REVIEW ARTICLEAnemergingexampleoftritrophiccoevolutionbetweenies(Diptera:Fergusoninidae)andnematodes(Nematoda:Neotylenchidae)onMyrtaceaehostplantsLEIGH A.NELSON1,KERRIE A.DAVIES2*,SONJA J.SCHEFFER3,GARYS.TAYLOR4,MATTHEWF.PURCELL5,ROBINM.GIBLIN-DAVIS6,ANDREWH.THORNHILL7andDAVIDK.YEATES11CSIROEcosystemSciences,CluniesRossStreet,Acton,ACT2601,Australia2AustralianCentreforEvolutionaryBiologyandBiodiversity,andSchoolofAgriculture,FoodandWine,TheUniversityofAdelaide,WaiteCampus,PMB1,GlenOsmond,SA5064,Australia3SystematicEntomologyLab,USDA-ARS,10300BaltimoreAv.,Beltsville,MD20705,USA4AustralianCentreforEvolutionaryBiologyandBiodiversity,andSchoolofEarthandEnvironmentalSciences,TheUniversityofAdelaide,NorthTerrace,Adelaide,SA5005,Australia5CSIROEcosystemSciences/USDAARSAustralianBiologicalControlLaboratory,GPOBox2583,Brisbane,Qld4001,Australia6FortLauderdaleResearchandEducationCenter,UniversityofFlorida,IFAS,3205CollegeAv.,FortLauderdale,FL33314,USA7AustralianTropicalHerbarium,JamesCookUniversity,Cairns,Qld4870,AustraliaReceived9September2013;revised14November2013;acceptedforpublication14November2013A uniqueobligatemutualismoccursbetweenspeciesofFergusoninaMallochies(Diptera:Fergusoninidae)andnematodes of the genus Fergusobia Currie (Nematoda: Neotylenchidae). These mutualists together form differenttypes of galls on Myrtaceae, mainly in Australia. The galling association is species-specic, and each mutualism inturndisplays host specicity. This tritrophic systemrepresents acompellingarenato test hypotheses aboutcoevolutionbetweenthehostplants, parasiticnematodesandthefergusoninidies, andtheevolutionof theseintimate mutualisms. We have a basic knowledge of the interactions between the host plant, y and nematode in thissystem, but a more sophisticated understanding will require a much more intensive and coordinated research effort.Summaries of the known Fergusonina/Fergusobia species associations and gall type terminology are presented. Thispaper identiesthekeyadvantagesof thesystemandquestionstobeaddressed, andproposesanumber ofpredictions about the evolutionary dynamics of the system given our understanding of the biology of the mutualists.Future research will protably focus on (1) gall cecidogenesis and phenology, (2) the interaction between the y larvaand the nematode in the gall, and between the adult female y and the parasitic nematode, (3) the means by whichthe y and nematode life cycles are coordinated, (4) a targeted search of groups in the plant family Myrtaceae thathave not yet been identied as gall hosts, and (5) establishment and comparison of the phylogenetic relationships ofthe host plants, y species and nematodes. Recently derived phylogenies and divergence time estimation studies ofthe Diptera and the Myrtaceae show that the y family Fergusoninidae is less than half the age of the Myrtaceae,discountingthehypothesisofcospeciationandcoradiationofthey/nematodemutualismandtheplantsatthebroadest levels. However, cospeciationmayhaveoccurredat shallower levels inthephylogeny, followingtheestablishment of the y/nematode mutualism on the Myrtaceae. 2014 The Linnean Society of London, BiologicalJournal of the Linnean Society, 2014, 111, 699718.ADDITIONAL KEYWORDS: cospeciation COI morphology tritrophic interaction tritrophicspecialization.*Correspondingauthor.E-mail:kerrie.davies@adelaide.edu.aubs_bs_bannerBiologicalJournaloftheLinneanSociety,2014,111,699718.With4gures2014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718 699INTRODUCTIONSpecies of Fergusonina Malloch ies (Diptera:Fergusoninidae) form unique species-specic associa-tions with nematodes of the genus Fergusobia Currie(Nematoda:Neotylenchidae),intheonlyknowncaseof obligate mutualism between nematodes and insects(Giblin-Davis, 1993). First reported by Morgan (1933),the Fergusonina/Fergusobia mutualists togetherform galls on plants of the family Myrtaceae, mainlyinAustralia, although some are fromIndia, NewGuinea, NewZealandandThePhilippines(Harris,1982; Siddiqi, 1986, 1994; Tayloret al., 2007). GallshavebeenrecordedpredominantlyfromEucalyptusspecies, although Angophora, Corymbia, Leptos-permum, Melaleuca, Metrosideros and Syzygiumarealsohosts(Currie, 1937; Tonnoir, 1937; Harris,1982; Siddiqi, 1986, 1994; Giblin-Daviset al., 2004b;Taylor, 2004; Taylor et al., 2007; Taylor &Davies,2008; Davies et al., 2010b; K. A. Davies et al., unpubl.data).Gallscompriseoneormoreseparatechambersorlocules, eachcontaininganindividual ylarvaandassociated nematodes (Giblin-Davis et al., 2004a).The y larvae feed on the cells lining the wall of theirassociatedlocule.The life cycles of the ies and their associatednematodesaresummarizedinFigure 1. Fergusobianematodes apparently induce galls on host plantsvia pharyngeal gland secretions produced duringfeeding on host cells (Currie, 1937; Giblin-Davis et al.,2001b). Fergusoninaiesfacilitatetransporttonewhost plantsandprovidenutritiontotheir parasiticnematodes(e.g.Currie,1937;Fisher&Nickle,1968;Giblin-Davis et al., 2001b; Taylor, Head &Davies,2005; Taylor & Davies, 2008). The system is thereforedescribed as a mutualism. The Fergusobia nematodesof the amphimictic generation mate within thelocules, and fertilized females enter the haemocoel ofthird-instar female Fergusonina larvae (Fig. 1) byan unknown mechanism. The parasitic female nema-tode has a highly modied, absorptive epidermis(Giblin-Davis et al., 2001a). It lays eggs withinthey haemocoel, giving rise to juvenile nematodeswhich are deposited by the y, together with itseggs, intomeristematicplant tissues(Currie, 1937;Giblin-Daviset al.,2001b).The origin of the y/nematode mutualism isunclear. Flies are known to be parasitized by varioustylenchid nematodes, e.g. Howardula and Parasi-tylenchus (Poinar, Jaenike & Shoemaker, 1998).Fergusobia is the only known tylenchid nematodethat has both insect- and plant-parasitic genera-tions(Siddiqi, 2000). However, tylenchidnematodeswith insect- and fungal-parasitic generations areknown(Siddiqi, 2000). Fergusobianematodesrepre-sent a large and potentially ancient radiation.Molecular evidence suggests a single originof theFergusonina/Fergusobiamutualism(Ye et al., 2007;Davies et al., 2010b). Possible scenarios for theevolution of its parasitism were discussed byGiblin-Davis et al. (2003) and Taylor et al. (2005), andfurther phylogenetic studies using suitable molecularmarkers could be used to understand the evolutionaryhistory of this unique relationship. Unfortunately, nomolecular phylogeny is available of the suborderHexatylina, towhichFergusobiabelongs, butwouldgreatly enhance understanding of its evolutionaryrelationships.Much of the research on the Fergusonina/Fergusobia mutualismover the last two decadesstemmed from its application as a potential biocontrolagentofMelaleucaquinquenervia,aseriousweedinFlorida, USA(Goolsby, Makinson&Purcell, 2000;Davies, Makinson & Purcell, 2001; Giblin-Daviset al., 2001b; Scheffer et al., 2004, 2013; Taylor, 2004;Center et al., 2011; Pratt et al., 2013). Morerecentresearch has taken advantage of these detailedstudiesandhasaddressedthebroaderevolutionaryIN FLYIN GALLParasitic EggsJuvenilesJuvenilesParthenogenetic