Syst. Biol. 48(2):233–253, 1999

CombinedMolecular Phylogeneti cAnalysisof theOrthoptera (Arthropoda,Insecta) and Implications for Their Higher Systematics

P. K. FLOOK,1 S. KLEE, AND C. H. F. ROWELL ZoologyInstitute, University of Basel, 4051-Basel,Switzerland

Abstract.—Aphylogenetic analysisof mitochondrial andnuclear rDNA sequences fromspecies of all the superfamilies of the insect orderOrthoptera (grasshoppers,crickets, andrelatives) conŽrmed thatalthough mitochondrial sequences provided goodresolution of the youngestsuperfamilies, nuclear rDNA sequences were necessaryto separatethe basalgroups. To try to reconcile these datasets into asingle, fully resolved orthopteranphylogeny ,we adoptedconsensus andcombined datastrategies. Theconsensus analysisproduced apartially resolved tree thatlacked several well- supported features ofthe individual analyses.However, this lackof resolution wasexplained by anexamination of resampled datasets, which identiŽed the likely source oferror asthe relatively short length of the individual mitochondrial datapartitions. Inasubsequentcomparison in which the mitochondrial sequences were initially combined,we observed less conict. Wethen used two approachesto examinethe validity ofcombining all ofthe datain asingle analysis:comparative analysisof trees recovered fromresampled datasets, andthe application of arandomizationtest. Be- cause the results did not point to signiŽcant levels ofheterogeneity in phylogenetic signalbetween the mitochondrial andnuclear datasets, we therefore proceeded with acombined analysis.Recon- structing phylogenies underthe minimum evolution andmaximum likelihood optimality criteria, we examinedmonophyly of the majororthopteran groups, using nonparametric and parametric bootstrapanalysis and Kishino– Hasegawa tests. Our analysissuggests that phylogeny reconstruc- tion underthe maximumlikelihood criteria is the most discriminating approachfor the combined sequences. Theresults indicate, moreover, thatthe caeliferan Pneumoroideaand Pamphagoidea, aspreviously suggested,are polyphyletic. TheAcridoidea is redeŽned to include all pamphagoid families other thanthe Pyrgomorphidae,which we propose should beaccorded superfamily status. [Combinedanalysis; insect phylogeny;molecular evolution; Orthoptera;ribosomal DNA.]

Wehaveused phylogenies reconstructed The failure ofthese analysesto generate fromnucleotide sequences toexamine the fully resolvedphylogenies maystem from evolutionaryhistory of the insectorder Or- the antiquityof the Orthoptera.Divergence thoptera(grasshoppers, crickets, and rel- datesof the majorgroups are estimated to atives)(Flook andRowell, 1997a, 1997b, range overa period of ~200million years, 1998).Of particularinterest are relationships withfossils of the oldestgroups appearing ofseveral of the higher taxa,and we have in the Permian(Carpenter andBurnham, attemptedto relate our results to existing 1985).Consequently ,althoughthe relatively systematicdisputes. The molecularphylo- rapidly-evolving mitochondrialsequences genies arewell suitedto this task and have haveproven valuablefor examining com- the potentialto resolve several outstanding parativelyrecent events(Flook andRow- problemsconcerning the ecology,character ell, 1997b),the basalbranching patternsof evolution,and biogeographic distributionof the Orthopteraare resolved only by the member taxa.However, the successof these moreslowly evolving nuclearribosomal analyseshas been limitedsince, on their RNAgene sequences (Flook andRowell, own,the different sequences haveproven in- 1998).In contrast,these nuclearsequences adequatefor reconstructing phylogeny over arealmost invariant in the morerecently the whole range oforthopteran evolution. evolved groups,and wewere previously un- Becauseof this, we haveso farbeen unable able todetect signiŽ cant phylogenetic signal toreduce the Žndings ofour work to a single amongthe four youngest caeliferansuper- schema. families.On the basisof these results,we ex- pect thatthe datasets contain enough phy- logenetic signalbetween themto determine 1 Address correspondence to Dr. P.K.Flook,Zool- ogisches Institut, Rheinsprung9, 4051-Basel, Switzer- mostof the majorfeatures of orthopteran land.E-mail:  [email protected]. phylogeny in asingle analysis.The purpose

