Robustness of Ancestral State Estimates - Systematic Biology

28 Pages·2002·0.7 MB·English

Syst.Biol.51(3):450–477,2002
DOI:10.1080/10635150290069896
Robustness of Ancestral StateEstimates: Evolutionof Life History
Strategy in Ichneumonoid Parasitoids
ROBERTBELSHAW1 ANDDONALDL.J.QUICKE2
1DepartmentofBiologicalSciences,ImperialCollegeatSilwoodPark,Ascot,BerkshireSL57PY,UK;
E-mail:[email protected]
2DepartmentofBiologicalSciencesandCentreforPopulationBiology,ImperialCollegeatSilwoodPark,
andDepartmentofEntomology,TheNaturalHistoryMuseum,LondonSW75BD,UK;E-mail:[email protected]
D
Abstract.—Wetesthypothesesfortheevolutionofalifehistorytraitamongagroupofparasitoid ow
wasps(Hymenoptera:Ichneumonoidea),namely,thetransitionamongkoinobiontparasitoids(par- n
lo
asitoidswhosehostscontinuedevelopmentafteroviposition)betweenattackingexposedhostsand a
d
attackinghoststhatareconcealedwithinplanttissue.Usingarangeofphylogenyestimatesbased e
d
oonds28toSersDtiNmAatseetqhueenancecse,swtraelulisfeemhiasxtoimryumtrapitasrfsoimrothneym(MaiPn)calnaddemsainximwhuimchlibkoetlihhotroadits(MocLc)umre(uths-- fro
m
ingtheprogramsMacCladeandDiscrete,respectively).Wealsoassesstherobustnessofthesees- h
timates;forMP,weusestepmatricesinPAUP¤ to(cid:142)ndtheminimumweightnecessarytoreverse ttp
einsgtimthaeteasncoersmtraaklneothdeemsaatmthbeigaultoerunsa,taivndesftoarteMs.LW,weealmsoemaseuarseutrheethdeiffreorbeunsctenseisnsolifktehliehoMoPdaanftceerst(cid:142)rxa-l s://a
c
stateestimateagainstuncertaintiesinthephylogenyestimate,manipulatingthemost-parsimonious a
d
treeinMacCladeto(cid:142)ndtheshortestsuboptimaltreeinwhichtheancestralstateestimateisreversed e
m
ormadeambiguous.Usingthesemethods,we(cid:142)ndstrongevidencesupportingtwotransitionsamong ic
koinobiontIchneumonoidea:(1)toattackingexposedhostsinacladeconsistingoftheHelconinae .o
u
andrelatedsubfamilies,and(2)thereversetransitioninacladeconsistingoftheEuphorinaeand p
relatedsubfamilies.Inexploringdifferentmethodsofanalyzingvariable-lengthDNAsequences,we .co
foundthatdirectoptimizationwithPOYgavesomeclearlyerroneousresultsthathadaprofound m
effectontheoverallphylogenyestimate.Wealsodiscussrelationshipswithinthesuperfamilyand /sy
s
expandtheMesostoinaetoincludeallthegall-associatedbraconidsthatformthesistergroupofthe b
Aphidiinae. io/a
rtic
le
-a
Estimatesofancestralstatesaresubjectto character, transitions in either direction are b
s
twosourcesoferror:uncertaintiesinestimat- equally probable (but see Maddison (1995) tra
c
ingtheancestralstategivenaphylogenyes- and Salisbury and Kim (2001) for discus- t/5
1
timate, and uncertainties in the phylogeny sions of the probability of MP-based ances- /3
estimateitself. tralstateestimations).Wecanmeasurethero- /4
5
0
Consideringthe(cid:142)rstsourceofuncertainty, bustnessofaMPestimatetothisassumption /1
for a given estimate of phylogeny, we can byusingasymmetricstepmatrices(Reeand 61
6
usemaximumparsimony(MP)ormaximum Donoghue,1998;Omland,1999).Byapplying 8
9
likelihood (ML) to estimate any ancestral increasing weights to one of the two trans- 0 b
character state. Cunningham et al. (1998) formations, we can (cid:142)nd the minimum ad- y g
compare these two methods and show that ditional weight necessary tochange the ob- ue
s
simple MP estimates may be misleading if served result.Severalauthors(Sperlingand t o
ratesofchangearehighortransitionratesare Feeny, 1996; Omland, 1997) have used this n 1
asymmetric.Particularlyvaluableformolec- method,generallybecausetheysuspectthat 4 A
uanlacrespthrayllosgtaetneeetsictismisatthesetaobitlaiktyeoinftMoLac-bcoasuendt geqauinasllyanpdrolboasbslees. of complex traits are not pril 2
0
branchlengths(changesaremorelikelytooc- Theimportanceofthesecondsourceofun- 19
curonlongbranchesifbranchlengthispro- certaintyforcomparativemethodshasbeen
portional to time). The robustness of a ML recognizedforsometime(HarveyandPagel,
ancestral state estimate can be assessed by 1991:203). We can gain a crude measure of
comparingthelikelihoodsofmodelsoftrait therobustnessofan ancestralstateestimate
change in which the trait is (cid:142)xed at the al- by repeating it on estimates of phylogeny
ternative states (Pagel, 1999a). As currently that use different methods and parameters.