Amphimictic +Fertilised preparasitic Nematode FlyEggs1st2nd3rdLarval instarsPupaeEggsOvipositionNematodes into larvaeFigure 1. Life cycles of Fergusonina ies and Fergusobianematodes. Femaleiesdepositeggsandjuvenilenema-todes intomeristematic plant tissue. Thephytophagousnematodesdepositedbytheydevelopintoparthenoge-netic females, which lay eggs in the gall. These eggs developinto phytophagous male and female nematodes, whichmate. By the time the y eggs hatch, adult male nematodesarepresent inthegall; femalesdeveloplater. Fertilizedpre-parasiticfemalenematodesenterthird-instarfemaley larvae and become entomoparasitic. There are three ylarval instars. During pupation (or following emergence ofthe y), female parasitic nematodes lay fertilized eggs intoy haemolymph. Juvenile nematodes hatch and move to yoviducts, awaiting oviposition.700 L.A.NELSONET AL.2014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718andcoevolutionaryaspectsof thistritrophicmutua-listic system(Ye et al., 2007; Davies et al., 2010b).Furthercollectinghasalsoextendedtheplant hostrange (Davies et al., 2010b), andmolecular genetictechniqueshavebeenusedtofurther elucidatethebiology of the system(Scheffer et al., 2004, 2013).Synthesizing this information, we make some predic-tionsaboutcoevolutionarypatternsandprocessesinthesystem.GALLTYPESSpecic pairs of Fergusonina ies and Fergusobianematodes cause different gall types on differentplant tissues. There are ve primary gall types basedon the location of the gall, and each of these types isdividedintounilocularversusmultilocularforms.Inaddition, owerbudgallersmayattackthestigma,stamenor ovaryof the host plant. The most com-monlycollectedgalls, becausetheyareeasilyseen,are large multilocular galls developing from terminalleafbuds. Themorecrypticuniloculargalls, occupy-ing positions such as small axial buds, may be under-represented in collections. In an effort to standardizeand normalize the terminologies used to delineategall types in the literature, a summary has beenprepared(Table 1),andcommongallformsareillus-tratedinFigure 2.Several gall typesweredenedbyCurrie(1937),andre-examinedbyTayloret al. (2005). Sincethen,histological study has revealed that differences in galltypesaredeterminedbytheplacement andtimingof oviposition by Fergusonina ies (Giblin-Daviset al., 2004a). Only a fewFergusonina/Fergusobiapairshavebeenfoundgallingmorethanonetissue.Examples are Fergusonina lockharti, which producesbothmultilocular axial andterminal leaf budgallson Eucalyptus camaldulensis (Taylor & Davies,2010), andF. turneri, whichproducesgallsonbothshootsandowerbudsonMelaleucaquinquenervia(Goolsby, Makinson & Purcell, 2000). Other separateFergusonina/Fergusobiaspecies have beencollectedfrom the stigma, stamen and ovary of ower bud galls(Giblin-Daviset al.,2004a).Molecular phylogenies so far indicate that whilegall type is conserved among some closely relatedFergusobia clades, gall types have evolved more thanonce(Yeet al.,2007;Davieset al.,2010b).Thekindsand frequencies of transitions between gall typesshouldbeassessedwhenaphylogenyof theiesisavailablethat includesreliableinformationonhostspeciesandgalltype.FLYANDNEMATODEDIVERSITYThe specicity of the mutualismto different hostspecies, and host tissue types, implies that there maybe many hundreds of Fergusonina/Fergusobia speciespairs across the Myrtaceae. Allopatric mutualistsare also knownindifferent parts of the range ofwidespread host species such as E. pauciora andE. camaldulensis (Fisher &Nickle, 1968; Taylor &Davies, 2010; Nelson, Scheffer &Yeates, 2011a, b;Davies et al., 2012a). This is a signicant case ofcryptic diversity inthe Australasianentomologicaland nematological fauna (Austin et al., 2004; Raven &Yeates,2007;Hodda&Nobbs,2008).Gaining insight into the Fergusonina/Fergusobiasysteminvolves the combinedanalysis of all threetrophiclevelsinvolved. However, obtaininginforma-tiononall threecomponents, namelytheidentica-tion and characterization of each species in theTable 1. A summaryofgalltypesinducedbytheFergusonina/FergusobiamutualismonMyrtaceaeOrgan Location Loculetype Gallform SchematicofcommongalllocationsLeaf Petiole(A) UnilocularMultilocularFlat(B) UnilocularMultilocularShootbud Axillary(C) UnilocularMultilocularTerminal(D) MultilocularFlowerbud(G)Stigma UnilocularStamen(primordialtissue)OvaryTRITROPHICCOEVOLUTIONBETWEENFLIES ANDNEMATODES 7012014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718tritrophic plant/nematode/y interaction, poses dis-tinct challenges. To date, records have been collectedforabout200tritrophicassociations(Tables 2and3;G.S.TaylorandK. A.Davies,unpubl.data).Descriptionof a Fergusobianematode is usuallyundertakenonlywhenalltaxonomicallyinformativestages of the life cycle have been obtained. Nematodesare only found in locules when eggs or the y larvaearepresent. Obtainingthetaxonomicallyimportantlife stages for the Fergusoninaies (larvae, pupaeandadult) iseasierthanfortheFergusobianema-todes. Theylarvaeandpupaeremaininthegallsforlonger, andprovidedthat third-stagelarvaearepresent, adult ies canusuallyberearedfromthegalls. The cuticular dorsal shieldis unique amongDipteralarvae. Itvariesbetweenspecies, fromcom-prising a few raised spicules (being almost absent) totransverse rows of raised, sclerotized spicules to largeplates with ridges or comb-like processes (Currie,1937; Taylor et al., 2005). Large multilocular galls canbe partially dissected to remove larvae and/or pupae,leaving the remainder of the gall intact for emergenceofadultspecimens.OftenaFergusonina/Fergusobiagallisfoundonamyrtaceous host plant, but insufficient material isavailable for positive identication of the plant, y ornematode species (Davies et al., 2010b). Of the 38described Fergusonina species (Currie, 1937; Fisher &Nickle, 1968; Siddiqi, 1986, 1994; Davies &Lloyd,1996; Davies &Giblin-Davis, 2004; Taylor, 2004;Taylor et al., 2007; Taylor &Davies, 2008, 2010;Nelson, Scheffer &Yeates, 2011a, b; Davies et al.,2012a, b; Purcellet al., 2013), only 14 have completerecords for corresponding myrtaceous host, gall type,y larval dorsal shield morphology and a namedFergusobia nematode (Table 2). However, informationFigure 2. Representative Fergusonina/Fergusobia gall forms. A, leaf pea galls (unilocular) from E. pauciora; B, shootbud gall from M. dealbata; C, axial leaf bud gall from E. camaldulensis; D, at leaf gall from E. leucoxylon; E, ower budgall from E. microcarpa; F, leafy leaf bud gall from E. aromaphloia; G, terminal leaf bud gall from E. obliqua. All gallsexcept A aremultilocular.NoteexitholesinCandG.Scalebars = 1 cm.702 L.A.NELSONET AL.2014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718Table2.SummaryofdescribedFergusoninaiesandFergusobianematodes,theirmyrtaceoushost,galltype,distributionbasedoncollectionrecordsandlarvaldorsalshieldtypeFergusoninasp.MyrtaceoushostGalltypeDistributionDorsalshieldFergusobiasp.atricornisMalloch1925UnknownUnknownSydney(NSW)Canberra(ACT)UnknownUnknownbisetaMalloch1932C.maculata(Malloch,1932;Morgan,1933)Flower-budBodalla(NSW)UnknownUnknownbrimblecombiTonnoir1937E.melanophloia(Tonnoir,1937)E.crebra(poss.)