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Home , Amathusia (butterfly), Nymphalidae

PhylogeneticrelationshipsamongtheNymphalidae ()inferredfrompa rtialsequencesof the wingless gene AndrewV .Z.Brower Department of Entomology,Oregon StateUniversity ,Corvallis, OR97331-2907,USA ([email protected] ) Acladisticanalysis was performed ona 378bp region of the wingless genefrom 1 03nymphalid species andthree pieridoutgroups in order to infer higherlevel patterns ofrelationship among nymphalid sub - familiesand tribes. Althoughthe dataare highly homoplastic, in many instances the most parsimonious cladogramscorroborate traditionally recognized groups. The results suggestthat this short generegion providesa usefulsource ofdata for phylogenetic inference, providedthat adequatee¡ ort is madeto samplea diversityof taxa. Keywords: cladistics; butter£y; molecularsystematics; taxonsampling

Wingshapes, patterns andcolours, which are 1.INTRODUCTION frequentlyused forthe recognitionof nymphalid species, Themorphologically and ecologically diverse nymphalid arenotoriously labile in response toselection forrecog- butter£ies havebeen the subjects ofprolonged and intense nitionby predators (Brower 1 984)and mates (Silberglied genetic,behavioural and ecological research forover a 1984)or even to di¡ erential weather conditions (e.g. century.Sincethe time ofDarwin and Bates, workers Brake¢eld 1 996).Nijhout( 1991)lamentedthat `The haverepeatedly turned tothis groupfor premier exam- freedomwith which butter£ y species areable to shift, ples ofnatural selection, polymorphismand realign,hide, unmask and alter the colorand shape of (Bates 1861;Poulton1 916;Goldschmidt 1945;Fisher 1958; their pattern elements suggests thatin principle there is Sheppard1963; T urner 1977).Thestudy of nymphalids nopattern towhich that ofanother species couldnot continuesto provide fundamental data for the diverse converge’(p. 24 1).Theoutlook for additional, subtler ¢elds ofecological genetics, chemicalecology ,community morphologicalcharacters is hardlymore sanguine. ecology,development,ethology and conservation biology Indeed,the recent cladistic analysisof adult - (Gilbert 1991;Nijhout1991 ;Carroll et al. 1994;W ahlberg logicalcharacters byDe Jong et al.(1996)o¡ ered less et al. 1996;Monteiro et al. 1997;DeVries et al. 1999;see also resolutionamong nymphalids than Ackery’s ( 1984)tree. the reviewsin V ane-Wright& Ackery( 1984)). Ackery( 1984)and De Vries et al. (1985)called for a Yet evenafter more thana centuryof taxonomic study , detailedanalysis of the larvalmorphology .Although the phylogeneticrelationships among higher groups of Harvey’s(1991)classi¢cation was partly based on exami- nymphalidsstill confoundsystematic biologists.U nam- nationof the larval¢ liformsetae, noresolution of the biguousmorphological synapomorphies, such astricari- relationshipsamong nymphalid subfamilies was implied nateantennae, diagnose the familyN ymphalidaeand there either.Ackery et al.’s(1999)most recent summary further suites ofcharacters permit the relativelyuncontro- concludedthat `most relationshipswithin and between versialrecognition of numerous well-de¢ ned subfamilies the variousgroups remain obscure . . . andcontinuing andtribes (Ehrlich 1958;Ackery 1 984;Scott 1985;De changesin higher classi¢ cation must beanticipated, Jong et al. 1996;Ackery et al. 1999).However,the relative includingalterations to limits ofmany of the subfamilies branchingorder among these intrafamilialclades remains themselves’ (p.287) . uncertainand has been presented bymost authors(e.g. Giventhe apparentintractability of the adult Ackery1984) as a largelyunresolved node in the clado- morphologyand the current lackof larval