implementedinavailablesoftware,estimat- However, for a more rigorous examination,
ingancestralstatesby usingMPis asimple we need to test whether trees supporting
optimizationandassumes that,fora binary the alternative ancestral state estimate are
450
2002 BELSHAWANDQUICKE—ROBUSTNESSOFANCESTRALSTATEESTIMATES 451
signi(cid:142)cantlyworse.Thisgeneralapproachis be correlated with this apparently funda-
used for testing a wide range of evolution- mental dichotomy (Sheehan and Hawkins,
aryhypothesesthatusephylogenyestimates 1991; Belshaw, 1994; Quicke, 1997; Mayhew
(HuelsenbeckandRannala,1997).Forexam- and Blackburn, 1999). In this paper we ex-
ple,toassessthesupport fordifferent num- amineonegroupofparasitoids,theIchneu-
bersofindependentdomesticationsoffungi monoidea(Hymenoptera),to(cid:142)ndthedirec-
byfungus-growingants,Muelleretal.(1998) tion of the evolutionary transition between
comparedoptimaltreeswithtreesfoundby koinobiont parasitoids whose hosts feed
searchesthatwereconstrainedtorecovercer- concealed in plant tissue, and koinobiont
tain fungus cultivars as monophyletic. Ap- parasitoids whose hosts feed exposed on D
o
plied to ancestral state estimates, this ap- plants. w
n
proachisusedby DumbacherandFleischer Evidence is strong that the common an- lo
a
(2001) to assess the support for a proposed cestor of the Ichneumonoidea was an idio- d
e
cieosnvcoermgpenacree tihnebriredsupltlsumobatgaein.eOdthbeyrustsuindg- bbiaosnistopfamraosirtpohidoloogficcoanlccehaalreadctheross,ttsh.iOsnistehse- d fro
m
optimal trees with those from suboptimal timatedtobetheancestralbiologyofthesis- h
trees, for example, Donoghue and Ackerly ter group of the Ichneumonoidea, the Ac- ttp
s
(1996) examining the evolution of branch- uleata (Hanson and Gauld, 1995), and it is ://a
c
ing architecture in Acer plants and Hibbett the biology of most taxa phylogenetically a
d
and Donoghue (2001) studying correlations close to the (Ichneumonoidea Aculeata) em
between life history traits of homobasid- clade (Ronquist, 1999). DowtonCand Austin ic
.o
iomycete fungi. Although most studies, in- (2001), using molecular data, also estimate u
p
cluding the one we present here, pursue this to be the ancestral biology of the Ich- .c
o
this only within a MP framework, the ap- neumonoidea(althoughtheydonotrecover m
/s
proach is applicable to other methods, for the Aculeata as the sister group). Within y
s
b
example, Bayesian inference (Huelsenbeck theIchneumonoideatransitionstokoinobio- io
etal.,2000). sisapparentlyhaveoccurredmultipletimes /a
Inthispaperweusetheabovemethodsto (Whit(cid:142)eld,1998;Belshawetal.,1998).Com- rticle
analyzetheevolutionofaparticularlifehis- monly, ichneumonoid koinobiont species -a
b
torytraitamongagroupofparasitoidwasps. oviposit in young hosts that feed exposed stra
Tosummarize,ourprocedureinvolves(1)es- on the surface of plants and kill them only c
timatingphylogeny,(2)estimatingancestral after the host has (cid:142)nished its larval growth t/51
states by MPand ML, (3)measuringthero- andmoved toits pupation site(Shaw, 1983; /3
/4
bustnessoftheseancestralstatesestimatesby Gauld, 1988). Many ichneumonoid koino- 5
0
usingstepmatrices(forMP)anddifferences bionts, however, attack hosts that are con- /1
6
1
inlikelihood(forML),and(4)measuringthe cealed in plant tissue. Quicke et al. (1999) 6
8
robustnessoftheancestralstateestimatesto suggestedthatthislattertraitismorelikelyto 9
0
uncertaintiesinthephylogeny(MPonly). haveevolvedthroughanintermediatestage b
y
Insect parasitoids oviposit onto, or into, ofbeing akoinobiont parasitoid of exposed g
u
e
their host, which typically is the immature hosts,ratherthandirectlyfromtheconcealed s
stageofanotherinsect.Theparasitoid larva idiobiontancestor.Theyarguedthat,despite t o
n
feeds on the host and eventually kills it theselectiveadvantagesproposedforkoino- 1
4
(Eggleton and Belshaw, 1992). Perhaps the biosisinparasitoidsattackingexposedhosts A
p
most striking feature of life history evolu- (forexample,inkillingthehostonlyafterit ril 2
tion among parasitoids has been that from has moved to a more protected site for pu- 0
1
idiobiosis to koinobiosis (Askew and Shaw, pation), such selective advantages are dif- 9
1986),thedistinctionbeingthatkoinobionts (cid:142)cult to attribute to koinobiosis when the
allow the host to continue development af- host is already concealed. Hence, among
ter oviposition, whereas idiobionts do not. koinobionts, attacking concealed hosts is
Commonly, koinobiont species oviposit in more likely to be a reversal to the ances-
immature hosts, which the parasitoid larva tral habit. Conversely, several authors have
kills only after the host has (cid:142)nished grow- suggested that—at least in certain ichneu-
ing.Incontrast,theovipositingidiobiontfe- monoid clades—koinobiosis in parasitoids
maleusuallypermanentlyparalyzesorkills attackingconcealedhostsarosedirectlyfrom
the host before laying an egg or eggs on theancestralidiobiontbiologywithoutanin-
it. Many other life history traits appear to termediate stageofattackingexposed hosts
452 SYSTEMATICBIOLOGY VOL.51
(Shaw,1983;Gauld,1988;Wharton,1993).In from the tree for ML estimates (see below).
thepresentstudyweattempttoresolvethis Taxawhosebiologywasdif(cid:142)culttocodein-
debate by using 28S rDNA sequence data, cludethetryphoninesmentionedaboveand
having previously shown that many mor- species such as the ichneutine Paroligoneu-
phological characters are related to the life rus Muesebeck, which attacks the eggs of
history trait investigated here (Quicke and leaf-mininghosts(eggsofleaf-minerscanbe
Belshaw,1999). pushedintoplanttissuetovariousdegrees).
