) = n ⋅ Var ( G ) 'We' performed' bidirectional' tests' against' this' distribution' to' test' if' the' SUMSTAT'statistic'for'a'given'gene'set'is'higher'or'lower'than'expected'by'chance,'corresponding'o'respectively'enrichment'or'depletion'for'positively'selected'genes'in'this'gene'set.'We'verified'the'accuracy'of'this'methodology'by'drawing'an'empirical'null'distribution'for'each'gene'set'of'size' n 'found'in'the'real'dataset,'based'on'scores'of'10,000'gene'sets'of'same' size' n& randomly' picked' from' the' whole' dataset.' The' distribution' of' SUMSTAT'scores'of'these'randomized'gene'sets'approximates'closely'a'normal'distribution,'even'when' the' set' size' is' small' (Figure' S6).' This' makes' the' SUMSTAT' test' less'computationally' intensive' than' other' gene' set' enrichment' approaches' (e.g.'GSEA)(Subramanian' et' al.' 2005)' where' the' null' distribution' cannot' be' inferred'mathematically' and' randomizations' have' to' be' performed' for' each' individual' test.' We'verified'that'a'GSEA'approach'gave'broadly'similar'results'(not'shown).''Because' different' gene' sets' sometimes' share' many' genes' in' common,' the' list' of'significant' gene' sets' resulting' from' enrichment' tests' is' usually' highly' redundant.' We'implemented'the'“elim”'algorithm'from'the'Bioconductor'package'topGO,'to'decorrelate'the'graph'structure'of'the'Gene'Ontology'(Alexa,'Rahnenfuhrer,'Lengauer'2006).'Briefly,'the' GO' categories' are' tested' recursively' starting' from' the' deeper' levels' of' the' GO' tree,'and' the' genes' annotated' to' these' significant' categories' are' removed' from' all' their'parent'categories.'As'the'tests'for'different'categories'are'not'independent,'it'is'not'clear'if' classical' approaches' to' assess' the' False' Discovery' Rate' (e.g.,' Benjamini,' Hochberg'1995)'are'accurate.'Thus'we'calculated'empirically'an'FDR'at'each'pEvalue'threshold'by'performing' 100' randomizations' where' the' scores' of' gene' families' were' permuted' and'the'gene'set'enrichment'test'rerun.'The'FDR'is'estimated'as:' FDR = FPFP + TP = N N t 'where' at' a' given' pEvalue' threshold' N ' represents' the' mean' number' of' false' positives'obtained'in'the'randomizations'and' N t 'represents'the'number'of'positives'obtained'with'the'real'dataset.'The'FDR'obtained'with'this'approach'was'in'good'agreement'with'the'BenjaminiEHochberg'FDR'(Benjamini,'Hochberg'1995).'Gene'Ontology'categories'with'a'FDR'<'20%'are'reported.'Functional'categories'depleted'in'positive'selection'reflect'the'most' conserved' sets' of' functional' categories,' under' the' action' of' purifying' selection.'These'are'not'discussed'in'this'manuscript.'The' gene' set' enrichment' test' ran' on' each' individual' branch' of' the' tree' with'results' of' the' branchEsite' test' yields' heterogeneous' results,' probably' resulting' from'differences' in' power' of' the' branchEsite' test' on' different' branches' of' the' phylogeny'Table'S9;'only'branches'Sinv,'Pbar,'Hsal,' D.&melanogaster
Tests of phenotypic category enrichment
Mutant' phenotype' annotations' of'
D.& melanogaster& & Expression data
Microarray'expression'data'from'
Solenopsis&invicta '(Ometto'et'al.'2011)'were'provided'by' the' authors' upon' request.' These' included' expression' levels' of' clones' of' the' spotted'microarray' used,' as' well' as' the' list' of' genes' identified' to' be' overEexpressed' in' each' of'the' three' castes' (workers,' queens' and' males),' both' at' pupal' and' adult' stages.' The'mapping'of'clones'to'the'gene'model'of'
Solenopsis&invicta& (OGS_2.2.3)(Wurm'et'al.'2011)'was' provided' by' Y.' Wurm,' and' is' similar' to' the' mapping' used' in' (Hunt' et' al.' 2011).' If'multiple' clones' mapped' to' the' same' gene,' the' average' signal' was' used' for' expression.'For' differential' expression,' we' used' the' results' of' the' original' study' (BAGEL' analysis,'where' a' clone' was' considered' to' be' differentially' expressed' between' conditions' if' the'Bayesian'posterior'probability'was'p<0.001,'corresponding'to'a'FDR~5%)(Ometto'et'al.'2011).'A'gene'was'considered'differentially'expressed'if'at'least'one'clone'mapped'to'it'was'found'differentially'expressed.'Expression'data'was'available'for'1,327'genes'of'the'singleEcopy' orthologs' dataset,' including' 603' genes' overEexpressed' in' at' least' one'condition.'We'ran'a'SUMSTAT'gene'set'enrichment'test'on'the'sets'of'genes'with'casteEspecific'expression'(pupal'male,'pupal'queen,'pupal'worker,'adult'male,'adult'queen'and'dult' worker).' PEvalues' were' obtained' by' comparison' to' an' empirical' distribution'created'with'10,000'randomizations'of'gene'scores.'
Aging genes
Aging' and' oxidative' stress' associated' genes' were' obtained' from' a' microarray' study' in'
Drosophila&melanogaster 'comparing'the'expression'of'genes'in'10'day'old'flies'to'61'day'old' flies,' and' flies' exposed' to' 100%' O ' for' 7' days' to' controls' (Landis' et' al.' 2004).' We'tested'the'enrichment'for'positively'selected'genes'(SUMSTAT'test)'in'four'gene'sets'to'constituted' of' up' and' downEregulated' genes' in' both' contrasts.' PEvalues' were' obtained'by'comparison'to'an'empirical'distribution'created'with'10,000'randomizations'of'gene'scores.' Genes with mitochondrial function
Genes' with' mitochondrial' function' were' identified' as' those' mapped' to' any' of' the' 310'Gene' Ontology' categories' including' “mitochondria*”' in' their' names' or' synonym' names'(using' the' search' engine' on' http://amigo.geneontology.org/).' 313' of' the' identified'genes'had'available'microarray'expression'data'in'
S.&invicta .' Data availability
Raw'and'filtered'alignments'used'in'these'analyses,'track'files'for'the'alignment'editor'Jalview' (Clamp' et' al.' 2004),' Codeml' control' files' and' result' files' can' be' downloaded' at'http://bioinfo.unil.ch/supdata/Roux_positive_selection_ants/Roux_et_al_datasets.tar.gz.''A'simple'web'interface'displaying'gene'families,'Gene'Ontology'mapping,'Codeml'results'and' alignments' (through' a' Jalview' applet)' is' available' at'http://bioinfo.unil.ch/supdata/Roux_positive_selection_ants/families.html.' Jalview'tracks'display'the'regions'used'or'filtered'out'in'the'original'protein'alignments,'as'well'as' the' residues' found' to' be' under' positive' selection' by' Bayes' Empirical' Bayes' (Yang,'Wong,' Nielsen' 2005)' in' all' the' branches' tested' for' each' of' the' 3' replicate' runs' (Figure'2).' cknowledgements
References
Abouheif' E,' Wray' GA.' 2002.' Evolution' of' the' gene' network' underlying' wing'polyphenism'in'ants.'Science'297:249E252.'Ackermann'M,'Strimmer'K.'2009.'A'general'modular'framework'for'gene'set'enrichment'analysis.'BMC'Bioinformatics'10:47.'Adams' MDCelniker' SEHolt' RA,' et' al.' 2000.' The' Genome' Sequence' of'
Drosophila&melanogaster .'Science'287:2185E2195.'Alexa'A,'Rahnenfuhrer'J,'Lengauer'T.'2006.'Improved'scoring'of'functional'groups'from'gene' expression' data' by' decorrelating' GO' graph' structure.' Bioinformatics'22:1600E1607.'Allison'DB,'Cui'X,'Page'GP,'Sabripour'M.'2006.'Microarray'data'analysis:'from'disarray'to'consolidation'and'consensus.'Nat'Rev'Genet'7:55E65.'Amdam' GV,' Omholt' SW.' 2002.' The' regulatory' anatomy' of' honeybee' lifespan.' J' Theor'Biol'216:209E228.'Anisimova' M,' Yang' Z.' 2007.' Multiple' Hypothesis' Testing' to' Detect' Lineages' under'Positive'Selection'that'Affects'Only'a'Few'Sites.'Mol'Biol'Evol'24:1219E1228.'Ashburner' M,' Ball' CA,' Blake' JA,' et' al.' 2000.' Gene' ontology:' tool' for' the' unification' of'biology.'The'Gene'Ontology'Consortium.'Nat'Genet'25:25E29.'Bakewell' MA,' Shi' P,' Zhang' J.' 2007.' More' genes' underwent' positive' selection' in'chimpanzee' evolution' than' in' human' evolution.' Proc' Natl' Acad' Sci' USA'104:7489E7494.'ard' F,' Casano' L,' Mallabiabarrena' A,' et' al.' 2006.' Functional' genomics' reveals' genes'involved'in'protein'secretion'and'Golgi'organization.'Nature'439:604E607.'Bazin' E,' Glemin' S,' Galtier' N.' 2006.' Population' size' does' not' influence' mitochondrial'genetic'diversity'in'animals.'Science'312:570E572.'Bazykin' GA,' Kondrashov' AS.' 2012.' Major' role' of' positive' selection' in' the' evolution' of'conservative'segments'of'Drosophila'proteins.'Proc'R'Soc'B'279:3409E3417.'Bechstedt' S,' Albert' JT,' Kreil' DP,' MullerEReichert' T,' Gopfert' MC,' Howard' J.' 2010.' A'doublecortin' containing' microtubuleEassociated' protein' is' implicated' in'mechanotransduction'in'Drosophila'sensory'cilia.'Nat'Commun'1:11.'Beller'M,'Sztalryd'C,'Southall'N,'Bell'M,'Jäckle'H,'Auld'DS,'Oliver'B.'2008.'COPI'Complex'Is'a'Regulator'of'Lipid'Homeostasis.'PLoS'Biol'6:e292.'Benjamini' Y,' Hochberg' Y.' 1995.' Controlling' the' False' Discovery' Rate:' A' Practical' and'Powerful' Approach' to' Multiple' Testing.' Journal' of' the' Royal' Statistical' Society.'Series'B'(Methodological)'57:289E300.'Bernt'M,'Donath'A,'Juhling'F,'Externbrink'F,'Florentz'C,'Fritzsch'G,'Putz'J,'Middendorf'M,'Stadler' PF.' 2012.' MITOS:' Improved' de' novo' metazoan' mitochondrial' genome'annotation.'Mol'Phylogenet'Evol'69:313E319.'Blanke' S,' Jackle' H.' 2006.' Novel' guanine' nucleotide' exchange' factor' GEFmeso' of'Drosophila' melanogaster' interacts' with' Ral' and' Rho' GTPase' Cdc42.' FASEB' J'20:683E691.'Bodily' KD,' Morrison' CM,' Renden' RB,' Broadie' K.' 2001.' A' Novel' Member' of' the' Ig'Superfamily,' turtle,' is' a' CNSESpecific' Protein' Required' for' Coordinated' Motor'Control.'Journal'of'Neuroscience'21:3113E3125.'Bonasio' R,' Zhang' G,' Ye' C,' et' al.' 2010.' Genomic' Comparison' of' the' Ants'