233 234 SYSTEMATICBIOLOGY VOL. 48 ofthe workreported here wasto identify hood(ML) optimality,weobtaina phyloge- aneffective strategyfor resolving the or- netic scheme in which the majorityof nodes thopteranphylogeny fromthe three data areresolved. sets. Twofundamentally different approaches fortreating multiple phylogenetic datasets arecommonly applied: consensus,and com- MATERIALS AND METHODS bined analyses.Both of these strategieshave been criticized(Bull et al.,1993; de Queiroz, Samples 1993),and selection of one overthe otheris Samples fromall the orthopteransuper- complicated.An argumentin favorof com- familieswere included in thisstudy .In the bining datais that, because mostreconstruc- suborderCaelifera (shorthorned grasshop- tionmethods are consistent (for mostun- pers) seven superfamilies arecommonly derlying tree shapes),an analysis is more recognized(see Dirsh,1975; Rentz, 1991): likely torecover the correctphylogeny as Tridactyloidea(false molecrickets, sand dataare added. On the otherhand, phylo- gropers);T etrigoidea(pygmy grasshoppers, genetic reconstructioncan become compli- grouselocusts); Eumastacoidea (monkey catedwhen datathat evolve atvery differ- grasshoppers,false stick insects); Pneu- ent rates(e.g., mitochondrialand nuclear moroidea( ying gooseberries,desert long- DNA)arecombined in asingle analysis.As horned grasshoppers,razor-back bush- hasbeen demonstrated,when heterogene- hoppers); Pamphagoidea(rugged earth ityexists in phylogenetic signalfrom dif- hoppers, true bush-hoppers); Acridoidea ferent datapartitions, the overallsignal is (grasshoppers,locusts); and T rigonoptery- sometimesdiminished afterpooling ofdata; goidea.In the otherorthopteran suborder , in suchcases, data combination should be Ensifera(long-horned grasshoppers,katy- avoided(Bull et al.,1993; de Queiroz,1993). dids,crickets), we sampledall four super- Bull et al.(1993), discussing the alternatives families(following the higher classiŽcation foranalyzing multiple datasets, maintain ofGorochov ,1995b):Tettigonioidea (bush- thata combining ofdata should be preceded crickets,katydids); Hagloidea (hump- byasearchfor any con ict between the in- winged crickets);Stenopelmatoidea (cave dividualdata sets. crickets,Jerusalem crickets,wetas); and Here we estimaterelationships among Grylloidea(true crickets,mole crickets). In orthopteransfrom new andpreviously pub- additionwe used outgrouptaxa from three lished moleculardata and, in doing so, relatedorders: Phasmida (walking sticks); examine severalimportant aspects of phy- Blattodea(cockroaches); and Grylloblat- logeny reconstruction.First, we considerthe todea(ice-crawlers). A full listof the mate- choiceof reconstruction method in situa- rialused in thisstudy is given in Table 1.The tionswhere contrastingphylogenetic lev- seven taxain whichsequences were deter- els areexamined simultaneously.Second, mined forthe Žrsttime were Rhainopomma wecomparethe use ofboth consensus and montanum (Caelifera, Acridoidea,Lentu- datacombination approaches for analyz- lidae), anunidentiŽ ed species of Systella ing the mitochondrialand nuclear riboso- fromBorneo (Caelifera, Trigonopterygoid- malDNA (rDNA) sequences. Wesuggest ea,T rigonopterygidae), Tanaocerus koebeli thatconsensus methods are too conservative (Caelifera, Pneumoroidea,T anaoceridae), forthe treatmentof the orthopterandata. Xyronotus aztecus (Caelifera, Pneumoroidea, Wedemonstratethat combining nuclearand Xyrolnotidae), Physemacrisvariolosa (Caeli- mitochondrialDNA (mtDNA) sequences in fera,Pneumoroidea, Pneumoridae), Comi- asingle analysisis legitimate, in spite ofthe cus campestris (Ensifera,Stenopelmatoidea, factthat the sequences areevolving atdif- Stenopelmatidae),and Ceuthophiluscarlsbad- ferent rates.Reconstructing trees from the ensis (Ensifera,Stenopelmatoidea, Rhaphid- combined dataset under the criteriaof min- ophoridae).Procedures for collection and imumevolution (ME) andmaximum likeli- storageof material and for DNA isolation 1999 FLOOKET AL.—PHYLOGENETIC ANALYSIS OF THEORTHOPTERA 235 , a a a a a a a b b d a 1 0 3 1 8 0 9 0 2 7 6 6 8 5 4 8 3 7 1 1 4 2 5 4 2 4 0 3 6 5 1 9 2 e e 9 6 7 8 6 9 7 8 6 7 7 8 8 8 8 7 8 6 7 3 7 7 6 7 8 6 7 6 6 7 6 6 9 t — — d s 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 5 5 5 5 5 5 5 5 5 5 5 5 5 S i i l 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 8 o 1 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 e g Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z s a . o h s h p o t n m o n t a o P i a a a a a a a a a a a a a a a a d s , 5 2 3 8 6 7 9 6 2 3 5 1 3 0 9 4 5 5 4 2 3 5 2 8 4 6 3 7 8 7 9 0 1 6 1 s n a 8 1 9 1 9 9 9 1 0 0 1 2 2 2 1 0 0 2 1 1 1 1 1 2 1 2 1 1 1 1 2 1 2 2 2 e S o e c 2 6 2 6 2 2 2 6 3 3 6 6 6 6 6 3 3 6 6 3 3 3 3 6 6 6 3 6 3 3 6 3 3 3 6 6 c p d 3 7 3 7 3 3 3 7 3 3 7 7 7 7 7 3 3 7 7 3 3 3 3 7 7 7 3 7 3 3 7 3 3 3 7 1 a i s 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 e o L Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z r d B r i o r M c c E A e a a a a a a a a a a a a a a a r a s 7 6 5 1 8 9 1 0 4 5 9 5 7 4 3 6 7 9 8 4 3 5 7 6 2 8 8 7 9 0 1 2 3 0 e 1 e 4 9 5 0 5 5 6 0 6 6 9 0 0 0 0 6 6 0 9 7 7 7 7 0 0 9 7 9 7 8 1 8 8 8 1 h i S l 2 5 2 6 2 2 2 6 2 2 5 6 6 6 6 2 2 6 5 2 2 2 2 6 6 6 2 5 2 2 6 2 2 2 6 2 i 3 7 3 7 3 3 3 7 3 3 7 7 7 7 7 3 3 7 7 3 3 3 3 7 7 7 3 7 3 3 7 3 3 3 7 d 1 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 e m a Ž Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z i f t r n e e p d u i s s e s a d s i s e r i i s s s s a s n r h o i e s s s m a m s n u t i t e e i i f u s i s u e i s m c r r u c u a m n i s c i c t o t t t o i i s s o a o a d s r e r s t n s a a e l l a s r s t t e r a r i i e a o o a d n t l s t l i t l a s e e a l a s i l f n p u s u i a t b l i b a d i s r s t a i s i e r e s c i i e e p p E u o c s u s S s d e t t d i u t d c i t r n h b u u r n e s l n s i c m i h r  e d . . . s c f r m r r e t n c e n a t m r m f o u e b u r a o a t o r r d i a e i o o i n p o z p p u o o o r u a r a a r u c a u e e o o n n r t c m t s s c m i v k a r s t r c d s c a k v n c c c m c d b c r p o a p s a e r r e r f s a i a o l i u n c l s e i a p x a x u ) h a i a s r p l a s r r a m 5 i h t s a u t a i i C s t y i o r s l t s a t s r 7 p r t a e s i r m i u e t a d a r a e c a u t u x 9 d c e r r c a o n h r u i a i a n s i o n a r a o s a a i e l m 1 t i h t o h o d p p m e e e m n i h r a a e s ( h t r t d d b t o a u l e m u h o o l l o m m c o o a i a a h d p c r e d c r i G l l c o u o m o n i o e e t h o h p p r i h f l n t g i l l p a d a e e t h a n h i h t s e l i t p t l l u o c i i a m g l u t t a i r s s e o s t r m s a l e p s m d a o l m p r r y h y m y y o t s s r u a o o n r i d t e o e e o c l y r c g r r h y u o e y i a r y u n h r u a h s y y t a e n p G A O R G B P P B P P T X S S P S E H I P N C T R C H C C G A P A G G D u u o r s g n e e c s i a e e a d a a e m i e e e d t a o d a i e a i a d e e r d n a i d e g i d e a i a e o o e t i o e m d e a e y e y e a t a l i d h d e x a e h s a a i a h r i a i i a d a d d a a t t p i a h p d e e i e d i d d p t d t m i d r t i e c e i i d i d c o h r i a m e e g r d l a a i e r i i m r o i l a a a p r i l a o t p d a a F d a r o e y a r t e n o e p i o i d d b h t o o l m e c m l s d h i d i o d d p m o h c c h n i i o m i n o o h h a n d l u g l l p c d g o l o a a n g o p l i l l t c u c i a i o g i i s u r g r t n l a d r r g s m d m e y r y y t n m n n e t t o i i i i h e a c r r y y h e u u a a y n r h s a r a r e r e t e c A L P P P T X T P E E B T T C T S M R H G P P G P h A T e . h y . T a d y e . u d a t y d l e s i u t e o y d s a a s l i a v a i i g a e i e s e o e i t e h y t d m d t c d h r i i d d a a i t a i a e i e o f o a a o t n e r e o r o p c m i e g r l e i o e l p s d d a f a o i y d i t e p n d e o i t d i s h o r o u o d p . c n e o m o a e S g l p , d g o l a a o i n l u ) i i n i e g r t n i r g m d e y m b t t i i a e a c d r u a n t m 5 r e r e t i . b a 9 A P P T E T T T S H G o y o 9 x g d 1 e s y ( u e r t e c s l e v n t s p a e o r i p a r e u h h r m o e t d q c e f a r n e i f o n s l o i s o i r e s b g c o d d a i i u n e e r G S C E h d m T s i u o d l l d n n c b n o a n u i a x p ) t , a a n 5 o a e T u 7 n e d 9 s y d o l 1 i e t . a s ( c t o r 1 a u r a a n e h l o d e t e E o i r i s b u L e p v d r o i m q B e d m o o l r e r l t s u A h D t S P e t a y O T a r r a b l n h y b P O P G B 236 SYSTEMATICBIOLOGY VOL. 48 havebeen published previously (Flook and used likelihood ratiotests (LRT s)to assess Rowell,1997a, 1998). the statisticalsigniŽ cance of differences be- tween likelihood scoresobtained for a given Sequence Data phylogeny under variousassumptions. The Weobtained3,177 bp (totalalignment phylogeny in questionwas estimated by length) ofsequence fromthree genes: 393 using equally weighted parsimony.Likeli- bp of the 30 half ofthe mitochondrialsmall- hoodswere calculatedwith the computer subunit rRNAgene (12S);558 bp fromthe programP AUP *4d61(test version provided 30 half ofthe mitochondriallarge-subunit by D.Swofford,Smithsonian Institution). rRNAgene (16S);and the complete se- Wecalculatedthe teststatistic as 2(ln L 0– ln quence (2,226bp) ofthe nuclearsmall- L1) = –2 ln L, where L0 and L1 arethe likeli- subunit rRNAgene (18S). Fordetails of hoodsunder the null andalternatehypothe- the PCRampliŽ cation of the mitochon- ses,respectively .The signiŽcance of the LRT drialgene sequences, see Flookand Rowell statisticwas testedby using a x 2 distribution (1997a).Nuclear 18S sequences were ampli- with n degrees offreedom, where n is the Žed, cloned,and sequenced asdescribed in number ofparameters that differ between Flookand Rowell (1998). All sequences have substitutionmodels. been depositedin the EMBLdatabase;a list Phylogenies were reconstructedand an- ofthe correspondingaccession numbers is alyzedby using PAUP *.Heuristicsearches given in Table 1. were conductedunder the maximumparsi- Rawdata were analyzedwith the Li-Cor mony(MP), ME,andML optimalitycriteria. Image-Analysissoftware (version 2.3) and ForMP weperformed equally weighted and contigassembly was performed by using characterstate– weighted heuristicsearches. the AssemblyLign package(Oxford Molec- Under the ME optimalitycriterion, after the ularGroup). New sequences were addedto resultsof the preliminary likelihood analy- the existingsequence alignmentsand edited sessearches, we used the generalized nu- manuallywith use ofthe SeqApp program cleotidesubstitution model of Hasegawa (D.Gilbert,Univ .Indiana).W eattemptedto et al.