specimens for gramof butter£ y relationships.I tisnotclear how existing manytaxa, molecular data may provide a new,comple- datamay be used toresolve these polytomies:despite mentaryapproach to deciphering phylogenetic patterns in morphologicalstudies usinga varietyof data sets and this largeand complex group. Martin &Pashley( 1992) analyticalmethods (Clark1 947;Ehrlich1 958;Kristensen and Weller et al. (1996)performed the onlymolecular 1976;Scott &Wright1990; Harvey 1 991;Ackery et al. systematic studies onhigher-levelrelationships among the 1999),the relationshipsamong nymphalid tribes and butter£ies publishedto date. Martin &Pashley( 1992) subfamiliesremain poorly understood. Both Harvey’ s examinedthe relationshipsamong eight of the 13recog- (1991)classi¢cation and De Jong et al.’s(1996)cladogram nizednymphalid subfamilies by examining 2 12sites ina (¢gure 1 )su¡er froma frustrating lackof basalresolution. regionof the 28Sribosomal RNA genefrom 1 5species, Importantly,the lackof a stablephylogenetic hypothesis and Weller et al. (1996)followed this upwith a combined fornymphalids leaves in question the widelycited hypoth- analysisof the 28Sdata, 320 bases ofthe mitochondrial eses onhost use andcoevolution (Brower & Brower1 964; ND-1 geneand a morphologicaldata matrix of 50 char- Ehrlich& Raven1 965,Ackery 1 988),whichrely on the acters compiledfrom Kristensen (1976),Scott (1985)and apparentlyconcordant taxonomic distribution of these Robbins( 1988,1989).Althoughboth papers were primarily butter£ies andtheir foodplants. focusedon familial and superfamilial relationships, the

Proc. R.Soc.Lond. B (2000) 267, 1201^1211 1201 © 2000The RoyalSociety Received 25January 2000 Accepted 9 March 2000 1202A. V.Z. Brower Phylogenetic relationships among the

Libythea LIB DAN LIM Basilarchia LIM Biblis LIM Agraulis HEL 8 LIM HEL AP HEL Megalura LIM LIB 7 HEL NYM NYM NYM 3 6 HEL 4 AP NYM 3 AP SAT 8 DAN CHA Anaea CHA Danaus DAN 5 Morpho MOR Heliconius HEL 3 Caligo BRA MOR 2 SAT BRA 5 SAT SAT Morpho MOR Figure2. Strict consensus of thethree trees for 16 nymphalidtaxa pruned from the combined cladistic analysis CAL ofmtDNAND-1, 28S rDNA and morphological characters for29 ,redrawn from Weller et al. (1996,their Figure1. The nymphalidclade recovered as partof thestrict ¢g.3b).Length 1058, CI 0.44 and RI 0.56. The ˆ ˆ ˆ consensusof the1580 most parsimonious cladograms from numbersbeneath the branches represent unambiguous De Jong et al. (1996,their ¢ g.4)basedon 59exemplar synapomorphies.Subfamilyabbreviations as in¢gure1. butter£y speciesand 103 morphological characters. Length 473, CI 0.321 and RI 0.723.The subfamilies, ˆ ˆ ˆ asrecognizedby Harvey(1991) and Ackery et al. (1999), areindicated as follows: AP, ;BRA, Brassolinae arenot demonstrably monophyletic and require revision. (includedin theMorphinae by Ackery et al. (1999));CAL, Itis currently beyondthe scopeof any single study to Calinaginae;CHA, ;DAN, (including incorporateall the species andit is evenimpractical to theIthomiinae according to Ackery et al.(1999));HEL, examinerepresentatives ofeachputative . However, ;LIB, ;LIM, ;MOR, asnotedby Wheeler (1992)and Judd ( 1998),appropriate ;NYM, ;SAT, . samplingof taxa is fundamentalto the inference ofstable phylogenetichypotheses. I havetherefore sampledthe diversityof the nymphalidbutter£ ies rather intensively, more recent paperincluded 1 6species representing 11 obtainingtaxa considered to be relevant to higher-level nymphalidsubfamilies, which were sampled for at least patterns ofhierarchical grouping based on the most onegene (¢ ve species weremissing onegene or the recent andcomprehensive classi¢ cation (Harvey 1 991). other).Thestrict consensus topologyfrom combined