Camponotus&floridanus 'and'
Harpegnathos&saltator .'Science'329:1068E1071.'Boussau'B,'Gouy'M.'2006.'Efficient'Likelihood'Computations'with'Nonreversible'Models'of'Evolution.'Systematic'Biology'55:756E768.'Brady' SG,' Schultz' TR,' Fisher' BL,' Ward' PS.' 2006.' Evaluating' alternative' hypotheses' for'the'early'evolution'and'diversification'of'ants.'Proc'Natl'Acad'Sci'USA'103:18172E18177.'Bronstein' R,' Levkovitz' L,' Yosef' N,' Yanku' M,' Ruppin' E,' Sharan' R,' Westphal' H,' Oliver' B,'Segal' D.' 2010.' Transcriptional' Regulation' by' CHIP/LDB' Complexes.' PLoS' Genet'6:e1001063.'Brunet'FG,'Crollius'HR,'Paris'M,'Aury'JM,'Gibert'P,'Jaillon'O,'Laudet'V,'RobinsonERechavi'M.'2006.'Gene'loss'and'evolutionary'rates'following'wholeEgenome'duplication'in'teleost'fishes.'Mol'Biol'Evol'23:1808E1816.'BrunetERossinni' A,' Austad' S.' 2004.' Ageing' Studies' on' Bats:' A' Review.' Biogerontology'5:211E222.'BrunetERossinni' AK.' 2004.' Reduced' freeEradical' production' and' extreme' longevity' in'the' little' brown' bat' (Myotis' lucifugus)' versus' two' nonEflying' mammals.' Mech'Ageing'Dev'125:11E20.'Bulmer'MS.'2010.'Evolution'of'Immune'Proteins'in'Insects.'Encyclopedia'of'Life'Sciences'(ELS).'Chichester:'John'Wiley'&'Sons,'Ltd.'Burton'RS,'Barreto'FS.'2012.'A'disproportionate'role'for'mtDNA'in'DobzhanskyEMuller'incompatibilities?'Mol'Ecol'21:4942E4957.'Canal' L.' 2005.' A' normal' approximation' for' the' chiEsquare' distribution.' Computational'Statistics'&'Data'Analysis'48:803E808.'hen'CC,'Wu'JK,'Lin'HW,'Pai'TP,'Fu'TF,'Wu'CL,'Tully'T,'Chiang'AS.'2012.'Visualizing'longEterm'memory'formation'in'two'neurons'of'the'
Drosophila 'brain.'Science'335:678E685.'Clamp' M,' Cuff' J,' Searle' SM,' Barton' GJ.' 2004.' The' Jalview' Java' alignment' editor.'Bioinformatics'20:426E427.'Collier'S,'Chan'HYE,'Toda'T,'McKimmie'C,'Johnson'G,'Adler'PN,'O'Kane'C,'Ashburner'M.'2000.' The'
Drosophila ' embargoed' Gene' Is' Required' for' Larval' Progression' and'Encodes' the' Functional' Homolog' of'
Schizosaccharomyces ' Crm1.' Genetics'155:1799E1807.'Corley' LS,' Lavine' MD.' 2006.' A' review' of' insect' stem' cell' types.' Seminars' in' Cell' &'Developmental'Biology'17:510E517.'Corona' M,' Hughes' KA,' Weaver' DB,' Robinson' GE.' 2005.' Gene' expression' patterns'associated'with'queen'honey'bee'longevity.'Mech'Ageing'Dev'126:1230E1238.'Corona' M,' Velarde' RA,' Remolina' S,' MoranELauter' A,' Wang' Y,' Hughes' KA,' Robinson' GE.'2007.' Vitellogenin,' juvenile' hormone,' insulin' signaling,' and' queen' honey' bee'longevity.'Proc'Natl'Acad'Sci'USA'104:7128E7133.'Cortopassi' GA.' 2002.' A' neutral' theory' predicts' multigenic' aging' and' increased'concentrations' of' deleterious' mutations' on' the' mitochondrial' and' Y'chromosomes.'Free'Radical'Biology'and'Medicine'33:605E610.'Cronin'SJ,'Nehme'NT,'Limmer'S,'et'al.'2009.'GenomeEwide'RNAi'screen'identifies'genes'involved'in'intestinal'pathogenic'bacterial'infection.'Science'325:340E343.'Crozier'RH,'Crozier'YC.'1993.'The'mitochondrial'genome'of'the'honeybee'
Apis&mellifera :'complete'sequence'and'genome'organization.'Genetics'133:97E117.'Cui' H,' Kong' Y,' Zhang' H.' 2012.' Oxidative' stress,' mitochondrial' dysfunction,' and' aging.' J'Signal'Transduct'2012:646354.'Curtis' C,' Landis' GN,' Folk' D,' et' al.' 2007.' Transcriptional' profiling' of' MnSODEmediated'lifespan' extension' in'
Drosophila ' reveals' a' speciesEgeneral' network' of' aging' and'metabolic'genes.'Genome'Biol'8:R262.'Daub' JT,' Hofer' T,' Cutivet' E,' Dupanloup' I,' QuintanaEMurci' L,' RobinsonERechavi' M,'Excoffier' L.' 2013.' Evidence' for' polygenic' adaptation' to' pathogens' in' the' human'genome.'Mol'Biol'Evol'30:1544E1558.'Davis' JC,' Petrov' DA.' 2004.' Preferential' duplication' of' conserved' proteins' in' eukaryotic'genomes.'PLoS'Biol'2:e55.'Dawkins'R,'Krebs'JR.'1979.'Arms'Races'between'and'within'Species.'Proceedings'of'the'Royal'Society'B:'Biological'Sciences'205:489E511.'Didelot' G,' Molinari' F,' Tchenio' P,' Comas' D,' Milhiet' E,' Munnich' A,' Colleaux' L,' Preat' T.'2006.'Tequila,'a'neurotrypsin'ortholog,'regulates'longEterm'memory'formation'in'
Drosophila .'Science'313:851E853.'Drosophila' 12' Genomes' Consortium.' 2007.' Evolution' of' genes' and' genomes' on' the'
Drosophila 'phylogeny.'Nature'450:203E218.'Drysdale'R.'2001.'Phenotypic'data'in'FlyBase.'Brief'Bioinform'2:68E80.'Duret' L.' 2008.' Neutral' theory:' The' null' hypothesis' of' molecular' evolution.' Nature'Education'1.'Efron'B,'Tibshirani'R.'2007.'On'Testing'the'Significance'of'Sets'of'Genes.'The'Annals'of'Applied'Statistics'1:107E129.'Farris' SM,' Schulmeister' S.' 2011.' Parasitoidism,' not' sociality,' is' associated' with' the'evolution' of' elaborate' mushroom' bodies' in' the' brains' of' hymenopteran' insects.'Proc'R'Soc'B'278:940E951.'ehringer'G,'Liu'G,'Briollais'L,'Brennan'P,'Amos'CI,'Spitz'MR,'Bickeböller'H,'Wichmann'HE,' Risch' A,' Hung' RJ.' 2012.' Comparison' of' Pathway' Analysis' Approaches' Using'Lung'Cancer'GWAS'Data'Sets.'PLoS'ONE'7:e31816.'Finkel'T,'Holbrook'NJ.'2000.'Oxidants,'oxidative'stress'and'the'biology'of'ageing.'Nature'408:239E247.'Fischman'BJ,'Woodard'SH,'Robinson'GE.'2011.'Molecular'evolutionary'analyses'of'insect'societies.'Proc'Natl'Acad'Sci'USA'108'Suppl'2:10847E10854.'Fletcher' W,' Yang' Z.' 2010.' The' Effect' of' Insertions,' Deletions,' and' Alignment' Errors' on'the'BranchESite'Test'of'Positive'Selection.'Mol'Biol'Evol'27:2257E2267.'Force' A,' Lynch' M,' Pickett' FB,' Amores' A,' Yan' YL,' Postlethwait' J.' 1999.' Preservation' of'duplicate'genes'by'complementary,'degenerative'mutations.'Genetics'151:1531E1545.'Galtier' N,' Gouy' M.' 1998.' Inferring' pattern' and' process:' maximumElikelihood'implementation' of' a' nonhomogeneous' model' of' DNA' sequence' evolution' for'phylogenetic'analysis.'Molecular'Biology'and'Evolution'15:871E879.'Gentleman' R,' Carey' V,' Bates' D,' et' al.' 2004.' Bioconductor:' open' software' development'for'computational'biology'and'bioinformatics.'Genome'Biol'5:R80.'George' RD,' McVicker' G,' Diederich' R,' Ng' SB,' MacKenzie' AP,' Swanson' WJ,' Shendure' J,'Thomas' JH.' 2011.' Trans' genomic' capture' and' sequencing' of' primate' exomes'reveals'new'targets'of'positive'selection.'Genome'Res'21:1686E1694.'Gerber' AS,' Loggins' R,' Kumar' S,' Dowling' TE.' 2001.' Does' Nonneutral' Evolution' Shape'Observed' Patterns' of' DNA' Variation' in' Animal' Mitochondrial' Genomes?' Annual'Review'of'Genetics'35:539E566.'Gharib' WH,' RobinsonERechavi' M.' 2013.' The' branchEsite' test' of' positive' selection' is'surprisingly' robust' but' lacks' power' under' synonymous' substitution' saturation'and'variation'in'GC.'Mol'Biol'Evol'30:1675E1686.'Gotzek' D,' Clarke' J,' Shoemaker' D.' 2010.' Mitochondrial' genome' evolution' in' fire' ants'(Hymenoptera:'Formicidae).'BMC'Evol'Biol'10:300.'GouveiaEOliveira'R,'Sackett'P,'Pedersen'A.'2007.'MaxAlign:'maximizing'usable'data'in'an'alignment.'BMC'Bioinformatics'8:312.'Guindon' S,' Gascuel' O.' 2003.' A' simple,' fast,' and' accurate' algorithm' to' estimate' large'phylogenies'by'maximum'likelihood.'Syst'Biol'52:696E704.'Hall' DW,' Goodisman' MA.' 2012.' The' effects' of' kin' selection' on' rates' of' molecular'evolution'in'social'insects.'Evolution'66:2080E2093.'Hambuch' TM,' Parsch' J.' 2005.' Patterns' of' Synonymous' Codon' Usage' in'
Drosophila&melanogaster 'Genes'With'SexEBiased'Expression.'Genetics'170:1691E1700.'Harman'D.'1972.'The'biologic'clock:'the'mitochondria?'J'Am'Geriatr'Soc'20:145E147.'Harpur' BA,' Zayed' A.' 2013.' Accelerated' evolution' of' innate' immunity' proteins' in' social'insects:'adaptive'evolution'or'relaxed'constraint?'Mol'Biol'Evol'30:1665E1674.'He'X,'Zhang'J.'2006.'Higher'Duplicability'of'Less'Important'Genes'in'Yeast'Genomes.'Mol'Biol'Evol'23:144E151.'Hölldobler'B,'Wilson'E.'1990.'The'ants:'Belknap'Press'of'Harvard'University'Press.'Honeybee' Genome' Sequencing' Consortium.' 2006.' Insights' into' social' insects' from' the'genome'of'the'honeybee'
Apis&mellifera .'Nature'443:931E949.'Hovemann'BT,'Sehlmeyer'F,'Malz'J.'1997.'