Taxainwhichthefemaleentersthechamber
ofconcealedhoststoattackthemwerecoded
METHODS
as exposed. The ancestral node of a clade D
TheTaxaCategorizationofandTheirLife o
wastreatedasthe(cid:142)rstbifurcationwithinthat w
History n
clade,ratherthanthenodelinkingtheclade lo
a
Our previous molecular phylogenetic toitssistergroup. d
e
slatutidoniesshoipfsthtiossbuepperofoarmlyilyresshoolvweded(Bbaeslsahlarwe- ApTpheendtiaxxawitahnatlhyezierdEMarBeL/liGsteendBainnk tahce- d from
etal.,1998,2000;Quickeetal.,2000).There- cession numbers. Our taxon coverage is h
fore,ratherthanattemptingtodetermineall very good at the subfamily level: Among ttp
s
ancestral states in the superfamily, we fo- the noncyclostome Braconidae we lack rep- ://a
c
cusontheancestralstatesofwell-supported resentatives only of the Amicrocentrinae, a
d
koinobiont clades that display both life his- which attack lepidopteran stalk borers and em
torytraits:speciesattackingconcealedhosts have long ovipositors (van Achterberg, ic
.o
andspeciesattackingexposedhosts.Fiveof 1979a); the Ecnomiinae, of which the bi- u
p
thesekoinobiontcladesarewithinthenoncy- ology is unknown and the af(cid:142)nities are .c
o
clostomeBraconidae,andthesixthiswithin unclear (Park and van Achterberg, 1994); m
/s
theophioniformcladeoftheIchneumonidae and a few small microgastroid subfami- y
s
b
(the two families together form the Ichneu- lies,namely,theDirrhopinae,Khoikhoiinae, io
mcloosntooimdeeab)r.aWcointhidosnaendexocpehpitoionnif,otrhmeinchonnecuy-- atinvdes Mfoerndoneslyellifnoauer.icWhneeulamckonirdepsruesbefnamta-- /article
monids are also exclusively endoparasitoid ilies, and three of these probably belong -a
b
(eggsarelaidwithinthehosts,andthelarva in the (pimpliformesCichneumoniformes) stra
developsinternally—atleastforthe(cid:142)rstpart clade:theAgrioptypinae,Pedunculinae,and c
of its life). The exception is the tryphonine Claseinae. The only omission from the t/51
ichneumonids,whichareacladeofectopara- ophioniformescladeistheTatogastrinae;the /3/4
sitoidkoinobionts.Theotherichneumonoid Townesioninae has recently been shown to 5
0
taxa are in clades dominated by the ances- belong to the Banchinae (Gauld and Wahl, /1
6
1
tralidiobiont biology andarenot examined 2000a). More detail of the life history of 6
8
here, namely, the cyclostome clade in the the noncyclostome Braconidae is given in 9
0
Braconidaeandthe(pimpliformes ichneu- Table1; thecompositionofthesix analyzed b
moniformes) clade in the IchneuCmonidae. cladesissummarizedbelow. y g
u
Gauld(1988)providesanoverviewofthedi- The sigalphoid clade.—This contains the es
versityoflifehistorystrategieswithintheen- Sigalphinae and the Disophrini (Agathidi- t o
n
tireIchneumonoidea.Wecategorizetaxaac- nae)braconids,whichattackexposedhosts, 1
4
cording to whether the host is concealed at andtheremainingAgathidinae,allofwhich A
p
thestageat which it is attackedand weuse attackconcealedhosts. ril 2
thetermconcealedtoincludehostsinallde- The macrocentroid clade.—Two macro- 0
1
grees of concealment: from within wood to centrine genera (Austrozele Roman and 9
inside leaf mines or leaf rolls. The depth of Dolichozele Viereck) attack exposed hosts,
concealment of the host may not automati- whereas the remaining genera attack
cally re(cid:143)ect how dif(cid:142)cult it is to parasitize: concealedhosts.
Parasitoidsattackinghostsdeeplyconcealed The helconoid clade.—The Xiphozelinae
in wood often simply thread their oviposi- and some Homolobinae attack exposed
torsthroughcracks,whereasthoseattacking hosts, whereas the Helconinae (as previ-
miners may have to penetrate plant tissue. ouslyconstituted), Microtypinae, andChar-
Nonkoinobionts, and taxa whose biology is montinae attack concealed hosts exclu-
unknown or dif(cid:142)cult to code, were treated sively. Two genera currently placed in
as uncertain for MP estimates and pruned the Helconinae, Canalicephalus Gibson and
2002 BELSHAWANDQUICKE—ROBUSTNESSOFANCESTRALSTATEESTIMATES 453
TABLE1. Categorizationoflifehistoryfornoncyclostomebraconidtaxa.“Semiconcealed”referstohostssuch
asleafminersandleafrollers,whicharetreatedasconcealedinouranalyses;“other”isusedforalllifehistories
thatdonot(cid:142)tourcategorization(discussedfurtherintext).