Harvey’s(1991)arrangement(table 1 )serves asthe null cladistic analysisof all the data(¢ gure 2) is almostfully hypothesisof relationshipsfor comparison with the results resolvedand for the most partagrees with traditional ofmyanalyses. hypothesesof grouping, but the taxonsampling is so minimalthat the monophylyof most subfamiliescould 2.MATERIAL AND METHODS notbe tested. Of the foursubfamilies represented bymore thana singleexemplar ,Satyrinaeand Apaturinae appear (a) Specimens asmonophyletic, while Heliconiinae and N ymphalinae Individualbutter£ ies were netted in the¢ eldand preserved areparaphyletic. The traditional association of the in cryotubesin liquidnitrogen or in 2^7mlvialsof 100% ethyl `satyrid’subfamilies (Brassolinae plus Morphinae plus alcohol(EtOH) .Ifoundthe EtOH preservation technique to be Satyrinae)is recovered,but Ithomiinaeplus Danainae is equivalentto liquid nitrogen preservation in thequality and not.Finally ,it shouldbe noted that noneof the morpho- quantityof DNA obtained and superior from a logistical logicalcharacters employedby W eller et al. (1996) is perspective.Specimens collected by colleagues in remote relevantto the resolutionof intrafamilial relationships: regions,preserved in EtOHand sent through the mail vastly `Nymphalidae’was a singleterminal taxonin Kristenen’ s expandedthe taxonomic and biogeographical scope of thestudy . (1976),Scott’s(1985)and R obbins’( 1988,1989) data Localitydata on voucher specimens are presented in table1. matrices. Here Iaddress the problemof nymphalid phylogenetic (b) Outgroups relationshipsat the subfamilialand tribal levels using Becausethe Nymphalidae and L ycaenidaehave been consid- DNA sequence datafrom a codingregion of the develop- eredparaphyletic with respectto one another in variousways mental patterninggene wingless (wg)(see the discussion (Ackery1988; Robbins 1988; De Jong et al. 1996;Campbell et al. inBrower & DeSalle( 1998)).Thefamily N ymphalidae 2000),thePieridae were selected as the nearest unambiguous currently comprises some 6000^6500described species outgroupclade. Three pierid genera were included as outgroups, (Shields 1989;Ackery et al.1999)in 350^450 recognized althoughthe cladograms were rooted with Pierisrapae alone. genera(Harvey 1 991;Ackery et al. 1999),manyof which The problemof nymphalid ^lycaenidrelationships and the

Proc. R.Soc.Lond. B (2000) Phylogenetic relationships among the Nymphalidae A.V.Z. Brower1 203 ) . t N r n H H H H H H H H H H H H H H H H H H H H H H H H H e Z o C C C C C C C C h N N N N N N N N N N N N N N Z N N N N Z N N N N N Z N N C C I I I I I I I I c ( I C C C u M U U M M U U M U U M M U U M M M M M M M M M M M M M M M M M M M o r e v A C C A A C C A C C A A C C A A A A A A A A M A A A A M A A A A A M A A d n u ’ i t i - ` s ’ a a y e o o c c v r o o s a a o M M c H y n h h a s n n t t a e e e r r r l l m c o o i a a r o u i i a s G G a s p n l s N N d d a k u u ) ) ) B s a s s s s s s s s y y p d a c n n n r e e e e e e e e e e e e a , e y r p o o a m m e o a a o c c c r r o l a a h l l e e k k g g g r g r r C C m m m m m m m m m g r a c a a s g B e e e e e e a e e e e e e C n n n , n , s s 2 2 o H u i i i a e B ¨ t n n d . e e a e a u u u u u u u u u o u o o 6 6 i i c i s r B a n P P P b s s s s s t c c o k q q q q q q q q q i , , a a , s C s n d a i i i i i i i i i i i i r T T a a a a R a a r a o o y y r r r a r r r r r r t a a t , , u C u u D m l l w h h ) e y y m m m r L G a v v M a a t t b , A A A A , A A A A A a y L L L a a o o o u , , A b a , u U I U I r t ¨ ¨ h h l u E , , , , , , , , , n , s , , a a a J e i a a s , , , , a K c c a , m a m a a a a a a a a B o Q a h h h v t u t i i i i i i i i i k k k k : v t n n N N N i r u