Drosophila&melanogaster 'NADPH–cytochrome'P450' oxidoreductase:' pronounced' expression' in' antennae' may' be' related' to'odorant'clearance.'Gene'189:213E219.'unt' BG,' Ometto' L,' Wurm' Y,' Shoemaker' D,' Yi' SV,' Keller' L,' Goodisman' MAD.' 2011.'Relaxed' selection' is' a' precursor' to' the' evolution' of' phenotypic' plasticity.' Proc'Natl'Acad'Sci'USA'108:15936E15941.'Ingram'KK,'Oefner'P,'Gordon'DM.'2005.'TaskEspecific'expression'of'the'foraging'gene'in'harvester'ants.'Mol'Ecol'14:813E818.'Jemielity'S,'Chapuisat'M,'Parker'JD,'Keller'L.'2005.'Long'live'the'queen:'studying'aging'in'social'insects.'Age'27:241E248.'Jensen' TF,' HolmEJensen' I.' 1980.' Energetic' cost' of' running' in' workers' of' three' ant'species,'
Formica& fusca& L. ,' Formica& rufa& L. ,' and'
Camponotus& herculeanus& L. '(Hymenoptera,'Formicidae).'Journal'of'comparative'physiology'137:151E156.'Jobson' RW,' Nabholz' B,' Galtier' N.' 2010.' An' Evolutionary' Genome' Scan' for' LongevityERelated'Natural'Selection'in'Mammals.'Mol'Biol'Evol'27:840E847.'Jordan'G,'Goldman'N.'2012.'The'effects'of'alignment'error'and'alignment'filtering'on'the'sitewise'detection'of'positive'selection.'Mol'Biol'Evol'29:1125E1139.'Katoh'K,'Kuma'K,'Toh'H,'Miyata'T.'2005.'MAFFT'version'5:'improvement'in'accuracy'of'multiple'sequence'alignment.'Nucleic'Acids'Res'33:511E518.'Keller'L,'Genoud'M.'1997.'Extraordinary'lifespans'in'ants:'a'test'of'evolutionary'theories'of'ageing.'Nature'389:958E960.'Keller' L,' Jemielity' S.' 2006.' Social' insects' as' a' model' to' study' the' molecular' basis' of'ageing.'Exp'Gerontol'41:553E556.'Kim' HJ,' Morrow' G,' Westwood' JT,' Michaud' S,' Tanguay' RM.' 2010a.' Gene' expression'profiling' implicates' OXPHOS' complexes' in' lifespan' extension' of' flies' overEexpressing'a'small'mitochondrial'chaperone,'Hsp22.'Exp'Gerontol'45:611E620.'Kim'HS,'Murphy'T,'Xia'J,'Caragea'D,'Park'Y,'Beeman'RW,'Lorenzen'MD,'Butcher'S,'Manak'JR,' Brown' SJ.' 2010b.' BeetleBase' in' 2010:' revisions' to' provide' comprehensive'genomic'information'for'
Tribolium&castaneum .'Nucleic'Acids'Res'38:D437ED442.'Kim' SY,' Volsky' DJ.' 2005.' PAGE:' parametric' analysis' of' gene' set' enrichment.' BMC'Bioinformatics'6:144.'Kirkness' EF,' Haas' BJ,' Sun' W,' et' al.' 2010.' Genome' sequences' of' the' human' body' louse'and' its' primary' endosymbiont' provide' insights' into' the' permanent' parasitic'lifestyle.'Proc'Natl'Acad'Sci'USA'107:12168E12173.'Kishita'Y,'Tsuda'M,'Aigaki'T.'2012.'Impaired'fatty'acid'oxidation'in'a'
Drosophila 'model'of'mitochondrial' trifunctional' protein' (MTP)' deficiency.' Biochem' Biophys' Res'Commun'419:344E349.'Kiss'DL,'Andrulis'ED.'2010.'GenomeEwide'analysis'reveals'distinct'substrate'specificities'of'Rrp6,'Dis3,'and'core'exosome'subunits.'RNA'16:781E791.'Kosiol' C,' Vinar' T,' da' Fonseca' RR,' Hubisz' MJ,' Bustamante' CD,' Nielsen' R,' Siepel' A.' 2008.'Patterns' of' Positive' Selection' in' Six' Mammalian' Genomes.' PLoS' Genet'4:e1000144.'Kowald' A,' Kirkwood' TB.' 2011.' Evolution' of' the' mitochondrial' fusionEfission' cycle' and'its'role'in'aging.'Proc'Natl'Acad'Sci'USA'108:10237E10242.'Kuan' YS,' BrewerEJensen' P,' Bai' WL,' Hunter' C,' Wilson' CB,' Bass' S,' Abernethy' J,' Wing' JS,'Searles' LL.' 2009.'
Drosophila ' suppressor' of' sable' protein' [Su(s)]' promotes'degradation' of' aberrant' and' transposonEderived' RNAs.' Mol' Cell' Biol' 29:5590E5603.'Kucherenko'MM,'Marrone'AK,'Rishko'VM,'Magliarelli'Hde'F,'Shcherbata'HR.'2011.'Stress'and' muscular' dystrophy:' a' genetic' screen' for' dystroglycan' and' dystrophin'interactors'in'
Drosophila 'identifies'cellular'stress'response'components.'Dev'Biol'352:228E242.'ulmuni' J,' Wurm' Y,' Pamilo' P.' 2013.' Comparative' genomics' of' chemosensory' protein'genes' reveals' rapid' evolution' and' positive' selection' in' antEspecific' duplicates.'Heredity'110:538E547.'Landis'GN,'Abdueva'D,'Skvortsov'D,'Yang'J,'Rabin'BE,'Carrick'J,'Tavare'S,'Tower'J.'2004.'Similar' gene' expression' patterns' characterize' aging' and' oxidative' stress' in'
Drosophila&melanogaster .'Proc'Natl'Acad'Sci'USA'101:7663E7668.'Lawson' D,' Arensburger' P,' Atkinson' P,' et' al.' 2009.' VectorBase:' a' data' resource' for'invertebrate'vector'genomics.'Nucleic'Acids'Res'37:D583ED587.'Leboeuf' AC,' Benton' R,' Keller' L.' 2013.' The' molecular' basis' of' social' behavior:' models,'methods'and'advances.'Curr'Opin'Neurobiol'23:3E10.'Lee'HY,'Chou'JY,'Cheong'L,'Chang'NH,'Yang'SY,'Leu'JY.'2008.'Incompatibility'of'nuclear'and' mitochondrial' genomes' causes' hybrid' sterility' between' two' yeast' species.'Cell'135:1065E1073.'Lenaz' G.' 1998.' Role' of' mitochondria' in' oxidative' stress' and' ageing.' Biochim' Biophys'Acta'1366:53E67.'Lihoreau' M,' Latty' T,' Chittka' L.' 2012.' An' exploration' of' the' social' brain' hypothesis' in'insects.'Front'Physiol'3:442.'LindbladEToh' K,' Garber' M,' Zuk' O,' et' al.' 2011.' A' highEresolution' map' of' human'evolutionary'constraint'using'29'mammals.'Nature'478:476E482.'Linksvayer' TA,' Wade' MJ.' 2009.' Genes' with' social' effects' are' expected' to' harbor' more'sequence'variation'within'and'between'species.'Evolution'63:1685E1696.'Löytynoja' A,' Goldman' N.' 2008.' PhylogenyEaware' gap' placement' prevents' errors' in'sequence'alignment'and'evolutionary'analysis.'Science'320:1632E1635.'Loytynoja' A,' Vilella' AJ,' Goldman' N.' 2012.' Accurate' extension' of' multiple' sequence'alignments' using' a' phylogenyEaware' graph' algorithm.' Bioinformatics' 28:1684E1691.'MarkovaERaina' P,' Petrov' D.' 2011.' High' sensitivity' to' aligner' and' high' rate' of' false'positives' in' the' estimates' of' positive' selection' in' the' 12'
Drosophila ' genomes.'Genome'Res'21:863E874.'McDonald' JH.' 2009.' Handbook' of' Biological' Statistics.' Baltimore,' Maryland:' Sparky'House'Publishing.'Meiklejohn' CD,' Montooth' KL,' Rand' DM.' 2007.' Positive' and' negative' selection' on' the'mitochondrial'genome.'Trends'Genet'23:259E263.'Mockett' RJ,' Orr' WC,' Rahmandar' JJ,' Benes' JJ,' Radyuk' SN,' Klichko' VI,' Sohal' RS.' 1999.'Overexpression' of' MnEContaining' Superoxide' Dismutase' in' Transgenic'
Drosophila&melanogaster .'Archives'of'Biochemistry'and'Biophysics'371:260E269.'Molnar'J,'Fong'KSK,'He'QP,'Hayashi'K,'Kim'Y,'Fong'SFT,'Fogelgren'B,'Molnarne'Szauter'K,' Mink' M,' Csiszar' K.' 2003.' Structural' and' functional' diversity' of' lysyl' oxidase'and' the' LOXElike' proteins.' Biochimica' et' Biophysica' Acta' (BBA)' E' Proteins' and'Proteomics'1647:220E224.'Molnar' J,' Ujfaludi' Z,' Fong' SF,' Bollinger' JA,' Waro' G,' Fogelgren' B,' Dooley' DM,' Mink' M,'Csiszar' K.' 2005.'