Lifehistory
Taxon category Furtherdetails
SigalphoidClade
Agathidinae Agathidini,Earinini,andMicrodini Concealed SemiconcealedLepidopteralarvaea
Disophrini Exposed Lepidopteralarvaea
Sigalphinae Concealed SemiconcealedLepidopteralarvaea
HelconoidClade
D
Helconinae(part) BrachistiniandDiospilini Concealed Seed-feedingColeopterab,cexcept o
w
forBrulleia,whichhasonerecord n
fromcerambycidlarvaed. loa
Egg-larvalwhereknown de
Eadyiini Exposed Ealdayravaaetthacksexposedchrysomelid d fro
m
Helconini Concealed Cerambycidandotherwood-boring h
Coleopteralarvaec ttp
Xiphozelinae Exposed Onerecordfromanexposed s
Lepidopteralarva;bothgenerahave ://a
shortovipositorse ca
Homolobinae Exposed Sequencedspecieshaveshort de
ovipositorsandattackcaterpillars m
ic
thatfeedexposedatnight;other .o
specieshavealongovipositor up
MacrocentroidClade .c
o
Macrocentrinae Macrocentrus,Hymenochaonia, Concealed SemiconcealedLepidopteralarvaeb m
andAulacocentrum /s
y
Austrozele Exposed Hostfeedsexposednocturnallyb. sb
Shortovipositor io
Dolichozele Exposed OnSehroerctoorvdipfroosmitoarnexposedhosta. /artic
le
Microtypinae Concealed SemiconcealedLepidopteralarvaea -a
Charmontinae Concealed SemiconcealedLepidopteralarvaeb b
s
EuphoroidClade tra
Euphorinae Exposed AdRuhlotpianlsoepchtosriunsaatnldeaCsotssmixooprdhoerrsinb;i ct/51
attackscolytidbeetles,butatleast /3
inCosmophorusthefemalegoesinto /45
thehostburrowandholdsthehost 0/1
withitsmandiblesb 6
1
Neoneurinae Exposed Ants 6
8
Meteorinae Meteorus Exposedor LepidopteraandColeopteralarvae; 9
0
concealed exposedandconcealedtovarious b
y
degrees,includingleafminersand g
barkborersb ue
MicrogastroidClade Zele Exposed NocturnalLepidopteralarvaeb st on
Cardiochilinae, Exposed, WiderangeofLepidopteralarvae, 1
4
Microgastrinae, concealed, bothexposedandsemiconcealeda; A
ChaenlodnMiniareacinae Otohrerother Egsgo-mlaervMalicpraorgaassittoriindaseoafrLeeepgidg-olpartevraali pril 2
(chie(cid:143)ysemiconcealed).Degreeof 01
exposureofeggs,andlengthof 9
ovipositor,varya
Adelius Other Leaf-miningLepidopteralarvae,
especiallyNepticulidae;assumed
tobeegg-larvalb
Othertaxa
Orgilinae Mimagathidini Concealed Leaf-miningLepidopteralarvae,but
probetheworkingsratherthan
piercingtheplanttissueb
Orgilini Concealed Leaf-miningLepidopteralarvae
Sources:a Whartonetal.(1997),b ShawandHuddleston(1991),c HansonandGauld(1995),d ChenandvanAchterberg
D D D D
(1993),e vanAchterberg(1979b),f Belshawetal.(2000),g Gibson(1972),h HuddlestonandShort(1978),i Rubersonand
D D D D D
Whit(cid:142)eld(1996),andj Odaetal.(2001).
D
454 SYSTEMATICBIOLOGY VOL.51
TABLE1. Continued.
Lifehistory
Taxon category Furtherdetails
Ichneutinae Other Egg-larvalparasitoidsofsaw(cid:143)ylarvaea,bexceptfor
OligoneurusandParoligoneurus,whichattack
leaf-miningLepidopteraandDipterabutprobably
alsoegg-larvala,b
Helconinae(part) Canalicephalusand Concealed Urosigalphusattacksseed-feedingColeopterag;assume
Urosigalphus thesameforCanalicephalus
Cenocoeliinae Concealed Coleopteralarvaewithvariousdegreesofconcealmentb
Aphidiinae Exposed Aphids Do
Mesostoinae Other Phytophagous,idiobiont,orunknownf,j w
n
lo
a
d
Urosigalphus Huddleston,wererarelyrecov- 2000); Panteles Fo¨rster is treated as missing e
d
ered in this clade. Eadya, previously placed data, although its host is a gall former, be- fro
intheEuphorinae,clearlyappearstobelong causeofitsputativecloserelationshiptoStil- m
h
intheHelconinaeandattacksexposedhosts. bops Fo¨rster, which oviposits into the host ttp
phTohreineauephaonrdoidNceloadnee.—uriTnhaies,caolnltoafinwshthicehEaut-- egg. s://ac
LaboratoryProtocols a
tackexposed(adult)hosts,plustheMeteori- d
e
nae, some of which attack concealed hosts. DNA was extracted from single speci- m
ic
In some analyses the Cenocoeliinae, which mens (or parts of specimens weighing no .o
u
attack concealed hosts, are also recovered morethan 30mg)byusingtheDNeasykit p
» .c
amongthisclade. (Qiagen)with(cid:142)nalelutioninto100¹lofwa- o
m
The microgastroid clade.—This contains ter.Allpolymerasechainreactionswerecar- /s
y
many genera that attack exposed hosts and riedoutinABI2400or9600thermalcyclersin s
b
othersthatattackconcealedhosts.Unlikein 50 ¹l reaction solutions containing1.0¹l of io/a
theabove clades,manygenerainthisclade, DNA extract, 20 pmol of primers, 10 nmol rtic
representing both life history states, are not of dNTPs (Amersham Pharmacia Biotech; le
-a
representedinourdataset. APB), 1.5 units of Taq polymerase (Roche), b
s
The Campopleginae clade.—This clade con- 5.0 ¹l of Taq buffer (containing 1.5 mM tra
c
sistsofthetaxacurrentlyplacedintheCam- MgCl2). Cycling conditions (35 cycles, plus t/5
popleginae but excluding Chriodes Fo¨rster, an initial denaturation for 2min anda (cid:142)nal 1/3
Hellwigia Sve´pligeti, and Nonnus Cresson, extension for 7 min) were 94±C for 30 sec, /45
which are misplaced within this subfamily. 50 Cfor30sec,and72 Cfor1min.Products 0
± ± /1
NotallofthevariationintheIchneumonidae werecleanedbyusingtheGFX bandpuri(cid:142)- 6
1
6
is represented by the taxa sequenced here, cationkit(APB)andweresequenceddirectly 8
9
and we assumed that life histories are con- with dye terminators on ABI 373 and 3700 0
b
stantwithingenera.Insomecases(Chriodes, automated sequencers (using one-half and y
g
Hellwigia, Nonnus, Oxytorus Fo¨rster, tersilo- one-fourth the recommended volumes, re- u
e
s
chinesp.)thehostisunknown,andwehave spectively).TheD2–D328SrDNAregionwas t o
inferred life history from the length of the ampli(cid:142)edasasinglefragment,andD4–D10 n
1
ovipositor and, in the case of Nonnus, the 28S rDNA was ampli(cid:142)ed in a further three 4
A
swollen foretibia often associated with lo- fragments.Allprimersequencesaregivenin p
cating concealed hosts (Broad and Quicke, Table2. ril 2
0
1
9
TABLE2. Primersequences.Allsequencesarewritten5’to3’.Variableregionsin28SrDNAmatchthosein
Drosophilamelanogaster(Hancocketal.,1988).18SrDNAprimersarefromSanchisetal.(2000):forwardisV4.up1,
reverse is 18S.lo1. In a few taxathese wereused in combination withoneof two internalprimers (forward is
NS58 2;reverseisNS58 3)toamplifythegeneintwocontiguousfragments.