u m m , l s , i a a a n i i r r r r n n n n n n n n n t a t t ¨ g l . . . W e a a e a Q o a o b b o o b o o h h o o o o i o o o o o f p n u u o n x x x S , n n z n c c i a a a D d d d d n d d d d d e a e a e Y Y Y Y i i P P B W o a a a i g a i : N S S h ( S ( ( c n n n n n n n n n : : : : c r P P s g P P u n a : : : : : : : w w w w o o o o o o o o o z m a a a i a r l : : n a a : : a i a i i a a a a a i i e e e e i r r e u G i i i i i i i d s P R a A a R R R R R R R R l b b b s s s s s s o o u e V N N W N N n y d : : : : : : : : : : : C h a y y y y y y l l l l l l l l l l l t z m m d d e e : : : : : : n m m m i : c r i i i i i i i i i i i a a a a a a e r l a a a a t o ’ z o l z o z z z o l z l z z l l z z l z u n A A A A A A s l l l a n a u u n n r a a a a a a a a d a a a a a a e a a a a S S S S S S e e c u o o o c c r r a r a r r r e r r r r r r n n r o i l A B C M B C P B P E B B E C M I B U U U U P M B B M ( M U U F B B M V B - ` e e h a d T i l . ) a 7 8 9 5 7 0 9 1 2 3 8 0 5 3 2 1 0 9 0 2 9 0 9 2 0 3 3 4 9 0 1 3 4 5 8 8 8 h k 2 2 3 8 3 0 0 2 0 0 1 2 3 8 8 4 4 1 3 4 6 9 0 4 7 9 5 9 2 4 4 3 3 9 9 3 n 6 p 5 5 1 5 1 9 9 9 9 9 9 9 1 8 5 1 1 9 5 5 5 5 6 1 5 5 5 5 5 5 5 5 5 5 5 5 n o 9 i a 6 6 4 6 4 9 9 9 9 9 9 9 4 9 6 4 4 9 6 6 6 6 6 4 6 6 6 6 6 6 6 6 6 6 6 6 m s 1 4 4 1 4 1 6 6 6 6 6 6 6 1 6 4 1 1 6 4 4 4 4 4 1 4 4 4 4 4 4 4 4 4 4 4 4 s y B ( e 2 2 0 2 0 1 1 1 1 1 1 1 0 1 2 0 0 1 2 2 2 2 2 0 2 2 2 2 2 2 2 2 2 2 2 2 n N c r e F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F c e f l G a A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A l o i n o M i t n a o c e 4 0 7 ¢ d 1 2 7 i d s o 3 3 1 e s s c 7 Y Y Y a - a 5 1 2 3 2 4 e 3 9 5 8 - 6 8 - 3 3 - 9 l 4 4 1 7 l - - - - 0 - - b 7 c - - - 8 2 7 4 5 4 9 5 0 4 5 1 3 5 1 A 6 p 2 2 1 2 7 7 - - 2 2 6 1 1 5 - 4 0 1 S l 2 3 2 2 9 2 2 9 3 2 9 3 s W - - - 5 - 3 3 1 1 1 4 4 e 5 2 1 1 e m B S B B B B A Y Y A P B B P Y Y - B B P 2 B ------E v A A A B V b a e i s N B C S R C P B P E R R E J S J R V N N V P N R R S H N N N G R R N V R l r - t r e n h i g i d h e c ) a 1 l 9 p 9 1 e ) . r ( s ) i a s n 9 ’ o e 9 y i e a t 9 a e n i v a a n s 1 i a t a o h i r i e i h g t s e a s i u a n t c p e a r c i o e e e a a a i i i u i g s a x p h o s l t r a l m o a h l y r t a s r s i n a o l d l c t e y n t r d a r t a n e e o e r a H s l o h e l a r e u e u l o s i e m u l a a l h t i n n a i a s t a a b i e a t c e t s t e p l b b a o i d e l i r m e i a i l a l a o t i o n h r a h a n p t i a n s r r e e l r c a a S l h b s n t t i h i s a p i a i e m c r t a a l l t r y p a c e b o a d e e m t y o s t i t i a d a n p a i a u e s v i h f e e o c h p t l c d y a i n s g s l a s g n y c i e r n a u a t h o v a m b i r o d s r e a y p l i t r n a u r o c n s n e e a u a h a i a n e a a m r a s s e n a m i i d h c t t j u n i i t d a i e l a e i h t a u i i s e e a o u g r s i o a e n o o o r r l m s y d s c e a s r r a d d s s e u n s t o e i d r i e i r o c g b i n t i a a o o n n o t e b h r m a a a h o u o n a i u l c i l i a o s s l i i y c y o h r r a e p p l r y y e r l p n p o o l i p p n y r o r t t e d l l e t e e b l n m r i r r e e y p y a c n c c c g u u o a u r i i e h o a i u o r u h o a e p y i c N e g A A A C P P D A D D E N L H C C E S B V P H S S P J H K E P E C B L M V a l G a d . c e i r t s e g i a d o l l r n ) a , i a o o a a y n n y n o i i n i n d s a r a i i i ’ a Z u d l e n n d n t n t y i i e r o y b i o s n e n t i i d e r c h o i v r c y y s i c l o t r l i p r y g l n n b b e h a r u u o h i t u o u i s H ( A B E P B E H s n s i i n i d m e d d m e u i t o l i i s n c i i i n C i n l l n i n i i i i i i l n a r i e e n n n a h u a r i i a o n m p x e a b t d i c o i i l a a i e o i l l s l l m t l r t b e e b e a y i c o r i a b r a i n t A H N K M C B l r r t p e t m n n e i e I e x h ( a e t a i E n a n 2 i . i n w l . d i 9 y i i a l 1 t a 2 i n i h r e o e n e p m l l c e n a i c b f l m i e m t a b e y i r G u T ( a s H N L