Drosophila ' lysyl' oxidases' DmloxlE1' and' DmloxlE2' are'differentially'expressed'and'the'active'DmLOXLE1'influences'gene'expression'and'development.'J'Biol'Chem'280:22977E22985.'Montooth' KL,' Rand' DM.' 2008.' The' Spectrum' of' Mitochondrial' Mutation' Differs' across'Species.'PLoS'Biol'6:e213.'MontoyaEBurgos' JI.' 2011.' Patterns' of' positive' selection' and' neutral' evolution' in' the'proteinEcoding'genes'of'Tetraodon'and'Takifugu.'PLoS'ONE'6:e24800.'oreau' CS,' Bell' CD,' Vila' R,' Archibald' SB,' Pierce' NE.' 2006.' Phylogeny' of' the' Ants:'Diversification'in'the'Age'of'Angiosperms.'Science'312:101E104.'Moretti' S,' Laurenczy' B,' Gharib' WH,' et' al.' 2014.' Selectome' update:' quality' control' and'computational' improvements' to' a' database' of' positive' selection.' Nucleic' Acids'Res'42:D917E921.'Munch' D,' Amdam' GV,' Wolschin' F.' 2008.' Ageing' in' a' eusocial' insect:' molecular' and'physiological' characteristics' of' life' span' plasticity' in' the' honey' bee.' Funct' Ecol'22:407E421.'MunozETorres'MC,'Reese'JT,'Childers'CP,'Bennett'AK,'Sundaram'JP,'Childs'KL,'Anzola'JM,'Milshina' N,' Elsik' CG.' 2011.' Hymenoptera' Genome' Database:' integrated'community'resources'for'insect'species'of'the'order'Hymenoptera.'Nucleic'Acids'Res'39:D658ED662.'Neumuller' RA,' Richter' C,' Fischer' A,' Novatchkova' M,' Neumuller' KG,' Knoblich' JA.' 2011.'GenomeEwide' analysis' of' selfErenewal' in'
Drosophila ' neural' stem' cells' by'transgenic'RNAi.'Cell'Stem'Cell'8:580E593.'Niven'JE,'Scharlemann'JP.'2005.'Do'insect'metabolic'rates'at'rest'and'during'flight'scale'with'body'mass?'Biology'Letters'1:346E349.'Notredame'C,'Higgins'DG,'Heringa'J.'2000.'TEcoffee:'a'novel'method'for'fast'and'accurate'multiple'sequence'alignment.'J'Mol'Biol'302:205E217.'Nozawa' M,' Nei' M.' 2007.' Evolutionary' dynamics' of' olfactory' receptor' genes' in'
Drosophila 'species.'Proc'Natl'Acad'Sci'USA'104:7122E7127.'Nygaard' S,' Zhang' G,' Schiøtt' M,' et' al.' 2011.' The' genome' of' the' leafEcutting' ant'
Acromyrmex& echinatior ' suggests' key' adaptations' to' advanced' social' life' and'fungus'farming.'Genome'Res'21:1339E1348.'Oliveira' DC,' Raychoudhury' R,' Lavrov' DV,' Werren' JH.' 2008.' Rapidly' evolving'mitochondrial' genome' and' directional' selection' in' mitochondrial' genes' in' the'parasitic' wasp' nasonia' (hymenoptera:' pteromalidae).' Mol' Biol' Evol' 25:2167E2180.'Oliver' TA,' Garfield' DA,' Manier' MK,' Haygood' R,' Wray' GA,' Palumbi' SR.' 2010.' WholeEGenome'Positive'Selection'and'HabitatEDriven'Evolution'in'a'Shallow'and'a'DeepESea'Urchin.'Genome'Biol'Evol'2:800E814.'Ometto' L,' Shoemaker' D,' Ross' KG,' Keller' L.' 2011.' Evolution' of' Gene' Expression' in' Fire'Ants:' The' Effects' of' Developmental' Stage,' Caste,' and' Species.' Mol' Biol' Evol'28:1381E1392.'OsumiESutherland'D,'Marygold'S,'Millburn'G,'McQuilton'P,'Ponting'L,'Stefancsik'R,'Falls'K,' Brown' N,' Gkoutos' G.' 2013.' The' Drosophila' phenotype' ontology.' Journal' of'Biomedical'Semantics'4:30.'Parker'JD,'Parker'KM,'Sohal'BH,'Sohal'RS,'Keller'L.'2004.'Decreased'expression'of'CuEZn'superoxide' dismutase' 1' in' ants' with' extreme' lifespan.' Proc' Natl' Acad' Sci' USA'101:3486E3489.'Penick' CA,' Prager' SS,' Liebig' J.' 2012.' Juvenile' hormone' induces' queen' development' in'lateEstage'larvae'of'the'ant'
Harpegnathos&saltator .'J'Insect'Physiol'58:1643E1649.'Penn' O,' Privman' E,' Landan' G,' Graur' D,' Pupko' T.' 2010.' An' alignment' confidence' score'capturing'robustness'to'guide'tree'uncertainty.'Mol'Biol'Evol'27:1759E1767.'PerezECampo' R,' LópezETorres' M,' Cadenas' S,' Rojas' C,' Barja' G.' 1998.' The' rate' of' free'radical' production' as' a' determinant' of' the' rate' of' aging:' evidence' from' the'comparative' approach.' Journal' of' Comparative' Physiology' B:' Biochemical,'Systemic,'and'Environmental'Physiology'168:149E158.'rivman' E,' Penn' O,' Pupko' T.' 2012.' Improving' the' performance' of' positive' selection'inference'by'filtering'unreliable'alignment'regions.'Mol'Biol'Evol'29:1E5.'Proux' E,' Studer' RA,' Moretti' S,' RobinsonERechavi' M.' 2009.' Selectome:' a' database' of'positive'selection.'Nucleic'Acids'Res'37:D404ED407.'Qi'H,'Rath'U,'Wang'D,'et'al.'2004.'Megator,'an'essential'coiledEcoil'protein'that'localizes'to' the' putative' spindle' matrix' during' mitosis' in' Drosophila.' Mol' Biol' Cell'15:4854E4865.'Rajakumar' R,' San' Mauro' D,' Dijkstra' MB,' Huang' MH,' Wheeler' DE,' HiouETim' F,' Khila' A,'Cournoyea' M,' Abouheif' E.' 2012.' Ancestral' developmental' potential' facilitates'parallel'evolution'in'ants.'Science'335:79E82.'Remolina' SC,' Chang' PL,' Leips' J,' Nuzhdin' SV,' Hughes' KA.' 2012.' Genomic' Basis' of' Aging'and'LifeEHistory'Evolution'in'
Drosophila&melanogaster .'Evolution'66:3390E3403.'Richly' E,' Chinnery' PF,' Leister' D.' 2003.' Evolutionary' diversification' of' mitochondrial'proteomes:'implications'for'human'disease.'Trends'Genet'19:356E362.'Robertson' HM,' Wanner' KW.' 2006.' The' chemoreceptor' superfamily' in' the' honey' bee,'
Apis ' mellifera :' expansion' of' the' odorant,' but' not' gustatory,' receptor' family.'Genome'Res'16:1395E1403.'Roth' P,' Xylourgidis' N,' Sabri' N,' Uv' A,' Fornerod' M,' Samakovlis' C.' 2003.' The' Drosophila 'nucleoporin'DNup88'localizes'DNup214'and'CRM1'on'the'nuclear'envelope'and'attenuates'NESEmediated'nuclear'export.'J'Cell'Biol'163:701E706.'Schabauer'H,'Valle'M,'Pacher'C,'Stockinger'H,'Stamatakis'A,'RobinsonERechavi'M,'Yang'Z,'Salamin'N.'2012.'SlimCodeML:'An'Optimized'Version'of'CodeML'for'the'BranchESite' Model.' 2012' IEEE' 26th' International' Parallel' and' Distributed' Processing'Symposium'Workshops'&'PhD'Forum:706E714.'Schneider' A,' Souvorov' A,' Sabath' N,' Landan' G,' Gonnet' GH,' Graur' D.' 2009.' Estimates' of'Positive' Darwinian' Selection' Are' Inflated' by' Errors' in' Sequencing,' Annotation,'and'Alignment.'Genome'Biol'Evol'1:114E118.'Schwander' T,' Lo' N,' Beekman' M,' Oldroyd' BP,' Keller' L.' 2010.' Nature' versus' nurture' in'social'insect'caste'differentiation.'Trends'Ecol'Evol'25:275E282.'Shen' YY,' Liang' L,' Zhu' ZH,' Zhou' WP,' Irwin' DM,' Zhang' YP.' 2010.' Adaptive' evolution' of'energy'metabolism'genes'and'the'origin'of'flight'in'bats.'Proc'Natl'Acad'Sci'U'S'A'107:8666E8671.'Simola' DF,' Wissler' L,' Donahue' G,' et' al.' 2013.' Social' insect' genomes' exhibit' dramatic'evolution' in' gene' composition' and' regulation' while' preserving' regulatory'features'linked'to'sociality.'Genome'Res'23:1235E1247.'Smith'B,'Ashburner'M,'Rosse'C,'et'al.'2007.'The'OBO'Foundry:'coordinated'evolution'of'ontologies'to'support'biomedical'data'integration.'Nat'Biotechnol'25:1251E1255.'Smith' CD,' Zimin' A,' Holt' C,' et' al.' 2011a.' Draft' genome' of' the' globally' widespread' and'invasive' Argentine' ant' (
Linepithema& humile ).' Proc' Natl' Acad' Sci' USA' 108:5673E5678.'Smith'CR,'Smith'CD,'Robertson'HM,'et'al.'2011b.'Draft'genome'of'the'red'harvester'ant'
Pogonomyrmex&barbatus .'Proc'Natl'Acad'Sci'USA'108:5667E5672.'Smith'CR,'Toth'AL,'Suarez'AV,'Robinson'GE.'2008.'Genetic'and'genomic'analyses'of'the'division'of'labour'in'insect'societies.'Nat'Rev'Genet'9:735E748.'Stamatakis' A.' 2006.' RAxMLEVIEHPC:' maximum' likelihoodEbased' phylogenetic' analyses'with'thousands'of'taxa'and'mixed'models.'Bioinformatics'22:2688E2690.'StroscheinEStevenson' SL,' Foley' E,' O'Farrell' PH,' Johnson' AD.' 2005.' Identification' of'
Drosophila ' Gene' Products' Required' for' Phagocytosis' of'