(1985), HKY85, with and without a resolvedifŽ cult regions of the DNAalign- gammacorrection for among-site rate vari- mentby referring tothe secondarystructure ation.W ecalculatedthe shape parameterby ofthe three genes (Flook andRowell,1997b, using the methodsof Sullivan et al.(1995) andunpubl. data);in severalregions, how- andY angand Kumar(1996) as implemented ever, the alignmentremained uncertain,and in PAUP*.Tocalculatephylogenies under we omittedthese regionsfrom subsequent the MLoptimalitycriterion, we used anit- analyses.A computerŽ le containingthe se- erativestrategy (Swofford etal., 1996). Ini- quence datain NEXUS formatis available tially,weused aMPtree asastartingtree for fromP .F.K.onrequest. the analysis.W ethen performed aheuris- ticsearch, using the optimalsubstitution Analyses modelidentiŽ ed in the preliminary like- Substitution patterns. —Preliminary anal- lihoodanalysis (see above),and simulta- ysesindicated that sequences differ from neously estimatingmodel parameters. W e eachother with respect to base composi- used the resulting phylogeny asthe starting tion(determined by comparisonof uncor- pointfor another search and repeated this rectedand LogDet nucleotide substitution processuntil the analysisconverged ona distances:Lockhart et al., 1994), variability , single phylogeny.ForME andMP trees,we anddistribution of among-site rate varia- used nonparametricbootstrap analysis to tion(through estimationof gamma distribu- assessthe conŽdence thatcould be attached tionshape parameters).To examine the un- tothe individualnodes. derlying patternsof nucleotide substitution Effectof sequence lengthon phylogenyestima- in moredetail, we adopteda MLapproach tion.—Weadopteda resamplingapproach (e.g., see Huelsenbeck andCrandall, 1997). tosimulate the effect ofincreasing sequence Tocomparethe Žtofsubstitution models, we length andexamined itsin uence onphylo- 1999 FLOOKET AL.—PHYLOGENETIC ANALYSIS OF THEORTHOPTERA 237 genetic reconstruction.W esampledthe orig- the samehistorical events. T operform the inaldata sets with replacement togenerate test,we then comparedthe observedtree-to- new samplesof various sequence lengths. tree distancefor phylogenies reconstructed Foreach sequence length we generated 50 fromtwo data sets against the correspond- new samples.New samplesof between 100 ing null distributions.This has three pos- and2,000 bp in sizewere generated in 100- sible outcomes.First, if the observedtree- bp increments;new samplesof between to-treedistance is less than the 95%value 2,500and 5,000 bp were generated in 500- ofboth null distributions,we donot reject bp increments.Resampled data sets were the hypothesisthat the observeddifferences generated withuse ofa programwritten by aredue tosampling error. Second, if the ob- P.K.F.(DOS programavailable from P .K.F. servedtree-to-tree distanceis greater than onrequest). Phylogenies were reconstructed the 95%value in only one ofthe compar- by twomethods: heuristic searches under isons,this indicates that the errorassociated the MPcriterion,and heuristic searches un- withone ofthe datasets is greater than that der MEcriterion(HKY85 distance). W edid withthe otherbut wouldnot be sufŽcient notperform analysesunder the MLopti- groundsto reject combination.However ,the malitycriterion because computationtimes thirdpossibility ,thatthe observedtree-to- were prohibitive. In caseswhen multiple tree distanceis greater than the 95%level in treeswere obtainedfor data sets, strict con- bothcomparisons, is explicable only fordata sensustrees were retainedfor analysis. The setsthat estimate signiŽ cantly different trees resultsof searches were then assessedby andso combinationof the datawould not be calculatingthe averagesymmetric differ- appropriate. ence (Penny andHendy ,1985)between all Parametric bootstrapanalysis of ME trees. — treesin different sizesamples (by using the Weused parametricbootstrapping to as- TREEDIST commandin PAUP *).Using this sessthe possibilitythat the presence ofa approach,we were then abletoobtain a mea- given grouping in atree mightbe the re- sure ofconvergence withresampled data sultof cumulative phylogenetic errorin setsupon anunspeciŽed tree. componentbranches, rather than a signiŽ- Randomizationtest ofphylogenetic hetero- cantphylogenetic signal.Although thisap- geneityamong data partitions. —The random- proachhas been previously applied in alike- izationtest of Rodrigo et al.(1993) was used lihoodframework (e.g., Huelsenbeck and toinvestigate the legitimacyof combining Rannala,1997), as indicatedabove, the com- the databy examining the hypothesisthat putationtimes for calculating ML treeswere differences between phylogenies estimated prohibitive. Insteadwe implemented the ap- fromdifferent datasets were due tosam- proachunder the criterionof ME optimality. pling error.The testhas the advantagein Togenerate parametricbootstrap samples, the present situationof being applicable un- weinitiallyreconstructed ME treeswith spe- der anyoptimality criterion; we used itto ciŽc constraintsenforced (i.e., monophyly of examine datacombination under ME.The agiven group) andused HKY85distances Žrststage of performing the testinvolved toestimate sequence divergence. Using the generating twosets of 500 nonparametric resulting topologiesand branch lengths, a bootstrapsamples from each of the individ- computerprogram simulated 100 new data ualdata partitions and from the 12S+ 16S setsof the samelength asthe originaldata datapartition. After estimatingtrees from set.The programincluded Csourcecode the bootstrapreplicates by using ME,wecal- fromthe Siminatorprogram (J. Huelsen- culatedsymmetric differences foreach of the beck, Univ.California-Berkeley) togener- 500pairs of trees, using the computepro- atethe transitionprobabilities under the gramComponent (Page, 1993).The result- HKY85model. Empirical estimates of base ing tree-to-tree distanceswere then plotted frequencies were used togenerate ancestral asfrequency histograms,which shouldcor- sequences in the simulation,and among- respondto the null distributionof tree-to- siterate variation was incorporatedby using tree distanceswhen twodata sets estimate agammashape parameterestimated from 238 SYSTEMATICBIOLOGY VOL. 48 the originaldata. Phylogenies were recon- perfamily (including Gryllus and Acheta), structedfrom the simulateddata under the but analysisof the 18Ssequences indicated MEcriterionand the resulting treessum- thatthey were extremely diverged fromthe marizedin amajorityrule consensustree. otherorthopterans. A preliminary analysis Wethen calculatedthe parametricbootstrap ofthe patternof variation in these genes support(PBS) forthe prespeciŽed node as indicatedthat any ensiferan phylogeny in- the percentage oftimes it was recovered in cluding the grylloid 18Sgene sequences is the consensus.W einterpreted the PBSvalue unlikely tocorrespond to the species phy- asindicative of whether adeparture from logeny (unpubl. results).However ,because the given hypothesis(i.e., nonmonophyly of the Grylloidearepresent animportant en- agroup) mightbe explicable by phyloge- siferangroup, wehaveattempted to recon- netic errorin the dataset. Thus, if we are cile theminto this analysis by performing a interestedin the absence ofa speciŽc group- separateanalysis of the mtDNA. ing in ourreconstruction, a very lowPBS The mainproperties ofthe DNAse- value wouldsignify high levels ofrandom quences aresummarized in Table 2.The errorin the datafor that hypothesis, and lit- alignmentlengths of12S, 16S, and 18S se- tle importanceshould be attachedto the ob- quences were 393,558, and 2,226bp, respec- servationof polyphyly in the originalanal- tively.After removalof ambiguous align- ysis.Conversely ,ahigh PBSvalue would mentpositions, the lengths ofthe threegenes beindicativeof low levels ofphylogenetic were reduced to316, 448, and 1,773 bp, re- noiseassociated with an hypothesis,and its spectively,giving atotalof 2,537 bp. The observationwould be therefore signiŽcant. proporionof variable sites in the mtDNA Kishino–Hasegawa T ests. —To compare (67.1%)was three timesgreater than that in competingphylogenetic hypotheses un- the 18Sgene (21.6%).The nucleotide com- der the MLoptimalitycriterion, we used positionsof the sequences alsocontrast. T a- the Kishino–Hasegawa test (KHT; Kishino ble 2emphasizesthe high A+Tcontentin andHasegawa, 1989) as implemented in the mitochondrialsequences (69.5%)com- PAUP*.TotestspeciŽ c hypotheses ofmono- pared withthe 18Ssequences, whichhave phyly,wereconstructedphylogenies under effectively nobias (52.7%). V ariationin base the constraintof monophyly forthe given composition,particularly in the mtDNA,is group, using the CONSTRAINTS optionof alsopresent between different taxonomic PAUP*.WeestimatedML parametersand groups(see Table 3),although the phy- treesby using the iterativeapproach de- logenetic componentof this variation is scribedabove. In thisway we were able to minimal.For example, the highest average recoverthe besttree compatiblewith a given mtDNAA +Tcontent(72.4%) is recorded in hypothesisof monophyly . the Trigonopterygoidea,whereas the second lowestmtDNA A +Tcontentis recorded RESULTS AND DISCUSSION in the Pamphagoidea(67.5%), a relatively closelyrelated group. Becausesuch vari- Sequence Data ationcan be confounding in phylogeny In additionto data collected in the previ- reconstructionin the mtDNAsequences ousstudies, we determined new sequences (Flook andRowell, 1997b), we alsoused in atotalof 15 species. Both mitochondrial the LogDet transformation(Lockhart et al., andnuclearsequences were obtainedfor the 1994),which corrects for distortions in phy- Žrsttime in seven ofthose taxa, and in the logenetic signalthat are due tocomposi- othereight we sequenced thosefragments tionalbias. Although treesbased on uncor- notincluded in the earlierwork. One impor- recteddistances (p-distance, Jukes– Cantor tantomission from the datareported here distance)differed fromLogDet trees,we isthe 18Ssequences frommembers ofthe detected nosuchdifferences between trees ensiferan Grylloidea(crickets, mole crick- thatwere basedon distances corrected for ets,and related insects). W eobtainedse- unequal basecomposition (e.g., HKY85). quences fromseveral members ofthis su- Therefore werejected the conditionof non- 1999 FLOOKET AL.—PHYLOGENETIC ANALYSIS OF THEORTHOPTERA 239