(Tonnoir,1937)E.odorata(Currie,1937)E.hemiphloia(Currie,1937)Flower-budCanberra(ACT)&QldQldSAVic2hookswithscoop-likeprojectionsrisingfrombaseUnknownburrowsiTaylor2004M.viridiora(Taylor,2004)Nodular,terminaloraxialshootbudNorthcoastalQld5broadblacksclerotizedplatesviridioraeDavies&Giblin-Davis2004carteriTonnoir1937E.bridgesiana(Currie,1937;Tonnoir,1937)E.amygdalina(Tonnoir,1937)Euc.spp.(Tonnoir,1937)leafgallsleafyleafbudCanberra(ACT)&Southerntablelands(NSW)Emerald(Vic)Adelaide(SA)2patchesofheavilysclerotizedcuticletumifaciensCurrie1937(onE.bridgesiana)centeriTaylor2004M.leucadendra(Taylor,2004)Nodular,terminaloraxialshootbud;shinyCairns(Qld)Cardwell(Qld)5broadblacksclerotizedplates;rstanterior2conuentleucadendraeDavies&Giblin-Davis2004currieiTonnoir1937E.macrorryncha(Tonnoir,1937)LeafbudCanberra(ACT)Approx.6separatetransversesclerotizedbandsUnknowndavidsoniTonnoir1937Eucalyptusspp.UnknownAdelaide(SA)UnknownUnknowndaviesaeNelson&Yeates2011E.paucioraspecies-groupTerminalleafbudFollowshigh-elevationsnowgumdistribution9separatetransversesclerotizedbandsUnknowneucalyptiMalloch1932C.maculata(Malloch,1932;Morgan,1933;Currie,1937)FlowerbudBodalla&BatemansBay(NSW)4sclerotizedplates,withrstandsecond,andthirdandfourth,conuenttoform2almond-shapedareasUnknownevansiTonnoir1927E.meliodora(Tonnoir,1937)Euc.spp.(Currie,1937)leafgallsCanberra(ACT)Adelaide(SA)TwosclerotizedplatesUnknownavicornisMalloch1925E.camaldulensis(Tayloretal.,1996)TerminalleafbudWA,SA,Vic.&NSWPlatewith3,mostly4andrarely5anterior-projectingprongssimilartoF.lockhartiUnknownfrenchiTonnoir1937E.amygdalina(Tonnoir,1937)leafgallsEmerald(Vic)UnknownUnknowngiblindavisiTaylor2008C.ptychocarpa(Taylor&Davies,2008)FlowerbudEasternAustraliaBrisbane(Qld)Cairns(Qld)Raisedsclerotizedspiculesonthe2ndand3rdthoracicsegmentsptychocarpaeDavies2008goolsbyiTaylor2004M.nervosa(Taylor,2004)Rosettegallatbaseofaxialshootbuds,=basalgallsofaxialshootbudsNorthcoastalQldMareeba(Qld)3bands2ofthemconuent(blendedinto1)Fb.species1(Davies&Giblin-Davis,2004)TRITROPHICCOEVOLUTIONBETWEENFLIES ANDNEMATODES 7032014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718Table2.ContinuedFergusoninasp.MyrtaceoushostGalltypeDistributionDorsalshieldFergusobiasp.greavesiCurrie1937E.polyanthemos(Currie,1937)stem-tipCanberra(ACT)4chitinousplatesintwopairsUnknowngurneyiMalloch1932C.maculata(Malloch,1932;Morgan,1933)FlowerbudBatemansBay(NSW)UnknownUnknownlockhartiTonnoir1937E.rudis(Tonnoir,1937)E.camaldulensis(Taylor&Davies,2010)leafgallTerminalleafAxialshootbudMundaring(WA)SouthQLD,VIC,SAandWA.Blacksclerotizedplatewith35anteriorprojectingteethwithraisedsclerotizedspiculesbrittenaeDavies2010(onE.camaldulensis)makinsoniTaylor2004M.nervosa(Taylor,2004)Terminaloraxialshootbudgalls;denselyconvolutednelypubescentNorthernQld7broadblacksclerotizedplatesdealbataeDavies&Giblin-Davis2004metrosiderosaeTaylor2007Metrosiderosexcelsa(Tayloretal.,2007)UnilocularshootbudgallNewZealandSmallwithabout20sparseraisedsclerotizedspiculespohutakawaDavies2007microceraMalloch1924UnknownUnknownSydney(NSW)(Malloch,1932)UnknownUnknownmorganiTonnoir1937E.hemiphloia(Tonnoir,1937)FlowerbudVicUnknownUnknownnewmaniTonnoir1937E.gomphocephala(Tonnoir,1937)Smallpea-likeuniloculargallsonyoungstemsandleafbudsPerth(WA)HeavilysclerotizedwithtwoorthreeteethgomphocephalaeDavies2012anicholsoniTonnoir1937E.macrorryncha(Tonnoir,1937)FlowerbudCanberra(ACT)Clare(SA)HeavilysclerotizedwithplateslackingteethjuliaeDavies2012omlandiNelson&Yeates2011E.paucioraspecies-group(Nelson&Yeates,2011)TerminalleafbudFollowslow-elevationsnowgumdistribution7(sometimes8)separatetransversesclerotizedbandsUnknownpescottiTonnoir1937E.amygdalina(Tonnoir,1937)leafgallEmerald(Vic)UnknownUnknownpurcelliTaylor2004M.nervosa(Taylor,2004)Nodular,terminaloraxialshootbudgallswithnepubescenceCoastalnorthQld5broadblacksclerotizedplatescajuputiaeDavies&Giblin-Davis2004schefferaeTaylor2004M.nervosa(Taylor,2004)Nodular,terminaloraxialshootbudgallswithnedensepubescenceCoastalnorthQld5narrowblacksclerotizedplatesnervosaeDavies&Giblin-Davis2004scutellataMalloch1925UnknownUnknownSydney(NSW)UnknownUnknownsyzygiiHarris1982Syzygiumcumini(Siddiqi,1986)AxillarybudIndiaSeveralpatchesofsclerotizedcuticlewithpatternsofraisedridgesandspiculesjambophilaSiddiqi1986tayloriNelson&Yeates2011E.paucioraspecies-groupTerminalleafbudFollowshigh-elevationsnowgumdistribution9separatetransversesclerotizedbandsUnknownthomasiTaylor2008C.abbreviata(Taylor&Davies,2008;Daviesetal.,2010a)FlowerbudKimberleyRegion(WA)UnknownUnknownthornhilliNelson&Yeates2011E.dalrympleana(Nelson&Yeates,2011)TerminalleafbudAbercrombieRiver(NSW)Onlyknownfrompupa:2patchesheavilysclerotizedcuticleUnknowntillyardiTonnoir1937E.blakelyi(Tonnoir,1937)E.camaldulensis(Tonnoir,1937)E.tereticornis(Currie,1937)FlowerbudCanberraNaracoorte(SA)Vic.(Currie,1937)Heavilysclerotized,withraisedspiculesand78teethcurrieionE.camaldulensis)(Fisher&Nickle,1968)704 L.A.NELSONET AL.2014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718turneriTaylor2004M.quinquenervia(Davies&Giblin-Davis,2004)M.uviatilis(Davies&Giblin-Davis,2004)TerminaloraxialshootbudandowerbudCoastalQldNorthcoastalNSW6narrowblacksclerotizedplatesquinquenerviaeDavies&Giblin-Davis2004williamsensisNelson&Yeates2011E.baxterispeciescomplexTerminalleafbudGrampians(Vic)8separatetransversesclerotizedbandsUnknownUnknownC.tessellaris(Siddiqi,1986;Daviesetal.,2010a)TerminalandaxialbudstemgallsCoastalQldShieldrestrictedtoabroadareaofweaklysclerotizedspiculesalonganteriormarginTS2magnaSiddiqi1984sensuDavies2010UnknownE.leucoxylon(Davies&Lloyd,1996)FlatleafAdelaide(SA)2patchesheavilysclerotizedcuticlesheriDavies&Lloyd1996UnknownE.deglupta(Siddiqi,1994;Daviesetal.,2010b)FlowerbudBulolo(PNG)UnknownbrevicaudaSiddiqi1994UnknownE.deglupta(Siddiqi,1994;Daviesetal.,2010b)FlowerbudPhilippinesUnknownphilippinensisSiddiqi1994UnknownFoundinsoil(Siddiqi,1986)n/aIndiaUnknownindica(Jairajpuri1962)Siddiqi1986UnknownE.eugenioides(Daviesetal.,2012b)FlowerbudCanberra(ACT)3conuentpatchesofsclerotizedcuticleeugenioidaeDavies2012UnknownE.fasciculosae(Daviesetal.,2012b)StigmagallGoolwa(SA)CuticularplateofAS1conuentwithshortbroadplateonAS2andaheavilysclerotizedplateonTS1;2rowsof810shortteethonAS1fasciculosaeDavies2012UnknownE.brosa(Daviesetal.,2012b)FlowerbudShuteHarbour(QLD)3conuentcuticularplates,with2forwardlyprojectingteethandaridgeonposteriormarginofAS2morrisaeDavies2012UnknownE.camaldulensis(Daviesetal.,2012a)AxialbudstemgallsAdelaide(SA)BarsofspiculescamaldulensaeDavies2012UnknownCorymbiasp.(Daviesetal.,2012a)UnilocularleafpeagallsWoodburn(NSW)AbsentrileyiDavies2012UnknownE.microcarpa(Daviesetal.,2013b)FlatleafgallAdelaide(SA)2patchesheavilysclerotizedcuticlemicrocarpaeDavies2013UnknownE.porosa(Daviesetal.,2013b)FlatleafgallStrathalbyn(SA)2patchesheavilysclerotizedcuticleporosaeDavies2013UnknownE.cosmophylla(Daviesetal.,2013a)LargeleafbudgallMylor(SA)3conuentcuticularplateswith6to9shortforwardlyprojectingteethcosmophyllaeDavies2013UnknownE.delegatensis(Daviesetal.,2013a)LargeleafbudgallBenLomond(TAS)Sevenor8broadtransverserowsofsclerotizedraisedspiculesdelegatensaeDavies2013UnknownE.diversifolia(Daviesetal.,2013a)LargeleafbudgallMeningie(SA)Eightor9broadtransverserowsofsclerotizedraisedspiculesdiversifoliaeDavies2013UnknownAngophoraoribunda(Daviesetal.,2013a)LargeleafbudgallMudgee(NSW)AbsentoribundaeDavies2013UnknownE.tereticornis(Daviesetal.,2013a)LargeleafbudgallSydney(NSW)HeavilysclerotizedwithtwoorthreeteethminimusLisnawita2013UnknownAngophoranr.woodsiana(Daviesetal.,2013a)LargeleafbudgallPimpama(QLD)AbsentpimpamensisDavies2013*AS1,AS2=abdominalsegments1,2.