C ¡
Region Forwardprimer Reverseprimer
18S CAGCCGCGGTAATTCCAGC CTTCYGCAGGTTCACCTAC
18Sinternal TCCGATAACGAACGAGACTC GAGTCTCGTTCGTTATCGGA
28SD2-3 GCGAACAAGTACCGTGAGGG TAGTTCACCATCTTTCGGGTC
28SD4-5 CCCGTCTTGAAACACGGACCAAGG GTTACACACTCCTTAGCGGA
28SD6-7 GGAGTGTGTAACAACTCACCTGCCG GACTTCCCTTACCTACAT
28SD8-10 CCCATATCCGCAGCAGGTCTCC AGTCAAACTCCCTACCTGGC
2002 BELSHAWANDQUICKE—ROBUSTNESSOFANCESTRALSTATEESTIMATES 455
PhylogenyEstimation in choosing between them (Morrison and
Ourphylogenyestimatesarebasedonan Ellis, 1997; Sanchis et al., 2001). One ap-
approximatelythreefoldexpansionoftheD2 proach is to use congruence as a measure,
28S rDNA molecular data set used in pre- preferring alignment methods or align-
vious studies (Belshaw et al., 1998, 2000; ment parameters that maximize congru-
Dowton et al., 2002). Because this region is ence. This congruence can be with a tax-
dif(cid:142)culttoalignacrosstheentiresuperfam- onomic classi(cid:142)cation, morphological data,
ily,wethereforeanalyzetheIchneumonidae other genes, or even different regions of
andBraconidaeseparatelyandrootthemby the same gene (Wheeler, 1995; Giribet and
using the results ofa third (“rooting”) phy- Wheeler, 1999; Cognato and Vogler, 2001; D
o
logenetic analysis. This rooting analysis is Giribet, 2001). However, in the absence of w
n
based on a data set comprising represen- independent data, the approach we chose lo
a
tative braconid and ichneumonid taxa plus was to use a wide range of methods and de
d
soefvtehrealIcahcnueleuamteosn(othideeap;uatattoivtealsoisfte3r2gtraoxuap). caolimgnpmareenttshearreesauvlatsil.abRleeprineseNnetaxtuivsefoDrmNaAt from
Wofetsheequ28eSncgeedntehe(ssepfaonrnainmgutchhelaDrg2e–rDr1e0giroen- aIcthhntetupm://onwowidw..bio.ic.ac.uk/research/data/ https
gions;length2643–3260bp),whichincludes The traditional method for analyzing ://a
c
more conserved regions that are alignable variable length sequences is to produce a ad
between more distantly related taxa, plus multiplealignment,fromwhichatreeissub- em
a 1177–1223 bp region of the 18S rDNA sequently built. An appealing recent alter- ic.o
gene.Wealsoincludedinthisrootinganaly- native is the program POY by D. Gladstein up
sis the braconid Megalohelcon Turner, which and W. Wheeler (Wheeler, 1996), which im- .co
m
we excluded from our analysis of the Bra- plements a direct optimization, (cid:142)nding the /s
conidae because ofits unknown life history most-parsimonious tree (MPT) for variable ys
b
andhighlydivergent(bothinlengthandbase length sequences, given the cost of insert- io
ceotmal.p,o1s9i9t8io).n)D2–D328Ssequence(Belshaw iWngedaissciunsgslePOgYapberleolawtiavmeotongathseubalsitgitnumtieonnt. /article
The data sets for the Braconidae and Ich- methods. -ab
neumonidae consist of 121 and 114 species, Alignment by using POY.—We analyzed stra
rgeiospnescotifve2l8yS, rsDeqNuAen;cseedqufeonrctehleenDg2th–Ds 3werree- o(oubrtaisneeqduenfrcoems wftipth://fPtpO.aYmnvhe.rosrigo/npu2b./6 ct/51
593–716bp(Braconidae;bothextremeswere molecular/poy), running in parallel on a /3/4
foundwithintheaberrantsubfamilyAphidi- cluster of (cid:142)ve 486 MHz processors. In ad- 50
inae,discussedbelow)and625–664bp (Ich- dition to the directly optimized phylogeny /16
1
neumonidae). The small D3 region was not estimates, POY can also generate a mul- 68
sequenced in 45% of the genera, which are tiple alignment (using the command, im- 90
from earlier studies, mostly our own. In pliedalignment), which we used for sub- by
g
addition, we repeated some of the analy- sequent tree-building, treating the gaps as u
e
sis with an additional 21 braconid species missingdata.TheoriginaltreefoundbyPOY st o
(total 142); these species were additional can be recovered from this multiple align- n
1
Aphidiinae, which we included to help ment by searching with a step matrix that 4
overcome phylogenetic artifacts caused by givesgapsthesameweightasintheoriginal Ap
the group (see below), plus representatives POYsearch. ril 2
of novel taxa obtained near the end of POY was the only method we used with 01
9
the project. The Braconidae analysis con- theD2–D1028Sand18SrDNArootingdata
sistedofnoncyclostomesplussixrepresenta- set.Tospeedcomputationwiththe28Sdata,
tivesofthewell-supportedcyclostomeclade. and hence to (cid:142)nd shorter trees in heuristic
For the Ichneumonidae, where fewer as- searches, we made initial alignments of the
sumptionsaboutrelationshipscanbemade, original four ampli(cid:142)ed fragments, which
we included representatives of all available we then subdivided to give a total of 31
subfamilies. homologousregions.These31 regionswere
Several phylogenetic methods are avail- alignedindependentlyinPOY,using20ran-
able for analyzing DNA sequences of such dom additions with SPR (subtree pruning
variable lengths, and the main problem is and regrafting)and TBR (tree bisection and
456 SYSTEMATICBIOLOGY VOL.51
reconnection) branch swapping on a maxi- according to Kjer (1995). The initial align-
mumof10treesineachcase.The18SrDNA mentwasbyClustalasimplementedinSSE.