Candida ' albicans .' PLoS'Biol'4:e4.'tuder'RA,'Penel'S,'Duret'L,'RobinsonERechavi'M.'2008.'Pervasive'positive'selection'on'duplicated' and' nonduplicated' vertebrate' protein' coding' genes.' Genome' Res'18:1393E1402.'Suarez' RK.' 2000.' Energy' Metabolism' during' Insect' Flight:' Biochemical' Design' and'Physiological'Performance.'Physiological'and'Biochemical'Zoology'73:765E771.'Subramanian' A,' Tamayo' P,' Mootha' VK,' et' al.' 2005.' Gene' set' enrichment' analysis:' A'knowledgeEbased' approach' for' interpreting' genomeEwide' expression' profiles.'Proc'Natl'Acad'Sci'USA'102:15545E15550.'Suen' G,' Teiling' C,' Li' L,' et' al.' 2011.' The' Genome' Sequence' of' the' LeafECutter' Ant' Atta&cephalotes ' Reveals' Insights' into' Its' Obligate' Symbiotic' Lifestyle.' PLoS' Genet'7:e1002007.'Swanson' WJ,' Nielsen' R,' Yang' Q.' 2003.' Pervasive' Adaptive' Evolution' in' Mammalian'Fertilization'Proteins.'Mol'Biol'Evol'20:18E20.'Talavera' G,' Castresana' J.' 2007.' Improvement' of' Phylogenies' after' Removing' Divergent'and' Ambiguously' Aligned' Blocks' from' Protein' Sequence' Alignments.' Systematic'Biology'56:564E577.'Tan'HEW,'Liu'GEH,'Dong'X,'Lin'REQ,'Song'HEQ,'Huang'SEY,'Yuan'ZEG,'Zhao'GEH,'Zhu'XEQ.'2011.' The' Complete' Mitochondrial' Genome' of' the' Asiatic' CavityENesting'Honeybee'
Apis&cerana '(Hymenoptera:'Apidae).'PLoS'ONE'6:e23008.'Tauber'E,'Eberl'DF.'2001.'Song'production'in'auditory'mutants'of'
Drosophila :'the'role'of'sensory' feedback.' Journal' of' Comparative' Physiology.' A:' Sensory,' neural,' and'behavioral'physiology'187:341E348.'Tintle' N,' Borchers' B,' Brown' M,' Bekmetjev' A.' 2009.' Comparing' gene' set' analysis'methods' on' singleEnucleotide' polymorphism' data' from' Genetic' Analysis'Workshop'16.'BMC'Proceedings'3:S96.'
Tribolium ' Genome' Sequencing' Consortium.' 2008.' The' genome' of' the' model' beetle' and'pest'
Tribolium&castaneum .'Nature'452:949E955.'Trifunovic'A,'Hansson'A,'Wredenberg'A,'Rovio'AT,'Dufour'E,'Khvorostov'I,'Spelbrink'JN,'Wibom'R,'Jacobs'HT,'Larsson'NEGr.'2005.'Somatic'mtDNA'mutations'cause'aging'phenotypes'without'affecting'reactive'oxygen'species'production.'Proc'Natl'Acad'Sci'USA'102:17993E17998.'Trifunovic' A,' Wredenberg' A,' Falkenberg' M,' et' al.' 2004.' Premature' ageing' in' mice'expressing'defective'mitochondrial'DNA'polymerase.'Nature'429:417E423.'Tweedie' S,' Ashburner' M,' Falls' K,' et' al.' 2009.' FlyBase:' enhancing' Drosophila' Gene'Ontology'annotations.'Nucleic'Acids'Res'37:D555ED559.'Vamathevan'J,'Hasan'S,'Emes'R,'et'al.'2008.'The'role'of'positive'selection'in'determining'the'molecular'cause'of'species'differences'in'disease.'BMC'Evol'Biol'8:273.'Vilella' AJ,' Severin' J,' UretaEVidal' A,' Heng' L,' Durbin' R,' Birney' E.' 2009.' EnsemblCompara'GeneTrees:' Complete,' duplicationEaware' phylogenetic' trees' in' vertebrates.'Genome'Res'19:327E335.'Viljakainen' L,' Evans' JD,' Hasselmann' M,' Rueppell' O,' Tingek' S,' Pamilo' P.' 2009.' Rapid'evolution'of'immune'proteins'in'social'insects.'Mol'Biol'Evol'26:1791E1801.'Wallace'IM,'O'Sullivan'O,'Higgins'DG,'Notredame'C.'2006.'MECoffee:'combining'multiple'sequence'alignment'methods'with'TECoffee.'Nucleic'Acids'Res'34:1692E1699.'Wang' T,' Montell' C.' 2006.' A' phosphoinositide' synthase' required' for' a' sustained' light'response.'J'Neurosci'26:12816E12825.'Waterhouse' RM,' Zdobnov' EM,' Kriventseva' EV.' 2011.' Correlating' Traits' of' Gene'Retention,' Sequence' Divergence,' Duplicability' and' Essentiality' in' Vertebrates,'Arthropods,'and'Fungi.'Genome'Biol'Evol'3:75E86.'aterhouse' RM,' Zdobnov' EM,' Tegenfeldt' F,' Li' J,' Kriventseva' EV.' 2011.' OrthoDB:' the'hierarchical'catalog'of'eukaryotic'orthologs'in'2011.'Nucleic'Acids'Res'39:D283ED288.'Weng'MP,'Liao'BY.'2011.'DroPhEA:'Drosophila'phenotype'enrichment'analysis'for'insect'functional'genomics.'Bioinformatics'27:3218E3219.'Werren'JH.'1997.'Biology'of'
Wolbachia .'Annual'Review'of'Entomology'42:587E609.'Werren' JH,' Richards' S,' Desjardins' CA,' Niehuis' O,' Gadau' J,' Colbourne' JK,' The' Nasonia'Genome' Working' G.' 2010.' Functional' and' Evolutionary' Insights' from' the'Genomes'of'Three'Parasitoid'Nasonia'Species.'Science'327:343E348.'Wong' WS,' Yang' Z,' Goldman' N,' Nielsen' R.' 2004.' Accuracy' and' power' of' statistical'methods' for' detecting' adaptive' evolution' in' protein' coding' sequences' and' for'identifying'positively'selected'sites.'Genetics'168:1041E1051.'Woodard' SH,' Fischman' BJ,' Venkat' A,' Hudson' ME,' Varala' K,' Cameron' SA,' Clark' AG,'Robinson'GE.'2011.'Genes'involved'in'convergent'evolution'of'eusociality'in'bees.'Proc'Natl'Acad'Sci'USA'108:7472E7477.'Wurm' Y,' Wang' J,' RibaEGrognuz' O,' et' al.' 2011.' The' genome' of' the' fire' ant'
Solenopsis&invicta .'Proc'Natl'Acad'Sci'USA'108:5679E5684.'Yang' Z.' 1998.' Likelihood' ratio' tests' for' detecting' positive' selection' and' application' to'primate'lysozyme'evolution.'Mol'Biol'Evol'15:568E573.'Yang' Z.' 2007.' PAML' 4:' Phylogenetic' Analysis' by' Maximum' Likelihood.' Mol' Biol' Evol'24:1586E1591.'Yang' Z,' dos' Reis' M.' 2011.' Statistical' Properties' of' the' BranchESite' Test' of' Positive'Selection.'Mol'Biol'Evol'28:1217E1228.'Yang' Z,' Nielsen' R,' Goldman' N,' Pedersen' AEMK.' 2000.' CodonESubstitution' Models' for'Heterogeneous'Selection'Pressure'at'Amino'Acid'Sites.'Genetics'155:431E449.'Yang' Z,' Wong' WSW,' Nielsen' R.' 2005.' Bayes' Empirical' Bayes' Inference' of' Amino' Acid'Sites'Under'Positive'Selection.'Mol'Biol'Evol'22:1107E1118.'Yui'R,'Ohno'Y,'Matsuura'ET.'2003.'Accumulation'of'deleted'mitochondrial'DNA'in'aging'
Drosophila&melanogaster .'Genes'&'Genetic'Systems'78:245E251.'Zhang' G,' Cowled' C,' Shi' Z,' et' al.' 2013.' Comparative' analysis' of' bat' genomes' provides'insight'into'the'evolution'of'flight'and'immunity.'Science'339:456E460.'Zhang'J.'2004.'Frequent'False'Detection'of'Positive'Selection'by'the'Likelihood'Method'with'BranchESite'Models.'Mol'Biol'Evol'21:1332E1339.'Zhang' J,' Nielsen' R,' Yang' Z.' 2005.' Evaluation' of' an' Improved' BranchESite' Likelihood'Method' for' Detecting' Positive' Selection' at' the' Molecular' Level.' Mol' Biol' Evol'22:2472E2479.'Zhou' X,' Slone' JD,' Rokas' A,' Berger' SL,' Liebig' J,' Ray' A,' Reinberg' D,' Zwiebel' LJ.' 2012.'Phylogenetic'and'transcriptomic'analysis'of'chemosensory'receptors'in'a'pair'of'divergent' ant' species' reveals' sexEspecific' signatures' of' odor' coding.' PLoS' Genet'8:e1002930.'''' igure legends
Figure'1:'Phylogeny'of'the'seven'sequenced'ant'species'and'the'five'outgroups'used'in'this'study.''The' maximum' likelihood' phylogeny' was' computed' by' R.' Waterhouse' from' the'concatenated' alignment' of' the' conserved' protein' sequences' of' 2,756' singleEcopy'orthologs' from' OrthoDB' (Simola' et' al.' 2013).' The' scale' bar' indicates' the' average'number' of' amino' acid' substitutions' per' site.' The' phylogeny' is' consistent' with' a'previously'published'study'(Brady'et'al.'2006).'A'second'study'only'found'a'difference'in'the' branching' of'
P.& barbatus ' and'
S.& invicta& (Moreau' et' al.' 2006).' The' 15' different'branches'where'positive'selection'was'tested'are'highlighted'in'red'(the'seven'terminal'branches'leading'to'ant'species'and'the'branches'numbered'
A.&mellifera&
P.& humanus ' illustration' was'downloaded' from' Vectorbase,'
D.& melanogaster,& T.& castaneum ' and & N.& vitripennis 'illustrations'were'downloaded'from'Wikipedia.'Illustrations'are'not'to'scale.''Figure'2:'Protein'alignment'view'of'positive'selection'signal'on'gene'family'11650.''See' Table' 1' for' description' of' the' potential' function' of' this' gene' family.' Protein'alignment' is' shown' partially,' from' position' 230' to' 350,' and'
D.& melanogaster ,' T.&castaneum ' and'