**TS1,TS2=thoracicsegments1,2.TRITROPHICCOEVOLUTIONBETWEENFLIES ANDNEMATODES 7052014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718Table 3. A listofFergusoninaeucalypthostssortedbythetaxonomictreatmentofBrooker,Slee&Connors(2006)Species Subgenus Section Subsection SeriesAngophoracostata AngophoraAngophoraoribunda AngophoraAngophorasubvelutina AngophoraCorymbiatrachyphloia Corymbia ApteriaCorymbiapapuana Corymbia Blakearia(ghostgums)Corymbiatessellaris Corymbia Blakearia(ghostgums)Corymbiatorelliana Corymbia CadagariaCorymbiacitriodora Corymbia Politaria(spottedgums)Corymbiamaculata Corymbia Politaria(spottedgums)Corymbiaabbreviata Corymbia Rufaria(redbloodwoods)Corymbiagummifera Corymbia Rufaria(redbloodwoods)Corymbiaintermedia Corymbia Rufaria(redbloodwoods)Corymbiaptychocarpa Corymbia Rufaria(redbloodwoods)Eucalyptusacmenoides Eucalyptus Amentum(whitemahoganies)Eucalyptusamygdalina Eucalyptus Aromatica(peppermints)Eucalyptuscoccifera Eucalyptus Aromatica(peppermints)Eucalyptuselata Eucalyptus Aromatica(peppermints)Eucalyptusnitida Eucalyptus Aromatica(peppermints)Eucalyptustenuiramis Eucalyptus Aromatica(peppermints)Eucalyptusbaxteri Eucalyptus Capillulus(stringybarks)Eucalyptuseugenioides Eucalyptus Capillulus(stringybarks)Eucalyptusligustrina Eucalyptus Capillulus(stringybarks)Eucalyptusmacrorhyncha Eucalyptus Capillulus(stringybarks)Eucalyptusdelegatensis Eucalyptus CineraceaeEucalyptushaemastoma Eucalyptus CineraceaeEucalyptuspauciora Eucalyptus CineraceaeEucalyptusracemosa Eucalyptus CineraceaeEucalyptussieberi Eucalyptus CineraceaeEucalyptusobliqua Eucalyptus Eucalyptus(theashes)Eucalyptusstricta Eucalyptus Eucalyptus(theashes)Eucalyptusplanchoniana Eucalyptus InsolitaeEucalyptusmarginata Eucalyptus Longistylus ArboreaeEucalyptusdiversifolia Eucalyptus Longistylus FruticesEucalyptusstellulata Eucalyptus LongitudinalesEucalyptusolsenii Eucalyptus NebulosaEucalyptuscloeziana IdiogenesEucalyptuscoolabah Symphyomyrtus Adnataria(theboxesandironbarks) Apicales AquilonaresEucalyptuspruinosa Symphyomyrtus Adnataria(theboxesandironbarks) Apicales AquilonaresEucalyptusalbens Symphyomyrtus Adnataria(theboxesandironbarks) Apicales BuxealesEucalyptusintertexta Symphyomyrtus Adnataria(theboxesandironbarks) Apicales BuxealesEucalyptuslargiorens Symphyomyrtus Adnataria(theboxesandironbarks) Apicales BuxealesEucalyptusmicrocarpa Symphyomyrtus Adnataria(theboxesandironbarks) Apicales BuxealesEucalyptusmoluccana Symphyomyrtus Adnataria(theboxesandironbarks) Apicales BuxealesEucalyptusodorata Symphyomyrtus Adnataria(theboxesandironbarks) Apicales BuxealesEucalyptuspolybractea Symphyomyrtus Adnataria(theboxesandironbarks) Apicales BuxealesEucalyptuspopulnea Symphyomyrtus Adnataria(theboxesandironbarks) Apicales BuxealesEucalyptusporosa Symphyomyrtus Adnataria(theboxesandironbarks) Apicales BuxealesEucalyptuscrebra Symphyomyrtus Adnataria(theboxesandironbarks) Apicales SiderophloiaeEucalyptusbrosa Symphyomyrtus Adnataria(theboxesandironbarks) Apicales SiderophloiaeEucalyptusmelanophloia Symphyomyrtus Adnataria(theboxesandironbarks) Apicales SiderophloiaeEucalyptussiderophloia Symphyomyrtus Adnataria(theboxesandironbarks) Apicales SiderophloiaeEucalyptusbaueriana Symphyomyrtus Adnataria(theboxesandironbarks) Terminales HeterophloiaeEucalyptusfasciculosa Symphyomyrtus Adnataria(theboxesandironbarks) Terminales HeterophloiaeEucalyptuspolyanthemos Symphyomyrtus Adnataria(theboxesandironbarks) Terminales HeterophloiaeEucalyptusleucoxylon Symphyomyrtus Adnataria(theboxesandironbarks) Terminales MelliodoraeEucalyptusmelliodora Symphyomyrtus Adnataria(theboxesandironbarks) Terminales MelliodoraeEucalyptussideroxylon Symphyomyrtus Adnataria(theboxesandironbarks) Terminales MelliodoraeEucalyptusyalatensis Symphyomyrtus Bisectae Destitutae(pithglandsabsent) SubulataeEucalyptuszopherophloia Symphyomyrtus Bisectae Glandulosae(pithglandspresent) Accedentes706 L.A.NELSONET AL.2014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718ongall type, host plant species and dorsal shieldmorphology of the third-instar y larva provides con-siderable information on which Fergusonina/Fergusobia pair is present within a given gall (Davieset al.,2010b).PLANTHOSTDIVERSITYA summary of plant host records for the Fergusonina/FergusobiamutualismonMyrtaceae was givenbyDavieset al. (2010b), andisupdatedhere(Table 3).Conrmed Fergusonina/Fergusobia galls have beenreported frommore than 80 species of eucalypts(Angophora, Corymbia and Eucalyptus), 12 Melaleuca,one Metrosideros, two Leptospermum and twoSyzygium species. These plant taxa represent only ve(Melaleuceae, Eucalypteae, Syzygieae, Leptospermeaeand Metrosidereae) of the 17 Myrtaceae tribes inWilsons (2011) classication.Figure 3, adapted from Thornhill et al. (2012),shows the relationships between these ve tribes, andthe divergence times between them. A number of veryTable 3. ContinuedSpecies Subgenus Section Subsection SeriesEucalyptusplatypus Symphyomyrtus Bisectae Glandulosae(pithglandspresent) ErectaeEucalyptusloxophleba Symphyomyrtus Bisectae Glandulosae(pithglandspresent) LoxophlebaeEucalyptusgomphocephalaSymphyomyrtus BolitesEucalyptuslesouei Symphyomyrtus Dumaria RuspermaeEucalyptusblakelyi Symphyomyrtus Exsertaria(redgumsandwhitegums)ErythroxylonEucalyptusdealbata Symphyomyrtus Exsertaria(redgumsandwhitegums)ErythroxylonEucalyptustereticornis Symphyomyrtus Exsertaria(redgumsandwhitegums)ErythroxylonEucalyptuslockyeri Symphyomyrtus Exsertaria(redgumsandwhitegums)PhaeoxylonEucalyptuscamaldulensis Symphyomyrtus Exsertaria(redgumsandwhitegums)RostrataeEucalyptusrudis Symphyomyrtus Exsertaria(redgumsandwhitegums)SingularesEucalyptuscupularis Symphyomyrtus Exsertaria(redgumsandwhitegums)Subexsertae(whitegums)Eucalyptuscosmophylla Symphyomyrtus IncognitaeEucalyptusrobusta Symphyomyrtus Latoangulatae Annulares(redmahoganies)Eucalyptusinterstans Symphyomyrtus Liberivalvae(redgums;discfreefromtheovaryroof)EucalyptusparramattensisSymphyomyrtus Liberivalvae(redgums;discfreefromtheovaryroof)Eucalyptusbridgesiana Symphyomyrtus Maidenaria Euryotae BridgesianaeEucalyptusglobulus Symphyomyrtus Maidenaria Euryotae GlobularesEucalyptusjohnstonii Symphyomyrtus Maidenaria Euryotae SemiunicoloresEucalyptusdalrympleana Symphyomyrtus Maidenaria Euryotae ViminalesEucalyptusviminalis Symphyomyrtus Maidenaria Euryotae ViminalesEucalyptusaromaphloia Symphyomyrtus Maidenaria Triangulares AcaciiformesEucalyptusnicholii Symphyomyrtus Maidenaria Triangulares AcaciiformesEucalyptusaggregata Symphyomyrtus Maidenaria Triangulares Foveolatae(swampgums)Eucalyptusovata Symphyomyrtus Maidenaria Triangulares Foveolatae(swampgums)Eucalyptusmannifera Symphyomyrtus Maidenaria Triangulares MicrocarpaeEucalyptusbrevifolia Symphyomyrtus PlatyspermaEucalyptusconuens