sequencesweremuchlessvariableinlength Theinitialfoldingestimatesforallsequences
and so were aligned as single fragments, were the single optimum folding structure
using the same search protocol (except for obtained from the program mfold version
Homolobus Foerster and Masona van 3.0(Zukeret al., 1999;Mathewset al., 1999)
Achterberg, which we were unable to as implemented at http://bioinfo.math.rpi.
amplifyforthisgene). edu/ mfold/rna with default settings. We
»
AlthoughthebraconidD2–D3regionwas didnotincludethesmallerD3regioninthe
ampli(cid:142)edasasinglefragment,wealignedit secondarystructure–basedanalyses. D
o
infourdata(cid:142)lessimultaneously.Wedidthis Alignment “by eye”.—We also aligned the w
n
toavoidobvious alignment artifacts caused sequences“byeye,”usingtheprimarystruc- lo
a
byadeletionof 50bpfoundinonegenus, ture only. This method can give defensible d
Egipohnedfrours,manandybeo»cfatuhseetwaxeal.acLkaerdgethgeapDs3arpe-- reetsaull.t,s2(0B0e0l;shSaawnchanisdeQt uali.c,k2e0,0119)97b;uBteilsshhainw- ed fro
m
pear to cause problems for all of the align- deredbysubjectivity,especiallyintheorder h
ment programs we have come across, such inwhichtaxaarealignedtoeachother,and ttp
s
that obviously homologous regions at the bynotbeingreplicable.Itisausefuladjunct ://a
c
beginning and ends of gaps are not being toalignmentprograms,especiallyinhelping a
d
aligned together. This problem canbe over- tospotartifacts. em
come by creating multiple data sets so that ic
.o
the regions homologous to large gaps are u
aligned separately. In addition to the de- Tree-BuildingMethods p.c
o
faultgap:substitutioncostinPOYof2:1,we Unfortunately,MLsearcheswerenotpos- m
/s
also explored the robustness of results to sible, given the computational demands of y
s
b
thealignmentprotocolbyrepeatinganalyses our large data set. Using Modeltest version io
wtioitsh,1tw:1,oaanddd3it:i1o.nEaalcghapse:asrucbhstciotuntsiiosntecdosotfr2a0- 3th.0at(PinocsoardpaoraantdingCrraantedahlel,te1r9o9g8e)n,ewiteyfionutonda /article
randomadditionsfollowedbybranchswap- modelimproved the(cid:142)tofthat modeltothe -a
b
pingbySPRandTBRonamaximumoftwo datamuchmorethandidincorporatingany stra
trees (we used only the (cid:142)rst multiple align- otherparameter.Forexample,thedifference c
ment where two were found). To align the in log likelihoods between a Jukes–Cantor t/51
ichneumonidD2–D3regionintwodata(cid:142)les (1969) model (JC) and JC plus a shape pa- /3/4
(D2 and D3), we used the default gap cost rameter (0) was more than twice that be- 5
0
onlyand50randomadditions. tween JC andgeneraltimereversible(GTR) /1
6
Alignment by using ClustalX.—We also models. However,evenafter settingJC 0 16
C 8
used the distance-based program ClustalX parametersfromtheMPT,heuristicsearches 9
0
(Thompsonetal.,1997),againusingarange consistingofasinglerandomadditionwith- b
y
ofgapcosts: We used gapopening costs of out branch swapping took 1 week on a g
u
» e
20, 10, 5, and 2.5 with gap extension costs 250 MHz G3 Powermac and failed to (cid:142)nd s
of10,5, 1, and 0.5, respectively(contraryto more likely trees. We therefore used two t o
n
the default setting, we gave transitions the methods: unweighted MP, and minimum 1
4
same weight as transversions). The D2–D3 evolution(ME). A
p
sequences were subdivided as for the POY WebuiltMPtreesusingtheabovemultiple ril 2
analysis, but each was aligned individually alignments using PAUP (Swofford, 1998) 0
¤ 1
andthenpastedtogetherintoaNexus(cid:142)lefor and found the MPT by using 1,000 random 9
treebuilding. additions with the TBR method of branch
Alignment by using secondary structure.— swappingwithTBRonasingletree,followed
The use of the secondary structure of RNA by an additionalroundofbranch swapping
moleculescanimprovephylogenyinference, on the resulting trees with no restriction on
although the preferred methods are dif(cid:142)- thenumberoftreesheld.Searchesweremade
cult to implement (Page, 2000). Here, we withgapstreatedasmissingdata.