TABLE 2.Descriptive statistics forseparate and combined datapartitions.

Datapartition 12S 16S 18S 12S+ 16S12S + 16S+ 18S Initial length (bp) 393 558 2226 951 3177 Finallength (bp) a 316 448 1773 764 2537 No.parsimony-informative sites 175 225 176 400 576 No.variable sites (%of Žnal) 217(68.7) 296 (66.1) 383 (21.6) 513 (67.1) 896 (35.3) A 0.31 0.32 0.25 0.32 0.27 C 0.11 0.12 0.23 0.11 0.20 G 0.17 0.20 0.28 0.19 0.25 T 0.41 0.36 0.24 0.38 0.28 MPtree length ( n)b 1,086(16 ) 1,378( 4)735(24 ) 2,516( 3)3,274( 1) CIc 0.35 0.38 0.67 0.36 0.43 RId 0.41 0.38 0.66 0.37 0.43 Gammae MP, YK 0.733 0.676 0.166 0.670 0.204 MP, S 0.802 0.799 0.253 0.777 0.246 ME, YK 0.714 0.656 0.146 0.669 0.203 ME,S 0.784 0.782 0.205 0.771 0.244 a Length of sequenceafter removal of ambiguousalignment sites. b n =numberof treesrecovered. c Consistencyindex for MPtree. d Retentionindex for MPtree. e Estimateof gammashape parameter from MPandME treesby methods of Yangand Kumar (1996) and Sullivan et al. (1995). stationarityof base composition bias as re- indices (CIs) were relativelylow in the quiring anyspecial attention in thisanalysis. mtDNAsequences (average= 0.36),re ect- ing the decreasedlevels ofcharacter congru- Propertiesof Parsimony T rees ence atthe baseof the orthopteranmolecu- Weinitiallyreconstructed phylogenies by larphylogeny .The CIforthe 18Sdata was equally weighted parsimony.The resulting higher (0.67),although the number ofinfor- treesare not shown here but their lengths mativesites (210) was approximately half andcorresponding statistics are summa- thatin the mtDNAsequences (402).Non- rizedin Table 2.V aluesfor consistency parametricbootstrap support (NBS) forthe

TABLE 3.Summary of phylogenetic analysesof orthopteransuperfamilies andphasmids.

A + T% Group 12S + 16S 18S 12S+ 16S+ 18S AutapomorphiesNBS a Caelifera 0.6978 0.4953 0.5523 0 89 Acridoidea 0.6939 0.4947 0.5509 0 0 Pamphagoidea 0.6746 0.4963 0.5466 0 0 Pneumoroidea 0.6892 0.4940 0.5496 0 0 Trigonopterygoidea 0.7243 0.4931 0.5606 9 100 Eumastacoidea 0.7099 0.4971 0.5562 0 11 Tetrigoidea 0.7127 0.4993 0.5588 7 100 Tridactyloidea 0.7133 0.4924 0.5525 7 100 Ensifera 0.6773 0.4960 0.5471 0 97 Tettigonioidea 0.6709 0.4954 0.5450 2 100 Stenopelmatoidea 0.6827 0.4971 0.5498 1 52 Orthoptera 0.6934 0.4955 0.5512 0 87 Phasmida 0.7148 0.4950 0.5543 19 100 aNonparametricbootstrap support. V aluescorrespond to thenumber of timesa particulargroup was recovered as monophyletic in1000 replicates. 240 SYSTEMATICBIOLOGY VOL. 48

TABLE 4.Maximum likelihood analysisof hierarchical substitution models forthe combined sequence data set. Thelog likelihood of amaximumparsimony phylogeny was estimated underdifferent models of nucleotide substitution. Likelihoods were assessedwith likelihood ratio tests asdescribed in Materialsand Methods. Models compared:JC69, Jukes and Cantor (1969); F81, Felsenstein (1981);HKY ,Hasegawa,Kishino, andY ang(1985); GTR,general time-reversible model (Lanaveet al.,1984); GTR + G,generaltime-reversible model with gamma distribution correction foramong-site variation.

H0 vs. H1 ln L0 ln L1 –2 ln L df P JC69 vs. F81 –19621.64 –19518.29 206.7 3 < < 0.001 F81vs. HKY85 –19518.29 –18912.96 1210.66 1 < < 0.001 HKY85vs. GTR –18912.96 –18864.14 97.64 4 < < 0.001 GTR vs. GTR + G –18864.14 –18323.86 1080.56 1 < < 0.001

unweighted parsimonytrees was generally marizedin Table 4,indicate that as more low,particularlyamong the higher caelifer- parametersare added to the substitution ans.W ealsoestimated character transition model,the Žtofthe modelto the combined matricesfrom these treesand reconstructed sequences issigniŽ cantly improved as as- phylogenies byusing weighted parsimony. sessedby likelihood ratiotests. Therefore, Forcharacter-state weighting weperformed forsubsequent analyseswith ML, weused severalsearches, using arange oftransi- the GTRmodelwith a gammacorrection tion:transversionweightings varyingfrom foramong-site rate variation. However, for 1:2to 10:1. As in the equally weighted par- analysesunder theMEcriteriawe decided to simony,severalnodes were poorlysup- use the transformationbased on the HKY85 portedin nonparametricbootstrap analy- model.This model is still a goodapproxi- sis.W econcludedthat the lowoverall level mationto the underlying patternof substi- ofcharacter congruence in the individual tutionbut hasa smallervariance than the genes atspeciŽ c phylogenetic levels pre- GTRdistancebecause ofthe lowernumber ventsthe parsimonymethod from effec- ofparameters,— adesirable property forre- tively recovering phylogeny forthe entire constructionof phylogenies fromdistance order.Character-state weighting issimilarly data(Kumar et al., 1993). ineffective because ofthe contrastingpat- ternsof substitutionat the different phylo- genetic levels in the datasets, particularly Reconstruction andConsensus Analysisof ME in the mtDNA.For this reason we didnot Trees pursue parsimonymethods further . Phylogenies were reconstructedunder the MEoptimalitycriteria from HKY85 dis- Analysisof Substitution Methods tancematrices as described above(Fig. 1a). Amoreeffective wayof analyzing the Theshapesof the mtDNAtrees are very sim- datathan by parsimonyis to use model- ilar,with short branches between the basal basedapproaches, namely ,MEandML. groups,whereas the 18Sphylogeny shows However,toapply these optimalitycrite- the reverse pattern.The symmetricdiffer- riaeffectively ,one mustidentify anappro- ence, ameasureofthe amountof congruence priatesubstitution model. For this purpose between twotrees (Penny andHendy,1985), we used alikelihood approachto examine between the 12Sand 16S trees was 44, and ahierarchyof different models.W eŽrst the averagedistance between the mtDNA examined the assumptionof equal base treesand the 18Stree was38. Thethree trees frequencies, then the assumptionof equal were comparedin astrictconsensus, and transitionand transversion rates, then the the results(Fig. 1b) conŽrm that the level generaltime-reversible (GTR)model (where ofcongruence between datasets is low ,the allsubstitution probabilities are assumed to only signiŽcant feature oforthopteran phy- beindependent), andŽ nally the assumption logeny recoveredin thisanalysis being the ofequal ratesamong sites. The results,sum- monophyly ofthe Caelifera. 1999 FLOOKET AL.—PHYLOGENETIC ANALYSIS OF THEORTHOPTERA 241