Symphyomyrtus PlatyspermaEucalyptuscladocalyx Symphyomyrtus SejunctaeTRITROPHICCOEVOLUTIONBETWEENFLIES ANDNEMATODES 7072014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718diversetribesof Myrtaceaearenot yet recordedashostsof theFergusonina/Fergusobiamutualismandit is likely that only a fraction of the diversity of theassociationisknown. Forexample, onlytwoof 632speciesof Syzygiumhavebeenrecordedwithgalls,and a systematic survey of the genus would probablyreveal many more host species. Syzygium is an impor-tant component of Old World tropical rainforestora (Biffin et al., 2006), and its species richness andlineage diversity are centred in the Australasianregion. New Guinea has about 200 species ofSyzygium, Malaya 200 species and Borneo 150species (Biffin et al., 2006) but Australia and theSouthPacicarerelativelyspecies-poor. Fergusobiajambophila is recorded as galling shoot buds onS. cumini in India (Siddiqi, 2000). However, nofergusoninidies were foundinasurveyof fruitscollectedfromS. paniculatumorS. australeinNewSouthWales(Juniper&Britton,2010).Furthermore, Leptospermeae and its sister tribeChamelaucieae occur over much of Australia, yet onlytwo Leptospermumspecies are recorded as hosts.Other Myrtaceae tribes such as Backhousieae,Tristanieae, Kanieae, Syncarpieae, Lophostemoneaeand Xanthostemoneae could also be hosts. Thesetribesoccurintropicalorwetforestsof Australasia.If gallsoccuronanyof theserainforest tribestheymay provide important insights into the evolutionarydevelopmentofthemutualism.TheFergusonina/Fergusobiamutualismhas mostcommonlybeenfoundonAngophora, CorymbiaandEucalyptus, includingthetwolargest sub-generaofthe last named. There are some eucalypt groups,however, withfewor no species recordedas hosts(see Fig. 3andTable 3). Inparticular, none of the22 species of the Eucalyptus sub-genus Eudesmiahas beenrecordedas a host andonly four of the250species intheEucalyptus sub-genus Symphyo-Figure 3. Chronogramof the tribes of Myrtaceae adjusted fromThornhill et al. (2012). Shaded tribes highlightFergusonina hosts and show that no sister tribes in Myrtaceae have been found to host the ies. The gures next to eachtribe name indicate how many species have been recorded as hosts versus the number of species recognized in the tribe.*TribesthatdonothaveanyrepresentativetaxainAustralia.708 L.A.NELSONET AL.2014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718myrtus section Bisectae. Some unidentied physi-ological characteristics (e.g. semiochemicals, leaftoughness) of thesecladesmaypreventcolonizationby Fergusonina/Fergusobia. On the other hand,some eucalypt groups seem to favour the mutualism,suchas the Eucalyptus sub-genus Symphyomyrtussections Exsertaria (red gums), Adnataria (boxes andironbarks) and Maidenaria, and the sub-genus Euca-lyptussectionCineraceae(peppermints)(Table 3).GALLPHENOLOGYMature galls of most well-studied Fergusonina/Fergusobia mutualisms generally appear to occurannually, oftenduringthecoolerwinterandspringmonths, which in southernAustralia may also bewetter. In a two-year study of F. turneri onM. quinquenvervia in northern New South Wales andsouth-eastern Queensland, Australia, populationswerefoundtofollowanannualcyclecorrelatedwithtemperature and bud density, but not rainfall(Goolsbyet al., 2000). Gall numberswerehighestinthecoolerwintermonthsofAugustandSeptember,whennewbudgrowthis abundant. InSouthAus-tralia, the phenology of E. camaldulensis wasobservedover a2-year period, andthedensitiesofthree gall forms developing onthat host was alsohighly seasonal (Head, 2008). Greatest density ofgrowing points, axial leaf buds (galled by Fergusoninasp. withFb. camaldulensae)andowerbuds(galledby F. tillyardi with Fb. curriei) occurred in mid-wintertospring(JulytoSeptember). Incontrast, mostter-minal bud galls (F. lockharti with Fb. brittenae) werefound from mid-spring to summer (October to Febru-ary) (Head, 2008). The occurrence of Fergusoninagalls on high-elevation snow gums was also seasonal,withmaturegallsoccurringinspring(SeptembertoNovember) (Nelsonet al., 2011b). Thespatial distri-butionof gallswithinahosttreeisaffectedbythedistributionof suitable meristematic tissues (grow-ing points, axial leaf and ower buds). DifferentFergusoninaspeciesdifferintheirpreferredlocationon host trees. In a study of galling species onE. camaldulensis inSouthAustralia, most terminalleafbudgallsoccurredonthenorthernandeasternquadrants of trees, while axial leaf budgalls andower bud galls occurred more in western and south-ernquadrants(Head,2008).Thegallsof someFergusonina/Fergusobiamutua-lisms appear to be less seasonal and/or less abundantthanothers. Obviously, speciesmayvaryinlifehis-tories and abundances because of any number ofecological andevolutionaryfactors, but somevaria-tion could be due to climactic aseasonality and/orextremes caused either directly or indirectly througheffects onhost plants. Inaddition, the prevalenceand seasonality of species-specic (or gall-specic)parasitoids (generallyHymenoptera) andinquilines(generally Lepidoptera) attacking the fergusoninidsand their galls could inuence species abundances aswell as lead to natural selection on seasonality(Currie,1937;Taylor,Austin&Davies,1996).CECIDOGENESISTheprocessof gall formation, orcecidogenesis, isaneoplastic outgrowthof plant tissueas adefensivemechanismfollowingherbivory(Schick&Dahlsten,2003). It involves complex interactions betweenplantsandcausativeorganisms, withhighlyspecicreciprocal adaptationsbetweenplant host andgall-inducer (e.g. Stone & Schnrogge, 2003; Raman,2011). The chemical interactions between gall-forming insects and their host plants are poorlyunderstood (Raman, 2010). Gall-forming insectscommonly show preference for undifferentiatedmeristematic plant tissue as ovipositionsites (e.g.Mani, 1964; Fritz et al., 1987; Price, Fernandes &Waring, 1987; Raman, 2010). Littleisknownabouthow Fergusonina ies select an oviposition site.Theirovipositionbehaviourmaybeinuencedbyacombinationof visual and olfactory cues includingsemiochemicals and plant hormones (auxins, e.g.indole-aceticacid), whichareparticularlyprevalentin meristematic tissue (Raman, 2010). Ovipositionpreference inthe shoot y (Atherigonasoccata) inseedlingsorghumispositivelycorrelatedwithnitro-gencontentofthegrowingtip(Ogwaro&Kokwaro,1981). Most Fergusonina/Fergusobia galls occuronyoungfoliage, presumablywithhigher nitrogencontent (Larsson & Ohmart, 1988; Edwards &Wanjura,1991).Experimental injection of juvenile Fergusobianematodes into shoot buds (Giblin-Davis et al., 2001b)ledtosomegall andnematodedevelopment. Thus,the nematode appears to induce gall development, buttheylarvaisresponsiblefortheinternalstructureof thegall. Nematodesfailedtodevelopinvitrooncallus culture (Head, 2008). Given their strong stylet,itismostlikelythatFergusobianematodesfeedonplant material (Giblin-Davis et al., 2001b, 2004a),although Currie (1937) suggested that they may alsofeedonexcretionsoftheco-occurringylarvae.In recent years, considerable efforts have beenmade to understand the process of cecidogenesisin gall-forming pest nematodes. All plant-parasiticnematode genomes examined to