used Page’s alignment editor SSE (obtai- Within the 28S sequences of our taxa is a
ned from http://darwin.zoology.gla.ac.uk/ rangeofcompositionalbiases.Inadditionto
rpage/sse/) to align sequences manually the general reduction in GC content in Bra-
»
with reference to the secondary structure conidae compared with the Ichneumonidae
2002 BELSHAWANDQUICKE—ROBUSTNESSOFANCESTRALSTATEESTIMATES 457
and other Hymenoptera (Belshaw et al., includedmoreaphidiinesequencesinourex-
1998), further marked reductions are evi- pandeddataset (142taxa)toreducebranch
dent within the family. Therefore, because lengths among the Aphidiinae. As a mea-
ofthispotentialproblemofconvergentbase sureoftheaccuracyofthedifferentmethods
composition, we also built ME trees by us- of phylogeny estimation, we noted which
ing LogDet/Paralinear distances in PAUP . analyses failedtorecovertheAphidiinaeas
¤
TheuseofLogDet/Paralineardistancescan monophyletic.WealsoscoredtheAphidiinae
makephylogeneticestimatesmorerobustto asmonophyleticwhentheywere(occasion-
changesinbasecomposition(Swoffordetal., ally)recovered as paraphyletic with respect
1996)andhasreducedthisprobleminother totheirsistergroup,whichwerefertointhis D
o
studies(ChangandCampbell,2000).Inaddi- studyastheMesostoinae. w
n
tiontotheproblem ofcompositionalbiases, lo
a
amoderatedegreeofamong-siteratehetero- d
gseeqnueeitnyce(Ys;anfogr,e1x9a9m6)pilsea,pthpearPeOntYaamliognngmoeunrt UsingthAenpchesytrloagleSntayteesEtsitmimaatetisoonbtainedas ed from
ofbraconidsequenceswithgapcostof2has explainedabove,weestimatedtheancestral h
a shape parameter ® of 0.70 (obtained from statesofthemainkoinobiontcladesbyboth ttp
s
MPT with the JC 0 model; gaps treated MP and ML and assessed the robustness of ://a
C c
as missingdata).Thepresenceofinvariable these estimates by using step matrices and a
d
sitescanmisleadmodel-basedphylogenyes- differencesinlikelihood,respectively. em
timation (Lockhart et al., 1996) unless they First, we estimated ancestral life history ic
.o
are accommodated by the model (Swofford states by using unweighted MP as imple- u
p
etal.,1996),soweincorporatedaninvariable mentedinMacCladeversion4.0(Maddison .c
o
sites model into our ME search in PAUP¤. and Maddison, 1992). The character was m/s
Theproportionofinvariablesites(I)wascal- treated as binary: either koinobionts with y
s
b
culatedfromaMPTandasimpleMLmodel host concealed ( 0) or host exposed ( 1). io
(pJlCexCmI;oudseelosfachltaenrngaetdivtehtereeesstaimndatmesoroefcIomby- AwserdeistcrueassteeddaabsoDmvei,sositnhgerdliaftea.hiIsntoarynaslDtyasteess /article
<1%).Becauseofthevariationinbasecom- of the main braconid data set ( 121 taxa) -a
position,siteswereremovedinproportionto wedeliberately miscoded the lifDe history of bstra
basefrequencies,whichwereestimatedfrom one member of the genus Meteorus Haliday c
constantsitesonly(Swoffordetal.,1996). so as to represent the range of life history t/51
Weassessedtherobustnessofbranchesby states known to occur within this morpho- /3/4
usingbootstrapping(Felsenstein,1985),with logically well-supported genus. Late in the 5
0
1,000 pseudoreplicates for ME phylogeny project weobtainedamemberofthis genus /1
6
1
estimates; for MP phylogeny estimates, we with the relevant life history, so we could 6
8
used 100 pseudoreplicates each of 100 ran- avoid this arti(cid:142)ciality in the analyses of the 9
0
dom additions with branch swapping on a expanded data set ( 142 taxa). To assess b
singletree. the robustness of thDese MP estimates, we y g
u
e
Somepreliminaryphylogenyestimatesof used step matrices as follows. We (cid:142)rst cre- s
theBraconidaerecoveredtheaphidiinetribe atedadatasetconsistingonlyofthelifehis- t o
n
Aphidiini together with two of the ichneu- tory character repeated many times plus a 1
4
tines:MasonbeckiaSharkeyandWhartonplus correspondingnumberofstepmatriceswith A
p
Proterops Wesmael. This result is clearly an the0to1transformationincreasinginincre- ril 2
artifact because, in addition tothe morpho- mentsof0.1,holdingconstanttheweightof 0
1
logicalevidence(QuickeandBelshaw,1999), the reverse transformation. Using this (cid:142)le, 9
several studies using a range of genes have we estimated the state of an internal node
shown the Aphidiini to be a derived clade of interest under each step matrix in a sin-
withintheAphidiinae(BelshawandQuicke, gle PAUP execution, repeating the proce-
¤
1997;Smithetal.,1999; Sanchiset al.,2000). dureforeachphylogenyestimate(withboth
This problem maybe caused by convergent ACCTRAN and DELTRAN transformations).
GC content, with Masonbeckia having the We found the weight at which the estimate
lowestCGcontentinourdataset(0.31),out- ofaspeci(cid:142)cinternalnodebecameeitheram-
sideoftheAphidiinae(whereCGcontentde- biguousorreversedbyloggingtheoutputto
creasesto0.25).Toalleviatethisproblem,we a(cid:142)leandusingaprogramwritteninPython
458 SYSTEMATICBIOLOGY VOL.51
2.1. (PAUP and Python (cid:142)les are available 2. Givethelifehistorycharacteraweightof
¤
at http://www.bio.ic.ac.uk/research/data/ 0,sothetreelengthdisplayedcomesfrom
Ichneumonoid.) theDNAcharactersonly.