P.& humanus& genes' were' removed' by' MaxAlign' because' of' insufficient'alignment' quality' (Materials' and' Methods).' The' second' annotation' track' under' the'protein'alignment'(“branch'5'BEB'site”)'indicates'positively'selected'sites'on'the'tested'branch'
P.& barbatus ' in' blue,' 320' genes' from'
L.& humile ' in' green' and' 262' genes'from'
N.& vitripennis ' in' black.' The' topology' of' the' tree' depicts' the' assemblage' of' 16'subtrees' where' positive' selection' was' tested' using' the' branchEsite' test' of' Codeml'(Materials'and'Methods).'Tested'branches'are'depicted'in'gray'if'there'was'no'evidence'or'positive'selection'and'in'red'if'there'was'significant'evidence'for'positive'selection'at'10%' FDR.' Untested' branches' are' depicted' in' black.' Scale' bar' indicates' the' number' of'amino'acid'substitutions'per'site.' ables
Table&1:&Top&scoring&gene&families&at&branch5site&and&site&tests&for&positive&selection.&Gene&families&are&ranked&based&on&their&log5likelihood&ratios&(ΔlnL).&Results&of&the&branch5site&test&were&filtered&to&keep&only&internal&ant&branches&of&the&phylogenetic&tree&(branches& d S &on&the&tested&branch&below&1.&Results&of&both&tests&were&filtered&to&keep&families&with&a&good&support&for&the&detection&of&sites&evolving&under&positive&selection&(BEB&posterior&probability&>&0.9).&Manual&inspection& of& the& best& hits& confirmed& that& the& signal& of& positive& selection& seemed& genuine& for& all& cases,& except& for& family& 12370& in& the&branch5site&test&results,&which&was&removed&from&the&list.& Test%used% Gene%Family% Branch% ΔlnL% p7val% FDR% d S ω%(proportion)% D. gene%name% Function%annotated%in%Flybase%and%Uniprot% Duplicates%in%ants% Uniprot%ID% Refs%
Branch7site%test% % a & 5& 12.1& 8.4E57& 7.9E55& 0.059& 299&(2.5%)& CG17321& Unknow& 5& Q9VJ40& 5& % % % % % (cid:1) (0.6%)& embargoed& Protein& binding;& protein& transporter& activity;&protein& export& from& nucleus;& multicellular&organismal&development;¢riole&replication& 5& Q9TVM2& (Collier& et& al.&2000;& Roth&et&al.&2003)& % (cid:1) (11%)& ATP& synthase,& subunit& b,&mitochondria& Hydrogen5exporting& ATPase& activity,&phosphorylative& mechanism;& phagocytosis,&engulfment& 5& Q94516& (Stroschein5Stevenson& et&al.&2005)& (cid:1) (0.5%)& lysyl&oxidase5like&2& Protein5lysine&65oxidase&activity& 5& Q8IH65& (Molnar&et&al.&2003;&Molnar& et& al.&2005)& % (cid:1) (1.2%)& Cytochrome& P450&reductase& NADPH5hemoprotein& reductase& activity;&oxidation5reduction& process;& putative& function& in&olfactory&clearance& 5& Q27597& (Hovemann,&Sehlmeyer,&Malz&1997)& Site%test% % % % % % % % % % Example&used&in&Figure&2& able& 2:& Amount& of& positive& selection& detected& on& different& branches& of& the& analyzed&phylogeny&
Branch'name a ' Lineage'delineated' Fraction'of'positively'selected'gene'families' Number'of'positively'selected'gene'families b ' Acep&
A.
A.
S.
P.
C.
L.
H. a& As&illustrated&in&Figure&1& b& Branches& of& gene& families& trees& can& be& merged& if& genes& are& missing& (or& removed& for&quality& reasons),& and& the& resulting& branches& do& not& correspond& to& canonical& branches&defined& by& the& species& topology& (Figure& 1).& When& positive& selection& is& found& on& such&branches,&it&was¬&counted&in&branchUspecific&numbers&displayed&in&Table&2,&but&it&was&counted&when&a&whole&lineage&was&considered&(e.g.,&Hymenoptera).&& able&3:&Gene&Ontology&categories&enriched&for&positively&selected&genes,&based&on&scores&from&the&branch;site&test&from&Codeml&in&ants.&&The&enrichment&test&considers&a&combined&score&for&all&analyzed&branches&of&the&ant&lineage&(Materials&and&Methods).&The&full&table&of&results&is&shown&in&Table&S6.& setID& Ontology& setName& setSize& score& p6value& FDR&
GO:0000313& CC& organellar&ribosome& 59& 26.8& 1.4E;10& 0&GO:0006120& BP& mitochondrial&electron&transport,&NADH&to&ubiquinone& 18& 10.6& 1.1E;9& 0&GO:0005759& CC& mitochondrial&matrix& 98& 39.7& 1.6E;9& 0&GO:0005762& CC& mitochondrial&large&ribosomal&subunit& 36& 16.8& 1.1E;7& 0.0025&GO:0005746& CC& mitochondrial&respiratory&chain& 31& 14.6& 4.5E;7& 0.0033&GO:0005747& CC& mitochondrial&respiratory&chain&complex&I& 22& 11.0& 1.3E;6& 0.0033&GO:0008137& MF& NADH&dehydrogenase&(ubiquinone)&activity& 16& 8.0& 3.2E;5& 0.013&GO:0005763& CC& mitochondrial&small&ribosomal&subunit& 25& 10.9& 0.00018& 0.047&GO:0008038& BP& neuron&recognition& 19& 8.7& 0.00023& 0.047&GO:0008344& BP& adult&locomotory&behavior& 19& 8.4& 0.00082& 0.086&GO:0042254& BP& ribosome&biogenesis& 39& 15.0& 0.0011& 0.099&GO:0003735& MF& structural&constituent&of&ribosome& 107& 36.4& 0.0012& 0.099&GO:0044459& CC& plasma&membrane&part& 129& 42.9& 0.0016& 0.12&GO:0006508& BP& Proteolysis& 145& 47.4& 0.0022& 0.14&GO:0006412& BP& Translation& 191& 61.0& 0.0025& 0.15&GO:0016491& MF& oxidoreductase&activity& 127& 41.8& 0.0028& 0.15&GO:0004872& MF& receptor&activity& 90& 30.6& 0.0028& 0.15&GO:0055114& BP& oxidation;reduction&process& 129& 42.2& 0.0038& 0.16&GO:0008237& MF& metallopeptidase&activity& 36& 13.6& 0.0039& 0.16&GO:0061134& MF& peptidase®ulator&activity& 17& 7.2& 0.0046& 0.18&GO:0002520& BP& immune&system&development& 26& 10.2& 0.0053& 0.19&
O:0048534& BP& hemopoietic&or&lymphoid&organ&development& 26& 10.2& 0.0053& 0.19&GO:0016616& MF& oxidoreductase&activity,&acting&on&the&CH;OH&group&of&donors,&NAD&or&NADP&as&acceptor& 18& 7.5& 0.0053& 0.19&GO:0016836& MF& hydro;lyase&activity& 14& 6.1& 0.0055& 0.19& &Table& 4:& Gene& Ontology& categories& enriched& for& positively& selected& genes,& based& on& scores& from& the& branch;site& test& from& Codeml& in&
Drosophila .&Depletion&results&are&shown&in&Table&S4.& setID & Ontology & setName & setSize & score & p6value & FDR & GO:0006030& BP& chitin&metabolic&process& 29& 16.2& 4.0E;6& 0.0015&GO:0006022& BP& aminoglycan&metabolic&process& 36& 19.4& 5.7E;6& 0.0018&GO:0006952& BP& defense&response& 36& 19.2& 9.8E;6& 0.0018&GO:0008061& MF& chitin&binding& 24& 13.5& 2.0E;5& 0.0020&GO:0004252& MF& serine;type&endopeptidase&activity& 52& 26.0& 3.3E;5& 0.0023&GO:0008026& MF& ATP;dependent&helicase&activity& 18& 10.5& 4.0E;5& 0.0026&GO:0004872& MF& receptor&activity& 13& 7.9& 8.5E;5& 0.0048&GO:0006006& BP& glucose&metabolic&process& 13& 7.8& 0.00021& 0.0082&GO:0046486& BP& glycerolipid&metabolic&process& 17& 9.7& 0.00023& 0.0090&GO:0005819& CC& spindle& 20& 10.9& 0.00046& 0.012&GO:0004175& MF& endopeptidase&activity& 78& 35.9& 0.00048& 0.012&GO:0009607& BP& response&to&biotic&stimulus& 31& 15.8& 0.00060& 0.013&GO:0051707& BP& response&to&other&organism& 31& 15.8& 0.00060& 0.013&GO:0006508& BP& proteolysis& 136& 59.5& 0.00071& 0.014&GO:0006007& BP& glucose&catabolic&process& 12& 7.0& 0.00072& 0.014&GO:0019320& BP& hexose&catabolic&process& 12& 7.0& 0.00072& 0.014&GO:0030312& CC& external&encapsulating&structure& 12& 7.0& 0.00074& 0.014&
O:0015081& MF& sodium&ion&transmembrane&transporter&activity& 16& 8.9& 0.00088& 0.016&GO:0051649& BP& establishment&of&localization&in&cell& 14& 7.9& 0.00093& 0.017&GO:0007126& BP& meiosis& 34& 16.9& 0.00096& 0.017&GO:0003824& MF& catalytic&activity& 844& 337.0& 0.0011& 0.018&GO:0046488& BP& phosphatidylinositol&metabolic&process& 11& 6.5& 0.0012& 0.018&GO:0016490& MF& structural&constituent&of&peritrophic&membrane& 11& 6.4& 0.0013& 0.018&GO:0005975& BP& carbohydrate&metabolic&process& 64& 29.5& 0.0015& 0.019&GO:0004888& MF& transmembrane&receptor&activity& 49& 23.2& 0.0016& 0.020&GO:0051276& BP& chromosome&organization& 59& 27.2& 0.0020& 0.024&GO:0008270& MF& zinc&ion&binding& 173& 73.4& 0.0027& 0.029&GO:0002376& BP& immune&system&process& 43& 20.4& 0.0027& 0.029&GO:0002759& BP& regulation&of&antimicrobial&humoral&response& 11& 6.3& 0.0028& 0.029&GO:0004984& MF& olfactory&receptor&activity& 19& 9.9& 0.0030& 0.029&GO:0002697& BP& regulation&of&immune&effector&process& 11& 6.2& 0.0038& 0.035&GO:0000819& BP& sister&chromatid&segregation& 11& 6.2& 0.0038& 0.035&GO:0007143& BP& female&meiosis& 11& 6.2& 0.0047& 0.040&GO:0016021& CC& integral&to&membrane& 224& 93.1& 0.0047& 0.040&GO:0031347& BP& regulation&of&defense&response& 12& 6.6& 0.0055& 0.044&GO:0015370& MF& solute:sodium&symporter&activity& 12& 6.6& 0.0061& 0.047&GO:0000272& BP& polysaccharide&catabolic&process& 11& 6.1& 0.0065& 0.049&GO:0016810& MF& hydrolase&activity,&acting&on&carbon;nitrogen&(but¬&peptide)&bonds& 28& 13.6& 0.0065& 0.049&GO:0004521& MF& endoribonuclease&activity& 11& 6.1& 0.0069& 0.052&GO:0007291& BP& sperm&individualization& 14& 7.4& 0.0074& 0.053&GO:0010564& BP& regulation&of&cell&cycle&process& 30& 14.4& 0.0075& 0.053&GO:0005635& CC& nuclear&envelope& 17& 8.7& 0.0077& 0.054&