FIGURE 1.Consensus analyses of minimum evolution (ME)trees. Combineddata partitions areindicated by “+”symbol (e.g., 12S + 16S);different datapartitions included in consensus tree areindicated by“ &”symbol (e.g.,12S & 16S).(a) HKY85 ME phylogramsfor 12S, 16S, combined (12S+ 16S),and 18S data partitions. (b)Strict consensus for12 S&16S& 18Strees. (c) Strict consensus for(12S + 16S)& 18Strees. (d)Adams consensus for(12S + 16S)& 18Strees. (e) Agreement subtree for(12S + 16S)& 18Strees. Abbreviations fortaxa: Gom = Gomphocerippus ; Acr = Acrida; Oed = Oedipoda; Rha = Rhainopomma ; Gla = Glauia; Bat = Batrachotetrix ; Prosph = Prosphena; Pyr = Pyrogomorpha ; Bul = Bullacris; Pne = Pneumora; Phys = Physemacris ; Tan = Tanaocerus; Xyr = Xyronotus; SysR = Systellaraf esi ; SysB = Systella sp.; Pros = Prosarthria ; Sti =Stiphra; Eus = Euschmidtia ; Hom = Homeomastax ; Tetr = Batrachideidaesp.; Par = Paratettix; Neo = Neotridactylus ; Cyl = Cylindraustralia ; Tetti = Tettigonia; Rus = Ruspolia; Com = Comicus; Hem = Hemideina; Ceu = Ceuthophilus ; Cyp = Cyphoderris ; Phyl = Phyllium; Aga = Agathemera; Gry = Grylloblatta ; Gro = Gromphadorhina .

Previousanalysis suggests that the in- ference =30).The sourceof disagreement dividualmtDNA sequences aretoo short between the different treesis clariŽ ed by toprovide resolutionat deep levels, and adoptinga different comparisonmethod. In thatcombining sequences greatlyimproves anAdams (1986) consensus, where nestings the effectiveness ofreconstructions (Flook withinthe individualtrees are preserved, andRowell, 1997b). W etherefore performed manyof the morerecently evolved caelif- aMEsearchfor the combined 12S+ 16S erantaxa are represented in the Žnaltree data(Fig. 1a)and used the resulting tree (Fig. 1d);however ,severalunlikely group- in asecondstrict consensus analysis with ings arealso contained in thistree (e.g. the 18Sdata. The consensustree obtained Eumastacidae+ Tetrigoidea).A thirdcom- (Fig. 1c)contains several more bifurca- parisonmethod, agreement subtree anal- tionsthan does the tree recoveredfrom the ysis(Finden andGordon, 1985), excludes consensusof the 12S,16S, and 18S data the conicting clusters from the Žnalphy- partitions,although the overallcongruence logeny (Fig. 1e).This reveals that the incon- between the treesis still low (symmetric dif- sistenciesare precisely thoserelationships 242 SYSTEMATICBIOLOGY VOL. 48 previously demonstratedto be the result ofreconstruction failure attributableto systematicerror (Flook andRowell, 1997a, 1998):long branchattraction between eu- mastacoidand tridactyloid mtDNA se- quences, very shortinternodes between the pneumorid andpyrgomorph lineages in the mtDNAphylogeny ,andpoor resolution between tetrigoidand eumastacid 18S se- quences. Thus,the lowlevel ofcongruence between the individualphylogenies reects alackof resolution of the individualdata setsrather than serious con ict in phyloge- netic signal.

Effectof Sequence Lengthon Phylogeny Reconstruction Toinvestigateour concerns about the con- tributionof the length ofsequences tophylo- genetic recoveryof these data,we examined theeffect ofincreasing sequence length ofse- quences fromthe different datapartitions. Havingfound no“ correct”phylogeny for FIGURE 2.Analysis ofthe effect ofsequence length the whole 33-taxondata set, we used agree- onthe effectiveness of phylogenetic reconstruction for mentamong the recoveredphylogenies as different sequences. Eachdata point represents the av- eragesymmetric difference between 50phylogenies re- the criterionby whichto judge the successof constructed fromrandom data samples of eachindi- the searcheson different sequence lengths. cated length.Random samples were obtainedby re- Generatingrandom samples from 12S, 16S, samplingwith replacement fromthe alignmentof the and18S sequences, we reconstructedME individual genes. u= 12S; n = 16S;s=18S.Thick line phylogenies fordifferent sequence lengths (comb.)represents apower function Žtted forthe data andmeasured the degree ofcongruence by points estimated forthe combined dataset. calculatingthe averagesymmetric differ- ence between trees.The results(Fig. 2)show strongphylogenetic expectationand for thatcurves obtained for the different se- which the individualdata provided very quences arevery similar.This indicates that clearresolution. W ethen reconstructedtrees aftercompensating for sequence length, the by using equally weighted parsimonyand overallability of the sequences toresolve the ME.Forthe lattermethod we used equal- phylogeny isapproximately equal. None of rateHKY85 distances. The results(Fig. 3) the lines converge onan average symmetric showthat the MEanalysisconverged on difference of0, but thisis explained bythe arelativelysmall number ofclosely re- variousdeŽ ciencies ofthe individualdata latedtrees (i.e., averagesymmetric differ- setsdiscussed above. In addition,when we ence closeto 0). These treesmatched the generated samplesfrom the combined 12S expectationfor the 12taxa (see legend for +16S+ 18Sdata (Fig. 2),the curve Žttedto Fig. 3).In contrast,the parsimonyanalyses these datawas very similarto that obtained clearlyfailed toconverge ona single tree. forthe individualdata partitions. Wealsoused thissimulation strat- egy toexamine ourreservations about Combinationof Data Sets the use ofparsimony for reconstructing The resultsof the aboveanalyses indicate phylogenies fromthe data.W egener- thatthe limitedrange overwhich the dif- atedrandom samples for a subsampleof ferent sequences resolveorthopteran phy- the taxa(Fig. 3)for which there wasa logeny isdue moreto the shortlength ofthe 1999 FLOOKET AL.—PHYLOGENETIC ANALYSIS OF THEORTHOPTERA 243

nationfor the mitochondrialdata, using incongruency indices,and demonstrated thatcombining the 12Sand16Ssequences is justiŽed (Flook andRowell, 1997b). Further- more,the convergence in the phylogenetic reconstructionsobserved in the analysisof randomlysampled sequences providesone line ofsupport that legitimizes the com- binationof nuclear and mitochondrial se- quences. AŽnalsource of support comes fromapplying the testof Rodrigoet al. (1993)for heterogeneity in phylogenetic sig- nal.Comparison of the observedsymmetric differences (Table 5)withthe estimatednull distributions(Fig. 4)showsthat in none of the comparisonsdo the observedtree-to- tree distancesexceed the 95%level. Inother words,we donotreject the null hypothesis thatthe observeddifferences between phy- logenies reconstructedfrom different data setsare due tosampling error. However ,it

FIGURE 3.Analysis ofthe effect ofsequence length isnotable that the outcomeof the testfor onthe effectiveness ofphylogenetic reconstruction the 12Sversus 18S comparison is very close fordifferent methodsand optimality criteria. Com- tothe 95%level. Weattributethis to the bined datasets fromsequences fromthe follow- limitationsof the symmetricdifference in- ingtaxa: two acridoids ( Acrida, Gomphocerippus ), the dex,in which rearrangementsof only one or pamphagoids,the tetrigoids, the tridactyloids, and twotaxa can result in maximaldifferences two outgroups (the phasmids).The expected phyloge- netic relationships between these taxasare (Phasmida (Penny andHendy ,1985).W ealsonote that (Tridactyloid (Tetrigoidea (Pyrgomorphidae(Pam- manysuboptimal trees were very closeto phagidae,Acridoidea))))). Eachdata point represents the MEtree in reconstructionsfrom the mito- the averagesymmetric difference between 50phyloge- chondrialdata. Comparing the Žrst10 sub- nies reconstructed fromrandom data samples ofeach optimaltrees for the twodata sets, we found indicated length.Random samples were obtainedby that20% of the comparisonsproduced crit- resamplingwith replacement fromthe alignmentof the icalvalues that would not be rejected by combined sequences. Results areshown forsearches performed with MP( n) and ME (u). the estimatednull distributions(i.e., < 46). Thissuggests that the symmetricdifference mightbe aninadequate metric; therefore, individualsequences thanto con ict or ab- we recalculatedthe null distributions,us- sence ofphylogenetic signal.W ehaveal- ing analternative and potentially more readyexamined the issueof data combi- discriminatingcomparison measure: the

TABLE 5.Results of randomizationtests. Numberscorrespond to the percentage ofthe estimated null distri- bution (see Fig.4) lower thanthe calculated symmetric differences between the MEtrees forthe compareddata partitions.