Second,weestimatedancestrallifehistory 3. Movebranchestomaketheestimatedan-
states by using ML as implemented in Dis- cestralstateambiguousorreversedwhile
crete 4.0 (Pagel, 1994). Rates of transitions adding the least to the tree length. Al-
between the two states were not restricted though one cannot possibly check all
(estimatedvaluesofthetransitionfromcon- topologies, which even in smaller trees
cealed toexposedwere between 1.1 and 2.7 would be astronomically high, the range
times greater than the reverse on different of candidate topologies is actually man- D
o
phylogeny estimates). Discrete accepts only ageableandcanbecomparedinthisway. w
n
binarydata,andtaxacodedasmissingdata Wefound that only two orthree changes lo
a
for the MP ancestral state estimates were wererequired,movingtaxawiththealter- d
e
pwreurneetdhefnrocmontvheertterdeetsobthyeMfoarcmCalatdree.quTirreeeds nclaatdivee,olirfejohinisitnogrybanseaalreclratdoetshteobgaestheeorf.the d fro
m
forDiscretebyusingtheprogramMoreways 4. After each change, use the search op- h
(writtenbyR.Freckleton,DepartmentofZo- tion in MacClade to try to (cid:142)nd more- ttp
s
ology,UniversityofOxford).Thisformatin- parsimoniousrearrangementsbothabove ://a
c
cludes a basal node with two branches, so andbelowthealteredregionofthetree. a
d
one outgroup taxon was left in; after con- 5. Savethenewsuboptimaltree. em
version, the branch of the outgroup taxon 6. Repeatforeachnodeofinterest. ic
.o
was deleted, together with the branch lead- 7. Repeat,usingotheralignments. u
p
ing to the ingroup. Repeated analyses gave 8. Compare the suboptimal trees with the .c
o
identicalresultsforalldatasets,withnoev- original MPT from which they were m
/s
idenceoflocaloptima.Toassesstherobust- derived.Severaltestsareavailableforas- y
s
b
ness of these ML estimates, we calculated sessing the signi(cid:142)canceof differences be- io
thelikelihood withthenodeofinterest suc- tweentwotrees.WeusedtheShimodaira– /a
cessively set to state 0 and then to state 1, Hasegawa (Shimodaira and Hasegawa, rticle
following Pagel (1999a) in using “local” es- 1999)asimplementedinPAUP¤.Thistest -ab
timates.Weinterpreteddifferences between compares thelikelihoods ofthetwo trees stra
theloglikelihoodvaluesof2.0andaboveas andismoreappropriatetotheaposteriori c
showingsigni(cid:142)cantsupportfortheancestral hypothesis testing here (Goldman et al., t/51
state with the greatest likelihood (Schluter 2000; Buckley et al., 2001). However, for /3
/4
et al., 1997; Pagel, 1999b). All ML ancestral consistencyofapproachwiththeMPma- 5
0
stateestimationswerecarriedoutonME I nipulations,wealsopresenttheresultsof /1
6
C 1
trees, which should provide more accurate the parsimony-based Kishino–Hasegawa 6
8
branchlengths. test (Kishino and Hasegawa, 1989), also 9
0
implementedinPAUP . b
¤ y
g
u
e
RobustnessofMPEstimatetoUncertaintiesin The above manipulations in MacClade s
Phylogeny are very laborious, so here we present the t on
results of manipulating only a single MPT 1
Asmentionedabove,theconsistencyofan- fromeachalignment.Onlyminordifferences 4 A
cestralstateestimatesondifferentestimates p
of phylogeny gives us a crude measure of werefoundwhenother different MPT were ril 2
analyzed; for example, in preliminary anal- 0
their robustness. In addition, we manipu- 1
yses(notshown)wecompared10MPTwith 9
lated MP trees to (cid:142)nd the shortest tree in
theirindividualsuboptimaltrees.In12of13
which theancestralstateestimateforaspe-
comparisons,thelengthdifferencesbetween
ci(cid:142)c mode was reversed or rendered am-
the optimal and suboptimal trees were all
biguous. We could then test whether this
within 20% of the minimum difference
suboptimal treewas signi(cid:142)cantly worse. As
observed.
illustratedinFigure1,ourprocedurewasas
When this method found trees that were
follows:
not signi(cid:142)cantly longer, in which the an-
cestral state estimate had been reversed or
1. Mapunambiguouschangesinlifehistory ambiguous,thenclearlythenullhypothesis
ontoasingleMPTinMacClade. cannotberejected.Whenthedifferencesare
2002 BELSHAWANDQUICKE—ROBUSTNESSOFANCESTRALSTATEESTIMATES 459
D
o
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lo
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d
e
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fro
m
h
ttp
s
://a
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e
m
ic
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6
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1
9
FIGURE1. MethodofassessingrobustnessofMPancestralstateestimatetouncertaintiesinphylogenyestima-
tion.(a)Optimaltree:oneMPTfromtheexpandednoncyclostomedatasetaligned“byeye”(length5,683).For
clarity,thistreehasbeenprunedofadditionalbrancheswithincladesofuniformlifehistory.Notethattheestimated
ancestralstateofthehelconoidcladeisunambiguouslyconcealed.(b)Shortestsuboptimaltreefoundbybranch
manipulationinMacCladeinwhichtheestimatedancestralstateofthehelconoidcladewasnotunambiguously
concealed(length5,704).Twobranchesweremovedto(cid:142)ndthissuboptimaltree:First,outsidethehelconoidclade,
theCanalicephalusbranchwasmovedonenodenearertothebaseoftree;second,insidethehelconoidclade,the
Homolobinaebranchwasmovedonenodenearertothebaseoftheclade.Thesetwochangeslengthenthetreeby
23steps;useofthesearchingtoolinMacCladethenfoundatreeonly21stepslonger(length5,704).(Note:The
branchlabeledCanalicephalusincludesUrosigalphus.)