O:0016773& MF& phosphotransferase&activity,&alcohol&group&as&acceptor& 82& 36.0& 0.0077& 0.054&GO:0051253& BP& negative®ulation&of&RNA&metabolic&process& 35& 16.5& 0.0080& 0.055&GO:0007608& BP& sensory&perception&of&smell& 21& 10.5& 0.0080& 0.055&GO:0004222& MF& metalloendopeptidase&activity& 26& 12.7& 0.0082& 0.055&GO:0006807& BP& nitrogen&compound&metabolic&process& 298& 121.5& 0.0090& 0.059&GO:0005576& CC& extracellular®ion& 97& 41.9& 0.010& 0.066&GO:0006814& BP& sodium&ion&transport& 19& 9.5& 0.011& 0.068&GO:0045132& BP& meiotic&chromosome&segregation& 11& 5.9& 0.011& 0.070&GO:0034641& BP& cellular&nitrogen&compound&metabolic&process& 296& 120.4& 0.012& 0.072&GO:0010629& BP& negative®ulation&of&gene&expression& 42& 19.2& 0.013& 0.073&GO:0090304& BP& nucleic&acid&metabolic&process& 162& 67.6& 0.013& 0.075&GO:0016301& MF& kinase&activity& 91& 39.2& 0.013& 0.075&GO:0048584& BP& positive®ulation&of&response&to&stimulus& 11& 5.9& 0.015& 0.084&GO:0016798& MF& hydrolase&activity,&acting&on&glycosyl&bonds& 26& 12.4& 0.017& 0.086&GO:0006139& BP& nucleobase,&nucleoside,&nucleotide&and&nucleic&acid&metabolic&process& 236& 96.4& 0.017& 0.086&GO:0016491& MF& oxidoreductase&activity& 201& 82.6& 0.018& 0.090&GO:0009987& BP& cellular&process& 790& 310.5& 0.019& 0.095&GO:0007088& BP& regulation&of&mitosis& 16& 8.0& 0.019& 0.095&GO:0051783& BP& regulation&of&nuclear&division& 16& 8.0& 0.019& 0.095&GO:0006810& BP& transport& 200& 82.1& 0.021& 0.10&GO:0051234& BP& establishment&of&localization& 197& 80.9& 0.021& 0.10&GO:0006066& BP& alcohol&metabolic&process& 35& 16.1& 0.021& 0.10&GO:0004553& MF& hydrolase&activity,&hydrolyzing&O;glycosyl&compounds& 22& 10.6& 0.022& 0.11&GO:0008233& MF& peptidase&activity& 26& 12.3& 0.022& 0.11&GO:0070011& MF& peptidase&activity,&acting&on&L;amino&acid&peptides& 22& 10.6& 0.022& 0.11&
O:0046914& MF& transition&metal&ion&binding& 58& 25.5& 0.022& 0.11&GO:0050660& MF& flavin&adenine&dinucleotide&binding& 16& 8.0& 0.024& 0.11&GO:0045892& BP& negative®ulation&of&transcription,&DNA;dependent& 26& 12.2& 0.024& 0.11&GO:0032553& MF& ribonucleotide&binding& 161& 66.5& 0.024& 0.11&GO:0032555& MF& purine&ribonucleotide&binding& 161& 66.5& 0.024& 0.11&GO:0035639& MF& purine&ribonucleoside&triphosphate&binding& 161& 66.5& 0.024& 0.11&GO:0006396& BP& RNA&processing& 36& 16.4& 0.025& 0.11&GO:0031226& CC& intrinsic&to&plasma&membrane& 30& 13.9& 0.026& 0.11&GO:0035222& BP& wing&disc&pattern&formation& 11& 5.7& 0.026& 0.12&GO:0007346& BP& regulation&of&mitotic&cell&cycle& 31& 14.3& 0.028& 0.12&GO:0045017& BP& glycerolipid&biosynthetic&process& 11& 5.7& 0.030& 0.13&GO:0006955& BP& immune&response& 30& 13.8& 0.030& 0.13&GO:0044262& BP& cellular&carbohydrate&metabolic&process& 44& 19.6& 0.030& 0.13&GO:0017076& MF& purine&nucleotide&binding& 164& 67.5& 0.032& 0.14&GO:0016705& MF& oxidoreductase& activity,& acting& on& paired& donors,& with& incorporation& or&reduction&of&molecular&oxygen& 21& 10.0& 0.032& 0.14&GO:0005524& MF& ATP&binding& 163& 67.0& 0.034& 0.14&GO:0030554& MF& adenyl&nucleotide&binding& 163& 67.0& 0.034& 0.14&GO:0032559& MF& adenyl&ribonucleotide&binding& 163& 67.0& 0.034& 0.14&GO:0008237& MF& metallopeptidase&activity& 12& 6.1& 0.034& 0.14&GO:0007127& BP& meiosis&I& 18& 8.7& 0.035& 0.14&GO:0019730& BP& antimicrobial&humoral&response& 14& 6.9& 0.039& 0.15&GO:0005815& CC& microtubule&organizing¢er& 16& 7.8& 0.041& 0.16&GO:0055114& BP& oxidation;reduction&process& 167& 68.3& 0.043& 0.17&GO:0019899& MF& enzyme&binding& 14& 6.9& 0.044& 0.17&
O:0048232& BP& male&gamete&generation& 45& 19.8& 0.045& 0.17&GO:0008033& BP& tRNA&processing& 17& 8.2& 0.045& 0.17&GO:0005887& CC& integral&to&plasma&membrane& 29& 13.2& 0.046& 0.17&GO:0044281& BP& small&molecule&metabolic&process& 212& 85.8& 0.046& 0.17&GO:0008238& MF& exopeptidase&activity& 18& 8.6& 0.046& 0.17&GO:0051179& BP& localization& 236& 95.1& 0.047& 0.17&GO:0007283& BP& spermatogenesis& 44& 19.3& 0.047& 0.18&GO:0050662& MF& coenzyme&binding& 48& 21.0& 0.048& 0.18&GO:0034470& BP& ncRNA&processing& 27& 12.3& 0.049& 0.18&GO:0048515& BP& spermatid&differentiation& 24& 11.1& 0.050& 0.18&GO:0045786& BP& negative®ulation&of&cell&cycle& 11& 5.5& 0.050& 0.18&GO:0045934& BP& negative& regulation& of& nucleobase,& nucleoside,& nucleotide& and& nucleic& acid&metabolic&process& 41& 18.1& 0.052& 0.18&GO:0010639& BP& negative®ulation&of&organelle&organization& 12& 5.9& 0.055& 0.19&GO:0015631& MF& tubulin&binding& 12& 5.9& 0.056& 0.19&GO:0005549& MF& odorant&binding& 43& 18.8& 0.058& 0.20& &Table&5:&Gene&Ontology&categories&enriched&for&positively&selected&genes,&based&on&scores&from&the&branch;site&test&from&Codeml&in&bees.&&Depletion&results&are&shown&in&Table&S5.& setID& Ontology& setName& setSize& score& p6value& FDR&
GO:0005099& MF& Ras>Pase&activator&activity& 11& 6.0& 1.1E;5& 0.03&GO:0005083& MF& small>Pase®ulator&activity& 18& 8.1& 0.00010& 0.041&GO:0004872& MF& receptor&activity& 16& 7.2& 0.00020& 0.041&GO:0022836& MF& gated&channel&activity& 11& 5.4& 0.00021& 0.041&
O:0006399& BP& tRNA&metabolic&process& 26& 10.3& 0.00040& 0.053&GO:0071842& BP& cellular&component&organization&at&cellular&level& 190& 55.5& 0.0013& 0.065&GO:0006418& BP& tRNA&aminoacylation&for&protein&translation& 19& 7.6& 0.0021& 0.084&GO:0009725& BP& response&to&hormone&stimulus& 11& 4.9& 0.0022& 0.084&GO:0005635& CC& nuclear&envelope& 13& 5.6& 0.0022& 0.084&GO:0032507& BP& maintenance&of&protein&location&in&cell& 12& 5.3& 0.0023& 0.084&GO:0051336& BP& regulation&of&hydrolase&activity& 20& 7.9& 0.0026& 0.089&GO:0006629& BP& lipid&metabolic&process& 51& 17.0& 0.0031& 0.095&GO:0031072& MF& heat&shock&protein&binding& 17& 6.7& 0.0046& 0.11&GO:0008152& BP& metabolic&process& 335& 91.3& 0.0070& 0.14&GO:0004812& MF& aminoacyl;tRNA&ligase&activity& 19& 7.2& 0.0075& 0.14&GO:0016740& MF& transferase&activity& 211& 59.1& 0.0087& 0.14&GO:0019899& MF& enzyme&binding& 19& 7.1& 0.0097& 0.14&GO:0005216& MF& ion&channel&activity& 14& 5.5& 0.011& 0.14&GO:0022838& MF& substrate;specific&channel&activity& 14& 5.5& 0.011& 0.14&GO:0009308& BP& amine&metabolic&process& 47& 15.2& 0.011& 0.14&GO:0004222& MF& metalloendopeptidase&activity& 15& 5.8& 0.012& 0.14&GO:0005938& CC& cell&cortex& 15& 5.8& 0.012& 0.14&GO:0008237& MF& metallopeptidase&activity& 23& 8.2& 0.012& 0.14&GO:0007275& BP& multicellular&organismal&development& 274& 74.9& 0.013& 0.14&GO:0007165& BP& signal&transduction& 97& 28.8& 0.013& 0.14&GO:0044106& BP& cellular&amine&metabolic&process& 40& 12.9& 0.019& 0.19&GO:0044459& CC& plasma&membrane&part& 45& 14.3& 0.021& 0.19&GO:0006520& BP& cellular&amino&acid&metabolic&process& 32& 10.6& 0.021& 0.19&GO:0032879& BP& regulation&of&localization& 36& 11.7& 0.022& 0.19&