Sym.diff.(AS) a 12S 16S 12S + 16S 18S 12S vs. 16S 44 (16) 88.3 (82.2) 89.26 ( 0.8) — — 12S vs. 18S 40 (15) 47.0 (70.2) — — 93.8 (86.4) 16S vs. 18S 32 (13 — 10.0 (49.9) — 49.9 (62.6) 12S+ 16Svs. 18S 26 (12) — — 16.4 (50.8) 43.6 (40.8) aFiguresin parentheses correspond to results when thetree comparisons are made usingthe agreement subtree (AS) distance providedin Component (Page, 1993). 244 SYSTEMATICBIOLOGY VOL. 48

MEcriterionand used nonparametricboot- strappingto assess the conŽdence thatcould be associatedwith individual nodes. Recon- structionsbased on equal-rate and gamma- correctedHKY85 distances recovered very similartrees and levels ofbootstrap sup- port.The equal-ratetree (Fig. 5)isequidis- tantfrom the 12S+ 16Sand the 18SMEtrees (symmetricdifferences of20 and16, respec- tively). Wealsoreconstructed trees under the MLoptimalitycriteria, using the GTR modelwith a gammadistribution correc- tionfor among-site rate variation. The ML tree (Fig. 6)is very similarto the MEtree (symmetricdifference =16).The mostim- portantdifference isthat the Tetrigoideaare recoveredin acladewith the Proscopiidae. Anotherinteresting difference isthat the Ha- gloideaare recovered as the primitive en- siferanlineage.

Assessment ofIndividual Nodes Becausethe different approachesused in ouranalysis recovered similar trees, a re- mainingtask was to assess the conŽdence thatcould be attachedto those relationships. Wechosethree strategiesfor this. First, weuse nonparametricbootstrapping (Efron, 1982;Felsenstein, 1985)to examine the con-

FIGURE 4.Frequency distributions forsymmetric Ždence thatcould be attachedto speciŽ c differences between trees calculated frompairwise nodesin MEtrees.Second, we performed comparisons of bootstrapped datasamples. Solid bars parametricbootstrapping to examine the correspond tothedistribution of treedistances basedon signiŽcance of prespeciŽ ed groupsin the agreementsubtree differences; white barscorrespond MEtrees.This latter approach is particularly to tree distances calculated byusingsymmetric differ- attractiveby offering exibility in examina- ences. tionof phylogenetic hypotheses (Huelsen- beck etal., 1996; Huelsenbeck andRannala, agreement subtree distance(Finden and 1997).Third, we used the KHT tocompare Gordon,1985). The results(Table 5)demon- MLtrees.W eapplied the Žrstmethod, non- stratethat in allcombinations the null hy- parametricbootstrapping, to examine the pothesisof the differences being due tosam- different nodessuggested in the full or- pling erroris clearly not rejected. thopteranphylogeny (Fig. 5)andthe other twomethods to investigate the following MEandML Analysisof theCombined Data Set three controversialrelationships suggested Taken together,we interpret the results by the analysis. ofthe randomizationtests and of the sim- First,we examined the apparentpoly- ulationanalyses as a sufŽcient basisfor phyly ofthe Pamphagoidea.In the nonpara- combining the datain the subsequent metricbootstrap analysis, a monophyletic examination.W etherefore proceeded to Pamphagoideawas not recovered from any the phylogenetic reconstructionof the com- ofthe replicates(Table 6).For the paramet- bined 12S+ 16S+ 18Ssequences. Initially, ricbootstrap analysis we simulatedthe data weperformed heuristicsearches under the asdescribed, enforcing the single constraint 1999 FLOOKET AL.—PHYLOGENETIC ANALYSIS OF THEORTHOPTERA 245

FIGURE 5.Phylogram of MEtree reconstructed fromHKY85 distance matrixfor combined 12S+ 16S+ 18S dataset. Numbersabove nodes indicate NBS(basedon 1,000 replicates) forequal-rate/ gamma-correctedHKY85 distances. Values areindicated onlyfor nodes that received > 50%in atleast oneof the analyses.The phylogeny shown wasreconstructed fromequal-rate distances. Thegamma-corrected tree differs onlyin its placing ofthe Tetrigoidea,which arerecovered inside the eumastacids. ofa monophyleticPamphagoidea. The pam- informativeenough topositively reject pam- phagoidclade was recovered in 31%of the phagoidmonophyly under the MEoptimal- replicates,suggesting thatthe datawere not itycriteria. This inference wassupported

TABLE 6.Summary of results ofbootstrap analyses and Kishino– Hasegawa tests (KHTs)of speciŽ c hypotheses of monophyly.

Hypothesis NBS PBS ML(ln L)a KHTb (Xyronotidae,Trigonopterygoidea) 60 81 –18387.93 NDc (Xyronotidae,Tanaoceridae) 12 30 –18405.10 1.461(0.144) (Acridoidea, Pamphagidae,Xyronotidae, 34 89 –18400.04 1.976(0.048) Trigonopterygoidea,Pneumoridae) (Pamphagidae,Acridoidea) 74 78 –18387.93 NDc (Trigonopterygoidea,Pyrgomorphidae) 1 17 –18400.14 1.337(0.182) (Pamphagoidea) 0 31 –18401.38 0.855(0.393) (Pneumoroidea) 0 10 –18419.08 1.928(0.054) (Eumastacoidea) 11 32 –18392.34 0.817(0.414) a Log likelihoodscore for optimaltree under given constraints. Phylogenies were compared with the ML phylogenyrecovered from anunconstrainedsearch (ln L=–18387.93). b Numberindicates T valuefor differentcomparisons. Number in parenthesesis the probability of gettinga more extremevalue of T underthe null hypothesis (of no difference betweentrees). c ND=notdone; KHTnotperformed becauseconstrained tree identical to unconstrained tree. 246 SYSTEMATICBIOLOGY VOL. 48