date (e.g. Bert et al.,2008; Bird, Williamson&Abad, 2009; Dieterich&Sommer, 2009; van Megen et al., 2009; Kikuchi et al.,2011) havegenes codingfor secretedenzymes thatdegradecell walls, whichmayhaveoriginatedfromhorizontally transferred bacterial or fungal genesTRITROPHICCOEVOLUTIONBETWEENFLIES ANDNEMATODES 7092014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718(Smantet al., 1998; Blaxter, 2007; Davis, Hussey&Baum, 2009; Kikuchi et al., 2011). Understanding theprocess of cecidogenesis inpest nematodes suchasthe cyst and root-knot nematodes will provideamolecular model against whichgall-formationinFergusonina/Fergusobiacanbe examinedandcom-pared. Like Fergusobia, these pest nematodes aretylenchids. However, phylogenetic analyses suggestthat gall-formation has evolved independently onmorethanoneoccasionwithinthetylenchids(Bertet al., 2008; vanMegenet al., 2009), and differingmechanismsofcecidogenesismayoccur.The role of the dorsal shield of the larvalfergusoninids in cecidogenesis is unknown. It is prob-ably used as a mechanical scraper within gall locules(Taylor, 2004), expandingthelocule, possiblydeter-mininginternal gall architecture, and/or producingpelletized food material for the y larvae and/ornematodes (Giblin-Davis et al., 2004b). Plant materialisoftenseenwedgedunderthehooksoflarvaepos-sessingthem.COORDINATIONOFFLY/NEMATODELIFECYCLESClearly, the life cycles of the y and the nematode arecloselycoordinated(Fig. 1). Thecueswhichregulatethis coordination and its evolution are unknown, butmay be hormonal. For example, in the beetle parasiteContortylenchus brevicomi, fourth-stage juvenilenematodes were stimulated to moult while within thebodycavityof thehost larva(Gibb&Fisher, 1986,1989), but ecdysiswasinhibitedbyhighconcentra-tions of juvenile hormone in the host, and nematodesmoultedoncetheyleft thelarva. Fergusobianema-todes parasitic in the pupae of the ies may bestimulated to moult by the highconcentrations ofecdysteroidknowntobepresentindipteranpuparia(Walker & Denlinger, 1980), which could be tested invitro.Between one and 50 juvenile nematodes were asso-ciated with Fergusonina eggs deposited in ower buds(Giblin-Davis et al., 2004a). Apparently, nematodesaredepositedpassivelywitheggsduringoviposition(Currie, 1937) anditisnotclearwhethertheyisabletoregulatethenumberofnematodesdepositedwitheachegg.Nematodes occur only in female larvae, pupae andies (Davies et al., 2001; Giblin-Davis et al., 2001b;Schefferet al.,2013).Sexpheromonesmayberecog-nizedintheselectionof femaleFergusoninalarvaeand pupae. Nematodes select for female hosts in othersystems, including the tylenchid Sphaerularia bombiwhich only invades queen bumblebees (Poinar &Van Der Laan, 1972) and the g-waspnematodetritrophic interactions in Ficus sycones (Krishnanet al.,2010).How Fergusobia nematodes enter third-instarFergusoninalarvaeisunknown. Commonroutesfornematodeinvasionof insectlarvaeincludepenetra-tionthroughthecuticleor viaspiracles, mouthoranus (e.g. Triggiani & Poinar, 1976; Georgis & Hague,1981). Nematodeentryis not likelyviathehighlysclerotized, convoluted spiracles of Fergusoninalarvae. The nematode suborder Hexatylina, whichincludes Fergusobia, contains nematodes parasitizinginsects and other invertebrates. Those seen penetrat-ing host insects have enlarged pharyngeal glands andapparentlyusesecretionsfromthemtoweakenthehost cuticle, and stylet thrusts to cut a hole for entry(Welch, 1959; Poinar &Doncaster, 1965; Bedding,1972; Poinar et al., 1993). The reduced stylet andsmaller pharyngeal glands of pre-parasitic Fergusobia(Davies&Giblin-Davis, 2004)doesnotsuggestpen-etrationof the Fergusoninalarvae via the cuticle.Theymayenterviathelarvalanus.A female nematode developing with a male y larvainauniloculargall will beunabletodevelopintoaparasitic female and reproduce. However, it is unclearif nematodes within a multilocular gall in loculeswithmalelarvaeareatareproductivedead-end. Itislikelythat nematodescanmovebetweenlocules,especially given observations of coalescing locules(Giblin-Davis et al., 2004a) at later stages in the galllife cycle. Because adult male ies do not have nema-todes, therecanbenosexual transferof nematodesbetweenysexes(Schefferet al.,2013).Betweenone and15parasitic female nematodesdevelopperfemalelarvaory(Currie,1937;Davieset al., 2001; Giblin-Davis et al., 2001b; Pratt et al.,2013). It is not known if or howthe number ofnematodes per female is regulated to preventoverexploitationof thehost. Hundredstothousandsof infective juvenile nematodes andnematode eggsoccur in the haemocoel of the abdomens of adultfemale ies, but the juveniles have cuticles suggestingthattheydonotabsorbnutrientsfromthey. Vari-ation in nematode densities per female y could meanthat species interdependency is context-dependent(Pratt et al., 2013), i.e. that lownematodenumberscouldresult inlowor irregular transference ratesduring ovipositionand limit gall development andhenceyperformance, andthathighdensitiesmaynegatively inuence survival or tness of the host y.Thiswillbedifficulttotest.Themechanismunderlyingevasionorsuppressionof the y immune systemby the nematodes isnot known, but is a further indicationof complexcoevolutionary interactions between Fergusonina andFergusobia. The models developed to explain immunesuppression in parasitoid wasp/host interactions maywellproveusefulinstudiesofhowFergusobiapara-sitic females and juveniles in the haemocoel of710 L.A.NELSONET AL.2014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718Fergusonina ies escape or evade the y immunologi-cal system (Schmidt & Theopold, 2004; Schmidt et al.,2005; Schmidt, 2007). A major difference, however, isthat in the case of the Fergusonina/Fergusobiaimmuneinteractions,itisinthebestinterestofthey to facilitate immune suppressioninsome way,whileinthecaseofaparasitizedinsectitisnot. Analternative model would be that of the immuneevasionsystempresent insome larial nematodes(Maizelset al.,2001).HOST-PLANTINTERACTIONSWITHFERGUSONINA/FERGUSOBIAItiswellknownthatplantchemistrymayinuencetheecologyandspeciationof phytophagousinsects,and both inter- and intraspecic variation can beimportant (e.g. Jolivet, 1998). Recent work shows thatsusceptibility to herbivory canvary evenwithinasingleEucalyptustree(Padovanet al., 2013). About2025%ofinsectsassociatedwitheucalyptsareher-bivores(Majer, Recher&Keals, 1999), andmanyofthesehavehighdegreesof specicityforparticularsubgenera and groups of species, e.g. gall-formingeriococcidscaleinsects(Cook, 2001; Cook&Gullen,2004). Fergusonina/Fergusobia mutualisms are afurther example, with each usually specic to a singlehost species. However, very little is known about theconstraintsthatdeterminethisspecicity.The Myrtaceae are well known as sources of essen-tial oils and other plant secondary compounds(Lassak&McCarthy, 1983; Brophy&Doran, 1996;Wheeler, 2007). Such compounds typically providedefence against generalist insect and other herbivoresbybeingdistastefuland/ortoxic.However,specialistinsectsadaptedtotoleratethesecondarycompoundsof theirhostplantsmayinfactusethepresenceofthese compounds for host locationand recognition(Jolivet, 1998). Althoughtissuesof