O:0006140& BP& regulation&of&nucleotide&metabolic&process& 13& 5.0& 0.022& 0.19&GO:0030811& BP& regulation&of&nucleotide&catabolic&process& 13& 5.0& 0.022& 0.19&GO:0033121& BP& regulation&of&purine&nucleotide&catabolic&process& 13& 5.0& 0.022& 0.19&GO:0033124& BP& regulation&of>P&catabolic&process& 13& 5.0& 0.022& 0.19&GO:0043087& BP& regulation&of>Pase&activity& 13& 5.0& 0.022& 0.19&GO:0006793& BP& phosphorus&metabolic&process& 97& 28.3& 0.023& 0.19&GO:0006796& BP& phosphate&metabolic&process& 97& 28.3& 0.023& 0.19&GO:0042578& MF& phosphoric&ester&hydrolase&activity& 42& 13.4& 0.024& 0.19&GO:0016758& MF& transferase&activity,&transferring&hexosyl&groups& 26& 8.8& 0.024& 0.19& && upporting information Supplementary text, methods, figures and references in separate PDF file. Supplementary tables in separate Excel files:
Table& S1:& Results& from& the& branch5site& test& for& positive& selection& applied& on& the& ant&single5copy& orthologs& dataset& (4,256& families).& Family& numbering& as& in& OrthoDB& run&(http://bioinfo.unil.ch/supdata/Roux_positive_selection_ants/orthoDB_run.zip).& Branch&numbering& as& in& Figure& 1.& A& “comparable”& branch& means& that& in& this& particular& gene&family,& the& topology& of& this& particular& branch& is& identical& to& the& species& tree,& and& not&affected&by&missing&genes.&Table& S2:& Results& from& the& branch5site& test& for& positive& selection& applied& on& the& bee&dataset& (2,256& families).& Family& numbering& based& on&
Apis% mellifera & orthologs.& Branch&numbering&as&in&Figure&S5.&Legend&as&Table&S1.&Table& S3:& Results& from& the& branch5site& test& for& positive& selection& applied& on& the& twelve&Drosophila& dataset& (3,749& families).& Family& numbering& based& on&
Drosophila%melanogaster &orthologs.&Branch&numbering&as&in&Figure&S4.&Legend&as&Table&S1.&Table& S4:& SUMSTAT& gene& set& enrichment& test& on& Gene& Ontology& functional& categories&based& on& scores& for& positive& selection& in& 12& Drosophila& using& the& branch5site& test& from&Codeml&(combined&score&for&all&analyzed&branches&of&the&Drosophila&lineage).&Table& S5:& SUMSTAT& gene& set& enrichment& test& on& Gene& Ontology& functional& categories&based& on& scores& for& positive& selection& in& bees& using& the& branch5site& test& from& Codeml&(combined& for& all& analyzed& branches& of& the& bee& lineage,& with& the& exception& of& two& long&basal&branches&leading&to&
Megachile%rotundata &and&
Exoneura%robusta ).&Table&S6:&Gene&Ontology&enrichment&test&based&on&scores&from&the&branch5site&test&from&Codeml&in&ants.&This&is&the&full&table&of&all&GO&categories&tested.&See&Table&3&for&only&the&GO&categories&that&are&significantly&enriched&or&depleted.&Table&S7:&Biases&in&Gene&Ontology&functional&categories&for&genes&present&in&the&dataset&of&bee&single5copy&orthologs&gene&families&(10&species).&Table&S8:&Biases&in&Gene&Ontology&functional&categories&for&genes&present&in&the&dataset&of&single5copy&orthologs&gene&families&(7&ant&species&and&12&species&in&total).& able& S9:& SUMSTAT& gene& set& enrichment& test& on& Gene& Ontology& functional& categories&based& on& scores& for& positive& selection& using& the& branch5site& test& from& Codeml& (an&enrichment&test&is&run&for&each&branch&separately).&Table& S10:& Biases& in& Gene& Ontology& functional& categories& for& genes& present& in& the&extensive& ant& dataset& including& gene& families& that& experienced& duplications& (7& ant&species&and&12&species&in&total).&Table&S11:&Biases&in&Gene&Ontology&functional&categories&for&genes&present&in&the&dataset&of&Drosophila % single5copy&orthologs&gene&families&(12&species).&Table& S12:& SUMSTAT& gene& set& enrichment& test& on& phenotypic& categories& based& on&positive& selection& scores& using& the& branch5site& test& from& Codeml& (an& enrichment& test& is&run&for&each&branch&separately).&&Table& S13:& SUMSTAT& gene& set& enrichment& test& on& Gene& Ontology& functional& categories&based&on&scores&for&positive&selection&in&ants&using&the&site&test&from&Codeml.&Table&S14:&Results&from&the&site&test&for&positive&selection&applied&on&the&mitochondrial&genome&dataset&(13&families).&Table& S15:& Results& from& the& branch5site& test& for& positive& selection& applied& on& the&mitochondrial&genome&dataset&(13&families).&Branch&numbering&as&in&Figure&S3.&Legend&as&Table&S1.&Table& S16:& SUMSTAT& gene& set& enrichment& test& on& sets& of& aging& and& oxidative& stress&differentially&expressed&genes&in& D.%melanogaster ,&based&on&scores&for&positive&selection&using& the& branch5site& test& from& Codeml& (combined& scores& for& all& analyzed& branches& of&the&ant&lineage).&Table& S17:& SUMSTAT& gene& set& enrichment& test& on& sets& of& caste5specific& differentially&expressed&genes&in&
S.%invicta ,&based&on&scores&for&positive&selection&using&the&branch5site&test&from&Codeml&(combined&scores&for&all&analyzed&branches&of&the&ant&lineage).&&Table& S18:& Results& from& the& branch5site& test& for& positive& selection& applied& on& the&olfactory&receptors&dataset.&Clades&and&branches&IDs&are&similar&to&Newick&trees&show&in&Supplementary&text.&Full&phylogenetic&tree&is&displayed&as&Figure&3&and&Figure&S1.&Table&S19:&Results&from&the&site&test&for&positive&selection&applied&on&the&extensive&ant&dataset& (6,186& families).& Family& numbering& as& in& OrthoDB& run&(http://bioinfo.unil.ch/supdata/Roux_positive_selection_ants/orthoDB_run.zip).&Table& S20:& SUMSTAT& gene& set& enrichment& test& on& Gene& Ontology& functional& categories&based& on& scores& for& positive& selection& in& ants& using& the& branch5site& test& from& Codeml& combined& score& for& all& analyzed& branches& of& the& ant& lineage).& Only& results& on& internal&branches&were&considered&(no&leaves).&Table& S21:& SUMSTAT& gene& set& enrichment& test& on& Gene& Ontology& functional& categories&based& on& scores& for& positive& selection& in& ants& using& the& branch5site& test& from& Codeml&(combined& score& for& all& analyzed& branches& of& the& ant& lineage).& We& considered& only&results&on&branches&were&the&rate&of&synonymous&substitutions& d S &was&lower&than&1.&Table& S22:& SUMSTAT& gene& set& enrichment& test& on& Gene& Ontology& functional& categories&based& on& scores& for& positive& selection& in& ants& using& the& branch5site& test& from& Codeml&(combined&score&for&all&analyzed&branches&of&the&ant&lineage).&Only&positive&results&with&identified&sites&on&the&alignment&(BEB&posterior&probability&>&0.9)&were&considered.&Table& S23:& SUMSTAT& gene& set& enrichment& test& on& Gene& Ontology& functional& categories&based& on& scores& for& positive& selection& in& ants& using& the& branch5site& test& from& Codeml&(combined&score&for&all&analyzed&branches&of&the&ant&lineage).&Only&results&where&the&3&independent&runs&of&Codeml&did¬&display&convergence&issues&were&considered.&Table& S24:& SUMSTAT& gene& set& enrichment& test& on& Gene& Ontology& functional& categories&based& on& scores& for& positive& selection& in& ants& using& the& branch5site& test& from& Codeml&(combined&score&for&all&analyzed&branches&of&the&ant&lineage).&Only&branches&where&the&increase&in&G+C&content&at&third&codon&positions&was&lower&than&10%&were&considered.&Table& S25:& SUMSTAT& gene& set& enrichment& test& on& Gene& Ontology& functional& categories&based& on& scores& for& positive& selection& in& ants& using& the& branch5site& test& from& Codeml&(combined& score& for& all& analyzed& branches& of& the& ant& lineage).& Codeml& was& run& on&PAGAN&multiple&sequence&alignments&filtered&with&GUIDANCE.&Table& S26:& SUMSTAT& gene& set& enrichment& test& on& KEGG& pathways,& based& on& scores& for&positive&selection&in&ants&using&the&branch5site&test&from&Codeml&(combined&score&for&all&analyzed&branches&of&the&ant&lineage).&& .4 o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o oo