FIGURE 6.Phylogram of MLtree reconstructed forcombined 12S+ 16S+ 18Sdata set. Thephylogeny was reconstructed underthe GTRmodel of substitution. Thelog likelihood of the phylogeny= –18387.93. by the KHT,which failed todetect signif- andRowell, 1997a), but the resultsof the icantdifferences between the bestuncon- present analysesand those of the 18Sanaly- strainedand pamphagoid-constrained ML sissuggested thatthe Eumastacidaemight trees.A slightly different approachto exam- be basal.This initially seemed tobe sup- ining thisproblem wastoexamine the puta- portedby the nonparametricbootstrap anal- tivegrouping ofthe Pamphagidaewith the ysis,where amonophyleticEumastacoidea Acridoidea.Here, the relativelyhigh NBS wasrecovered from only 11%of replicates. forthis clade (73%) also corresponded to However,the resultsare complicated by the arelativelyhigh PBS value (78%),indicat- factthat the proscopiidswere grouped with ing thatthis observation was unlikely tobe the tetrigoidsin someof the analyses(e.g., due tocumulative random error. However, in the MLtree in Fig. 6).No suchgrouping the KHTwasnot particularly informative haspreviously been suggested in the liter- because the acridoid–pamphagid grouping ature,and the twogroups are morphologi- wasrecovered in allof the unconstrained callyvery distinct.As a resultof this discrep- MLsearches.Thus, although we areunable ancy,noclear alternative hypothesis is sug- toreject monophyly ofthe Pamphagoidea gested by ouranalyses of the combined data, outright,it obtains no support from our andwe canonly examine towhat extent analyses. the datareject monophyly.Using the same The secondrelationship investigated was strategyas outlined forthe Pamphagoidea, amongthe four eumastacoidtaxa. Previ- we performed asearchenforcing the con- ousanalyses with the mitochondrialdata straintof a monophyleticEumastacoidea, hadsuggested asistergroup relationshipof but the PBSvalue ofonly 32%indicated the Eumastacidaeand Proscopiidae (Flook there wasrelatively little informative phy- 1999 FLOOKET AL.—PHYLOGENETIC ANALYSIS OF THEORTHOPTERA 247 logenetic signalrelating to the monophyly siferais unquestioned andbecause exclu- ofthe Eumastacoideain the ME analysis. sionof these sequences greatlyreduced the Furthermore,in the KHT,the difference be- amountof computation. The MEtree for tween the unconstrainedML tree andthe one ofthe samples(Fig. 7)illustrates that eumastacoidtree wasnot signiŽ cant. There- the mtDNAsequences areunable toresolve fore,we conclude thatthe sequences donot the ensiferan phylogeny when the grylloid containenough phylogenetic signalto settle taxaare included. AlowNBS value isob- the issue ofeumastacoid monophyly . tainedfor a grylloid/tettigonioidgrouping Finally,we testedthe monophyly ofthe (54%),but the signiŽcance of this value is Pneumoroidea.The interrelationshipsof madeunclear by the factthat when dif- the three pneumoroidfamilies (T able 6) ferent combinationsof outgroups are used havebeen the subject ofmuch specula- (e.g., nophasmids; addition of dipterans), tion(e.g., Kevan,1982) and we initially the grylloid taxaare placed outsideof the testedthe hypothesisof the monophyly otherensiferans. However, in allthe recon- ofthis superfamily .Noevidence fora structions,the branchconnecting the gryl- monophyleticPneumoroidea was obtained loidsequences tothe otherensiferans (Fig. 7) fromthe nonparametricbootstrap analy- isthe longestinternal branch, exceeding sis,but alowPBS value (10%)indicated the lengths ofbranches connecting the out- thatthe observationof pneumoroid poly- groups.Combined withtheir highly un- phyly mightbe explained by randomer- usual18S sequences (which will be dis- rorin the data.However, large differences cussedelsewhere), thissuggests to us that were detected between the unconstrained the Grylloideaprobably represent asister tree andthe Pneumoroidea-constrainedML group tothe restof the extantEnsifera. tree.W etestedseveral other hypotheses in- volvingmembers ofthe Pneumoroidea(T a- Discussion ofPhylogenetic Findings ble 6)and obtained high PBS valuesfor a The mainphylogenetic resultsare sum- Xyronotidae+ Trigonopterygidaegrouping marizedin Figure 8.This phylogeny in- (81%)and for placing the Tanaoceridaeand cludes only thoseinternal branches that re- Pyrgomorphidaeoutside of the (Acridoidea, ceived highsupportfrom the nonparametric Pamphagidae,Pneumoridae, Xyronotidae, bootstrapanalyses or for which we believe Trigonopterygidae)(89%);these resultssug- phylogenetic supportobtained in thisanal- gestthat the occurrenceof these groupings ysisis compelling. Thisresults in alossof in the MEtree maybe well founded. We resolutioncompared with the tree shownin therefore reject the monophyly ofthe Pneu- Figure 5,for example, but allowsus to ad- moroideaon the basisfor the analysisof the dressconŽ dently severalspeciŽ c points. combined data. Caelifera.—The consensustree showsthe Caeliferaas the sistergroup ofthe En- PhylogeneticPosition of the Grylloidea sifera(node O),but withthe tridactyloids Intermsof taxonomic sampling, the main asthe sistergroup ofall other caelifer- deŽciency ofthis analysis was the absence ans(node C1)and widely separatedfrom ofany grylloid taxa.T orectify this,we at- them.Our previous analyses had shown tempted afurther combined mtDNAanal- aconict with respect to the relationship ysis,this time including sequences fortwo ofthe Tridactyloideato the restof the gryllids, Grylluscampestris and Acheta do- Caelifera.The mtDNAanalysis recovered mesticus.The aimshere were toestablish atetrigoid/tridactyloidgrouping, but this the monophyly ofthe Ensiferaand to de- mayhave been due toinadequate taxonomic termine the positionof the Grylloidea.Us- samplingwith respect to outgroups for the ing the mtDNAsequences, wereconstructed orthopterantaxa. In the present analysisour aphylogeny fromseveral subsamples of resultsŽ rmlyreinforce ourprevious inter- orthopterantaxa and their outgroups.W e pretationof the 18Stree: The tridactyloids omittedmost of the caeliferan taxabecause area single clade;there therefore appearsto their positionas a sistergroup ofthe En- be nojustiŽcation for splitting off the cylin- 248 SYSTEMATICBIOLOGY VOL. 48

FIGURE 7.Unrooted phylogramof MEtree reconstructed fromHKY85 distance matrixfor combined (12S+ 16S) dataset, including sequences fromtwo grylloid species. Thicklines indicate branchesfor which > 89%bootstrap support was obtained. drachetidsas a separatesuperfamily ,aspro- genes aremore likely toresolve short intern- posedby Kevan (1982). odesthan are nuclear genes (Moore,1995), Of the remaining caeliferans,we place wecanspeculate that the resultsdo notfavor the Tetrigoideaas the mostbasal (node amonophyleticEumastacoidea. This inter- C2),closely followed (node C3)by the Eu- pretationfavors recognizing the proscopi- mastacidaesensu latoand the Proscopiidae. idsas a superfamily,asproposed by (e.g.) All these groupsare monophyletic (and re- Descamps(1973). The remaining taxa(i.e., mainso in otheranalyses with much larger the Acridomorphoideasensu Dirsh[1975] taxonsamples; data not shown). The sepa- plus the Pneumoroideaand T rigonoptery- rationof the Tetrigoideaand T ridactyloidea goidea) constitutea fourthclade (node C4), isvery goodand clariŽ es earlier results withinwhich the branching orderis not cer- basedon mitochondrial sequences (Flook tainlyresolved in ouranalyses. andRowell, 1997a). The separationof the The variousresolved lineages withinthis Tetrigoideafrom the eumastacoidsis not lastclade, however, are of considerable sys- entirely clearin the present analysisbe- tematicinterest. causeof their occasionalgrouping withthe proscopiids.However, the Tetrigoideawere 1.The SonoranT anaoceridae(though un- very clearlyidentiŽ ed asbasal to the eumas- fortunatelyrepresented in ouranalysis tacoidtaxa in the mtDNAanalyses (Flook by only asingle genus) forma solitary andRowell, 1997a). At node C3,our data branchof their own(L1), and contrary donot allow us to decide whether the Eu- toKevan (1982)do not cluster with the mastacidaes.l. and Proscopiidae are sister SoutheastMexican Xyronotidae. W ecan groupswithin a monophyleticclade (the Eu- conŽdently exclude the possibilitythat mastacoideasensu Dirsh,1996) or whether the Tanaoceridaeare related to the eu- the latterindeed branchseparately from mastacids,as proposed by Rehn (1948). the maintrunk ofthe tree.The mitochon- These resultsseem toconŽ rm Dirsh’ s drialand nuclear data con ict on this point, (1955)decision to remove them from the formerrejecting asistergroup rela- the lattergroup andgive themindepen- tionship.However, because mitochondrial dent familystatus. As noted above, the 1999 FLOOKET AL.—PHYLOGENETIC ANALYSIS OF THEORTHOPTERA 249

FIGURE 8.Summary of the phylogenetic conclusions of this study,indicating proposed revisions of orthopteran classiŽcation. Ocorresponds to the ancestralorthopteran node. Nodes E1 and E2 correspond to the two resolved ensiferannodes. Nodes C1– C4 indicate the nodesclearly resolved within the Caeliferaon the basisof our molec- ularanalyses. Although the remainingcaeliferan lineages areseparated in individual analyses(e.g., Fig. 5), we donot consider their branchingorder to havereceived sufŽcient support fromthe bootstrappingand likelihood analyses.