hostplantsmaycontainhighlevelsofnoxiouscompounds, gallscon-tainingherbivorousinsectscommonlyhavemodiedlevels of plant secondaryas well as nutritivecom-pounds within their tissues, particularly in the modi-ed cells lining the gall upon which the gallersfeed(Hartley, 1998). Manyof thehostplantsof theFergusonina/Fergusobia mutualism are known tocontain high levels of secondary compounds. Forexample, inthefollowingknownhosts, leavesof E.loxophleba contain sideroxylonal (Foley &Lassak,2004); those of the Eucalyptus subgenus Symphyo-myrtus contain formylated phloroglucinal compounds(Eschler et al., 2000); E. cladocalyx, E. leucoxylon,E. polyanthemos, E. viminalis, E. diversifolia andE. ovata contain the glycoside prunasin (Gleadowet al., 2008); and M. quinquenervia has the terpenoidsE-nerolidol and viridiorol (Giblin-Davis et al., 2005).Infact, inFergusonina/Fergusobiagalls, oil glandsare usually external to the individual gall locules(Giblin-Davis et al., 2004b), suggesting that themutualists are able to avoid oil gland involvement orintegration during cecidogenesis. Thus, the secondarycompoundsaremorelikelytohavearoleasattract-ants for the ies and to function in host selection thantodetergalldevelopment.The only direct evidence that we have of resistanceto the Fergusonina/Fergusobia mutualism comesfrom a eld observation (Giblin-Davis et al., 2001b). InM. quinquenervia, hypersensitiveresponsestoovipo-sition by F. turneri followed large numbers of oviposi-tions in one bud. Thus, plants may react to ovipositionby the y/nematode mutualism, but whether this is aspecic response to Fergusonina oviposition or a gen-eralized wound response is not known.Leaf toughness (resistance to fracture per unitfracture area) (Ohmart & Edwards, 1991; Steinbauer,Clarke &Madden, 1998) may function in defenceagainstsomegall formers.Aseucalyptfoliageages,leaf toughness increases and nitrogen contentdeclines (Larsson & Ohmart, 1988; Edwards &Wanjura, 1991). However, as Fergusonina/Fergusobiamutualistsareusuallyfoundassociatedwithyoungfoliage or ower buds (Currie, 1937; Goolsby,Makinson & Purcell, 2000; Taylor & Davies, 2010) andy larvae and nematodes feed within galls, toughnessmay not have a role in defence in this system.Because many gall-forming nematodes are eco-nomically important plant pathogens, plant resist-ance to themhas been extensively studied (e.g.Williamson & Kumar, 2006). Sequencing of thegenomesofsomeofthese, followedbymapping, willleadtoidenticationof genesinvolvedinvirulenceand pathogenicity. This knowledge could be usedin studies of host selection by the Fergusonina/Fergusobiamutualists.FLYNEMATODECOSPECIATIONCospeciation(co-cladogenesis) betweencloselyinter-actingorganismshasbeenstudiedextensivelyoverthe past several decades, resultinginthe commonviewthatcospeciationismostlikelyincasesofver-tical (rather than horizontal) transmission of theinteraction(Moran&Baumann, 2000; Page, 2003;Hosokawaet al., 2006). TheFergusonina/Fergusobiamutualism appears to be an exceptional candidate forcospeciationgiventheapparentstrictverticaltrans-mission of nematodes between y generations (Davieset al., 2010b). However, recent molecular evidencesuggeststhatlargemultiloculargallscanhavemul-tipleconspecicyfoundresses, potentiallyallowingfor horizontal transfer of nematodes between off-springofdifferentfemales(Purcell,2012).SwitchingTRITROPHICCOEVOLUTIONBETWEENFLIES ANDNEMATODES 7112014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718of nematode lineages between conspecic y lineagescould occur frequently in the many Fergusonina/Fergusobiamutualismsthatformlargemultiloculargalls (Giblin-Davis et al., 2004b; Ye et al., 2007; Taylor&Davies, 2010; Davieset al., 2010b; Purcell, 2012).Potentialdeparturefromthetheoryofstrictverticaltransmissionofnematodeshasevolutionaryimplica-tionsfortheFergusonina/Fergusobiamutualismandaddscomplexitytotheevolutionarydynamics.Occa-sional host-switchesbythenematodemayalsohelpexplainanomaliesobservedincomparativeanalysesof molecular datasets(Yeet al., 2007; Davieset al.,2010b).Mating between nematodes associated with theoffspring of different y foundresses, even whenconspecic, could result in increased genetic diversityinthenematodesof multiloculargallsascomparedwith nematodes in unilocular galls, as Fergusobiaspecies forming unilocular galls froma single yfemalewouldneverhaveanopportunityformovingbetween y lineages (Taylor & Davies, 2010). Havingreducedgeneticvariationcouldinuencetheevolu-tionarypotential ofnematodespeciesforadaptationto environmental challenges as well as for host shiftsto new plant species. It should be possible to comparethe genetic variability of nematodes from multiloculargallswiththatofnematodesfromuniloculargalls.SPECIATIONANDEVOLUTIONOFHOST-SELECTIONHost range may affect diversication rates of ecologi-cally specialized gall-forming insects (Hardy & Cook,2010). In the case of the Fergusonina/Fergusobiamutualisms, most arehighlyspecializedandfoundonly on one or a few closely related host plant species(Davies et al., 2010b; Nelson et al., 2011a), suggestingthatspeciationeventstypicallyoccurinconjunctionwith a shift to a new host plant species or tissue type.Because the female Fergusonina y chooses newovipositionsitesforgalling, itisthebehaviourandhost plant/tissuespecicityof theiesthat providetheinitial micro-evolutionaryvariationuponwhichspeciation processes can act. The ability of the nema-todesbothtoinitiateandtofunctionwithinthegallis critical and a major source of selection on theoviposition choices made by the female ies. A femaleythat ovipositsontoanewplant speciesorplanttissuetypeinwhichthenematodescannotfunctionnormally will have lower tness than the ies choos-ingtheirnormalhost.Little work has been undertaken on host choice forthey/nematodemutualism. Intheonlyworkpub-lished to date, Wright et al. (2013) made a seriesof no-choice oviposition and development tests toassess host use by the F. turneri/F. quinquenerviaemutualisminFlorida. Ovipositionandgall develop-ment was assessed on eight species of Myrtaceaenative to Florida, eight species phylogeneticallyrelatedtoitsnaturalhostM. quinquenerviaandvenon-myrtaceous species. Femaleies didnot probeanynon-myrtaceousspecies, butdidprobe11of 16tested Myrtaceae. Fly eggs and nematodes weredepositedinfourof these. However, gallsdevelopedand matured only on M. quinquenervia. Thus, themutualismhasanarrowrangeof hostchoices, andevidencethat they/nematodemutualismishighlyhost-specic, withoneoratthemosttwohostplantspecies,issupported.Therearemanycircumstancesthat couldleadtoyovipositiononanon-hostplantspeciesortissue,mostnotablyalackofsuitableovipositionsites(e.g.Wright et al., 2013). The extreme and often unpredict-able weather patterns common in Australia maygreatlyinuencethetimingandgeneralavailabilityof ovipositionsites present onany particular hostplant species. Intheabsenceof suitablesitesonacertainhost plant species or tissue, afemale maydump her eggs onwhatever non-host is available(e.g. Kostal, 1993; Messina, Morrey&Mendenhall,2007; Wright et al., 2013). This may provide an initialimpetus for host shifting onto new host plant taxa ortissue types. Oviposition sites on non-traditional hostplantsortissuetypeswouldleadtostrongselectiononlarval andnematodeperformancetoincorporatethe plant as a newhost. Inturn, this couldleadto population divergence and speciation under awide variety of genetic models and scenarios, e.g.sympatric, parapatric and ecological speciation(Tilmon,2008;Butlin,Bridle&Schluter,2009).Oneof thekeyquestionsabout theFergusonina/Fergusobiamutualismiswhetherphylogeniesofthenematodey associations are congruent with theradiationoftheirhostplants. ThefamilyMyrtaceaecomprises 17 tribes, 130150 genera and nearly 6000species. It is widespread in the southern hemispherewith centres of diversity in Australia, South-East AsiaandSouthAmerica(Wilson, 2011). ThemostrecentestimatedcrownageofthefamilyistheLateCreta-ceous (85 Mya) (Thornhill et al., 2012), andmosttribes had split from each other by the Oligocene (seeFig. 4). Of the ve tribes that have beenfoundtobehostsof theFergusonina/Fergusobiamutualism,none are sister groups, and divergence times betweenthesehost tribesdatebacktotheLateCretaceous,Palaeocene and Eocene. Recent dating of the yradiations suggests that Fergusoninidae divergedfrom its sister family Asteiidae around 42 Mya(Wiegmann et al., 2011), and a crown age for thefamily is unknown but is younger than 42 Mya. Thus,the Fergusoninidae did not cospeciate with theMyrtaceae tribes onwhichtheyoccur andevolvedwhenmosttribesof Myrtaceaehadalreadydiversi-712 L.A.NELSONET AL.2014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718ed. The dating of the Fergusonina phylogeny shouldprovide us with a clearer timeframe of when thegroupradiatedaround Australia. Whilethecoevolu-tionof Fergusonina onMyrtaceae at the broadestlevel isprecludedbecauseofthedifferentgeologicalagesof thegroups, coevolutionwithinsmallersub-groups of ies andplants that have diversiedinmore recent times (e.g., in the last 1015 Mya) is stillpossible.Similarity in the molecular sequences and morphol-ogy of the dorsal shields of the Fergusonina ies andtheir associatedFergusobianematodes, respectivelyfromatleaf gallsonhostspeciesfromEucalyptussectionAdnataria and terminal shoot bud galls on hostspecies from section Exsertaria, suggests cospeciation(Davies et al., 2010b, 2013a, b). Dated phylogenies ofthesehostEucalyptusgroupsareneededtoconrmthis potential radiation with host species.Clearly, the development of a Fergusonina/Fergusobiagall dependsontheclosebiological rela-tionship between the mutualists themselves thathasevolvedovermillionsof years, andtheirabilityto initiate and produce galls on a particular hostMyrtaceae species and tissue. Speciation and diversi-cation in the Fergusonina/Fergusobia mutualismprobably involve some combination of cospeciationwiththehostplants, speciationtrackinghostplantphylogeny, and speciation via host-shifts and changestothetissuetypeselectedfor galling. Determiningthe relative importance of these and their associatedmechanisms will require a multidisciplinary approachincluding molecular systematics, phytochemistry,population genetics, behaviour and evolutionaryecology. Our understanding of speciation and diversi-cation in the Fergusonina/Fergusobia/host plantsystemisstillinitsearlieststages.CONCLUSIONSWhileunderstandingof theFergusonina/Fergusobiaassociation is limited, its life cycle is established, andit is known that ies and nematodes have a complexand obligatory mutualism, each of which is generallyhost-specic. Atpresent,littleisknownofybehav-iour, host choice, the process of cecidogenesis, factorsdetermining host resistance or the selection pressuresoperating onthe mutualism. However, the genetictools needed to analyse many of these are now avail-able, andmodels against whichhypotheses canbetestedarebecomingincreasinglyavailable.Plant-feeding insects comprise an extraordinaryproportionof extant biodiversity(Strong, Lawton&Southwood, 1984; Mitter, Farrell & Wiegmann, 1988;Schoonhoven, van Loon & Marcel, 2005), and provideimportant and compelling examples of evolution.Given the complex and obligate nature of theFergusonina/Fergusobia mutualism, and their rela-tionshipswiththeirmyrtaceoushosts, thedevelop-ment of robust, dated, phylogenies for all threetrophiclevelswillprovideauniquestudysystemforcospeciation and coevolution. A robust phylogenyof both ies and nematodes at deep levels is necessaryfor co-phylogenetic comparisons, but isonlyavaila-ble for the ies. Such phylogenies will also allowFigure 4. Dendrogram of the eucalypts based on A. H. Thornhill et al. (unpubl. data) showing how the major subgeneraand sections are related to each other and where the concentration of Fergusonina host species occurs in the phylogeny.TRITROPHICCOEVOLUTIONBETWEENFLIES ANDNEMATODES 7132014TheLinneanSocietyofLondon,BiologicalJournaloftheLinneanSociety,2014,111,699718us to further understand the evolutionary andcoevolutionary history of host-use, gall-formation andfactorssuchasdevelopmentofthedorsalshield.Theapparent vertical transmissionof FergusobiaacrossFergusoninagenerationsledtothepredictionof congruent phylogenies betweenFergusoninaandFergusobia (Giblin-Davis et al., 2004b; Ye et al., 2007;Davieset al., 2010b). However, thetwophylogeneticstudiesoftheiesandnematodespublishedtodate(Scheffer et al., 2004; Ye et al., 2007) didnot showstrict congruence when compared ad hoc. Geneticevidencethat multilocular gallsmayhavemultiplefoundresses possibly allowing for lateral transmissionofnematodesprovidesatleastonemechanismforadeparture from strict cospeciation, and others are nodoubtawaitingdiscovery. Wenowpredictthatstrictco-phylogenybetweentheyandnematodeswill befound more often in clades of unilocular gallers ratherthan multilocular gallers where nematodes can prob-ablymovebetweenlocules.Recent studies providing divergence time estimatesfor boththeMyrtaceaeandtheDipterashowthattheyfamilyFergusoninidaeismuchyoungerthanthe Myrtaceae, thus rejecting the hypothesis ofcospeciation of the ies and the plants at deep levels.Weexpect that tritrophicco-phylogenybetweentheFergusoninidae, their mutualist nematodes and theirmyrtaceoushostplantswill mostlikelybefoundatlow taxonomic levels in the Myrtaceae, such as withincladesof host EucalyptusorCorymbiaspeciesthathave been diversifying more recently than the 42 MyacrownageofFergusoninidae.ACKNOWLEDGEMENTSSpecial thankstoTedandBarbCenter, Paul Pratt,GregWheeler, ScottBlackwood, PhilTipping, SusanWright, WeiminYe and Dorota Porazinska amongmany others at the USDAIPRL (Invasive PlantResearchLab) andtheUniversityof Florida, IFASFLREC in Fort Lauderdale, Florida, for their help andsupportduringstudiesofthisamazinggroupofiesand nematodes for deployment as a biological controlagent against M. quinquenervia in the United States.Some of the research summarized in this article wassupportedbyUSDASpecial GrantsinTropical andSubtropical Agriculture (CRSR-99-34135-8478 andCRSR-03-34135-14078)toR.G.D.; andby AustralianBiological Resources Studies grants to K.A.D.REFERENCESAustin AD, Yeates DK, Cassis G, Fletcher MJ, La Salle J,LawrenceJF, McQuillanPB, MoundLA, Bickel DJ,GullanPJ, HalesDF, TaylorGS. 2004. 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