parametricbootstrap analysis (T able 5) withthe Lentulidae (e.g., Amed´ egnato,´ gives supportfor the propositionthat the 1993;Dirsh, 1956). Tanaoceridae,followed by the Pyrgomor- 3.The AfricanPneumoridae areresolved phidae, arethe mostprimitive branches asa monophyleticgroup (L3) withno ofthe Žfth clade. closerelationship to any other taxa. 2.The worldwidePyrgomorphidae (L2) The Tanaoceridaeand the Xyronotidae, forma monophyleticgrouping clearly grouped by Dirsh(1966) and Kevan distinctfrom all the others.There isno (1982)with the Pneumoridae in asu- supportfor the occasionalsuggestion of a perfamily Pneumoroideaon the basis closerelationship of the Pyrgomorphidae ofsimilar stridulatory mechanisms, are 250 SYSTEMATICBIOLOGY VOL. 48

excluded fromthis clade. W ealsoper- Thefailure ofthe Pamphagidaeand the Pyr- formeda KHTtoexamine the hypoth- gomorphidaeto cluster together in anyanal- esisthat the Pneumoridae were primi- ysisusing anyof the three sequenced parts tiveto the otherhigher caeliferansexcept ofthe genome hasbeen discussedin our forthe Tanaoceridae(i.e., Trigonoptery- previouspapers. The combined sequences goidea,Pamphagoidea, Acridoidea, Xy- clearlylink the pamphagidswith the acri- ronotidae).The difference between the didsrather than with the pyrgomorphids besttree consistentwith this hypothesis andindicate that the Pamphagoideasensu andthe unconstrainedML tree wassig- Dirsh(1975) or Kevan (1982)are a poly- niŽcant (P < 0.05),and we thusreject the phyletic grouping. Asthe Pamphagidaereg- hypothesis. ularlyemerge fromthe analysesembedded 4.The Xyronotidae,also represented in our withinan assemblage of acridid subfami- analysisby only asingle genus, donot lies andacridoid families, the Acridoideain clusterwith any Pneumoroidea sensu the usualmodern sense (i.e., excluding the Dirsh,but insteadare grouped with Pamphagidae) forma paraphyleticgroup- the SoutheastAsian Trigonopterygidae ing. Also,the Oedipodinae areinvariably in afurther distinctclade (lineage L4). separatedfrom the cladecontaining both These latterare deŽ nitely notassoci- Acrida and Gomphocerippus ,in accordance atedwith the eumastacidsor proscopi- withtheir rather basal and isolated posi- ids,contrary to the suggestionof Dirsh tionwithin the Acrididae,already noted in (1975).The hypothesisof Bol´ õ var (1909) ouranalyses of the mitochondrialsequences andKirby (1910), accordingto which the (Flook andRowell, 1997a). Trigonopterygidaeare allied tothe Pyr- Ensifera.—Ouranalyses split the Ensifera gomorphidae,also received nosupport intothree groups.The grylloidsmay be in ouranalysis. the mostbasal (node E1)and are also very 5.The lastlineage (lineage L5)includes highly diverged, rathersimilar to but far the OldW orldPamphagidae, the Cen- exceeding the situationof the tridactyloids tralAfrican Lentulidae, andthe world- withinthe Caelifera.In the MEtree (Fig. 5), wide Acrididae(for details,see Fig. 5). the remaining ensiferans fall intotwo sis- The unity ofthis assemblage is sup- tergroups: a tettigonioidclade (represented portedwith high NBS(73%)and PBS in oursample by Haglidae+ Tettigoniidae) values(78%), indicating that this ob- anda stenopelmatoidclade (in oursam- servationis unlikely tobe accounted ple, Mimnermidae +(Stenopelmatidae + forby randomerror alone. In other Rhaphidophoridae)).This agrees with the analyses(data not shown) this clade conclusionsof Gorochov (1995a), based on alsoincludes the New Worldfamilies morphology,but disagreesmarkedly with Pauliniidae, Tristiridae,Ommexechidae, the hypothesisof Gwynne (1995),which de- andRomaleidae. On the basisof their rivesfrom a cladisticanalysis of the mor- morphologywe wouldexpect the Old phologicaldata of Ander (1939)and other WorldLathiceridae and Charilaidae to authors.However, in the MLanalysis(Fig. 6) be included aswell, but we haveno the hagloidis recovered as the mostprimi- sequence datafor these twofamilies. tivegroup. Therefore, because ofthis con ict andthe lowNBS andPBS valuesobtained The identiŽcation of these lineages, and forthe ensiferan taxa(Fig. 5)our interpre- moresigniŽ cantly the rejectionsof several tationin Figure 8(node E2)is conservative previously proposedgroupings (Tables3, 5), andtentative. haveseveral important consequences for currentinterpretations of their relation- Implicationsfor the Systematic Nomenclature ships.W ewill discussfurther the interrela- oftheOrthoptera tionshipsof the Pneumoroideasensu Dirsh Many modernwriters have recom- in anotherpaper, but ourdata clearly do mended anode-based ora lineage-based notsupport the retentionof this superfamily . classiŽcation (see deQueriozand Gauthier , 1999 FLOOKET AL.—PHYLOGENETIC ANALYSIS OF THEORTHOPTERA 251

1994),and if the necessaryphylogenetic Eumasticoidea,and (4) anunnamed clade knowledge isavailable, the advantagesin- ofhigher Caelifera,containing Ž ve resolved deed seem compelling. Asour analysis has lineages ofuncertain branching order.How- notcompletely resolvedbranching orders,a ever, wehope thatfurther dataor improved node-based systemis still impracticable, but analysis(see below) will eventually succeed the lessrevolutionary lineage-based system in resolvingthe branching orderof these maywell be explored. Whatare the implica- lineages, andwe wouldthen havemax- tionsof the aboveŽ ndings fororthopteran imally(4) Tanaoceroidea new superfamily , classiŽcation? (5) Pyrgomorphoidea new superfamily (see Wehavenot investigated the Ensifera alsoVickery [1997],who, however ,gives withthe samedepth oftaxon sampling noreasons for this grouping), (6) Pneumor- asthe Caelifera.However, reconstructions oidea,(7) Trigonopterygoideasensu novo, basedon different taxonomicsamples of and(8) Acridoideasensu novo.Our Pneu- mtDNAsequences consistentlysupport a moroideawould, however ,include only one monophyleticEnsifera. Furthermore, al- family,notthe three conceivedby Dirsh though directcomparison of allthe ensiferan (1975);the Trigonopterygoideawould be en- superfamilies isdifŽ cult for the combined largedfrom two to three families;and our data,the long branchlength connectingthe Acridoideawould include allthe current grylloid taxato the otherensiferans, com- Pamphagoideaexcept the Pyrgomorphidae. bined withtheir highly diverged 18Sse- Itseems logical to treat resolved lineages quences (unpubl. data),suggests to us that withinthese superfamilies asfamilies. W e the Grylloidearepresent the basalensiferan arepreparing more-detailedanalyses of re- lineage. Thusin the Ensiferawe identify lationshipswithin both the Eumastacoidea only twonodes in oursummary: E1 andE2. andthe Acridoidea(both used in the sense Within the Caelifera,many of the resolved proposedabove), using much largernum- cladesalready bear classicalnames. Basal bers oftaxa. When these arecompleted, we lineages atnodes C1 to C3 in Figure 8cor- hope tobe able tosuggest further reŽne- respondto the Tridactyloidea,Tetrigoidea, mentsto our classiŽ cation. However, the andEumastacoidea, respectively .The Ž- present restricteddata set supports the re- nalclade (C4) ispractically identical with tentionof at leastthe Lentulidae, Pamphagi- the Acridomorphoideasensu Dirsh(1975), dae,and Acrididae as separate families with except thatit additionally encompasses the Acridoidea. the Trigonopterygoidea,which Dirsh erro- neously placed in the Eumastacoidea.The Prospects fora FullyResolved Molecular lineages we distinguishwithin this clade Phylogenyof the Orthoptera aremostly currently recognizedas fami- Although the resolutionafforded by our lies (Tanaoceridae,Pyrgomorphidae, Pneu- combined analysiscompares favorably with moridae,Lentulidae, Pamphagidae,and otherhigher-level studiesof insect molecu- Acrididae).The proposedgrouping ofthe larphylogeny (e.g., Campbell etal., 1995; Trigonopterygidaeand the Xyronotidaecor- Dowtonand Austin, 1994; Kambhampati, respondsto Kevan’ s original(1952) placing 1995),outstanding difŽ culties remain. In of Xyronotus,though he abandonedthis in particular,the basalrelationships of the laterpublications. Acridomorphoideaare still unclear, and the Howcan the existingnames be recon- lowPBS valuesassociated with several of ciled withthe phylogenetic relationshere the putativegroupings suggestthat we are described? Weproposethat the lineages de- unable todistinguish sufŽ cient phyloge- Žned by well-resolved nodeson the back- netic signalto recover the precise branching bone ofthe tree be allottedsuperfamily rank. patterns.Furthermore, simulations (Fig. 2) Strictapplication of this strategy to our cur- indicatethat the accumulationof longer rentdata would divide the Caeliferainto sequences cannotbe guaranteedto im- four superfamilies, ratherthan the existing proveour understanding of thispart of seven: (1) Tridactyloidea(2) Tetrigoidea,(3) the phylogeny.Comparingthe paleontolog- 252 SYSTEMATICBIOLOGY VOL. 48 icaldata (for asummary,see Flookand REFERENCES

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