Proc. R.Soc.Lond. B (2000) 1204A.V.Z. Brower Phylogenetic relationships among the Nymphalidae ) . t r n H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H e o h N N N N N N N N N N N Z N Z N N N N N N N Z N N N N N N N N N Z N N N N N N C c ( u C C C C M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M o v A A A A A A A A A A A M A M A A A A A A A M A A A A A A A A A M A A A A A A w o l l o H s a n i c g n a a d r e r n i n a a h B ) ) ) ) s s s s s s s s s s s s s s s s s s s s s s r b S e e e e e e e e e e e e e e e e e e e e e e e e e e , e s c c c c , t o i r r g r g r g m m m m m m m m m m m m m m m m m m m m m m e r r a e e e e e e e e e e e e e e e e e e e e e e e e e e e e l l n n n s n u i i i i B l l i w u u u u u u u u u u u u u u u u u u u u u u e o o o i i c l , P P P P s s s s o d q q q q q q q q q q q q q v q q q q q q q q v q a o s d i i i i i i i i i i i i i i i i i i i i i i i i i i a a a s s y a r y r r r r r r r r r r r r r r r r r r r r r r n e e a m u u u o w l a m m m m a r n n A A A A A A A A A A A A A A A A A A A A A A a L L l L o o o o A i i m C U s B , , , , , , , , , , , , , , , , , , , , , , , , , a a a a a a a , , K a a a a a a a a a a a a a a a a a a a a a a , a n f h h h t i i i i i i i i i i i i i i i i i i i i i i k k : l N N N N e G G l a a a r r n n n n n n n n n n n n n n n n n n n n n n a e . . . . W e , , A o b b b o o o o o o o o o o o o o o o o o o o o o o o n x x x u x a a S , a a a D d d d d d d d d e d e d d d d d e d d d d d d d e d a Y Y d d o i : ( ( S ( S Q ( S N i i n n n n n n n n n n n n n n n n n n n n n n c r r u a : : : : : : : : : w w o o o o o o o o o o o o o o o o o o o o o o z l o o a a a a a a a a a e e e u G l l i i i i i i i i i R R R R R R R R R R R R R R R R R R R R R R l l s s s s s s s u N F F N y : : : : : : : : : : : : : : : : : : : : : : C h a a y y y y y y y t z l l l l l l l l l l l l l l l l l l l l l l : : : : i c : i i i i i i i i i i i i i i i i i i i i i r r i e a a a a a a a l t t z z z z z z z z l z l l z z z z z l z z z l z z z z l l z u n A A A A s s n a r a a a a a a a a e a a a a a a a a a a a a a a a a a a a a a S S S S c e u u r r r r r r r r r r r r r r r r r r r r r r e r o l V B B B B B B B B U F M B M M B B B B U B M B B B M B B B B A M M A U P B U 0 6 3 2 7 5 2 1 3 1 4 0 2 6 5 4 7 9 1 6 5 8 4 5 0 3 4 5 9 8 8 6 0 6 7 2 8 5 k 8 3 7 5 3 3 7 8 4 3 7 1 3 8 0 5 5 3 1 5 5 8 4 7 5 8 4 4 4 6 7 9 0 4 4 0 4 4 n 5 5 5 5 5 5 5 5 1 5 5 6 5 5 6 5 5 5 6 5 5 5 1 5 5 5 5 5 5 5 5 5 6 5 5 6 5 1 n o i 6 6 6 6 6 6 6 6 4 6 6 6 6 6 6 6 6 6 6 6 6 6 4 6 6 6 6 6 6 6 6 6 6 6 6 6 6 4 a s 4 4 4 4 4 4 4 4 1 4 4 4 4 4 4 4 4 4 4 4 4 4 1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 s B e 2 2 2 2 2 2 2 2 0 2 2 2 2 2 2 2 2 2 2 2 2 2 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 n c e F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F c G a A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A e 6 7 5 1 d 5 2 3 9 o 1 2 2 1 c Y Y Y 4 7 4 8 1 e 2 4 2 7 5 1 8 2 - 0 Y 6 7 6 1 3 - 5 8 5 7 9 3 6 - 4 3 l - - - - - 3 8 4 6 8 2 2 4 4 5 5 5 5 1 2 6 7 5 9 1 5 4 5 5 0 5 9 p 6 2 2 2 6 3 8 1 2 2 2 3 2 2 3 2 9 2 9 2 3 2 2 2 9 2 3 3 2 2 3 3 9 2 W 2 - - - - 3 0 6 m L 2 B B B B B B B B Y - P B P B B B B B P B B B B B B B P S B Y 2 2 E A A A a s V R R R R R R R R N G N R N S R R R R F R N R R R S R R R R Q N S N F P R N s s s u a u u a n p s i a r s a a a i h o n t s r t i p t a s e u t a i i n c n s s i a r n o i a e e s a o m u n r e y o l r o e a e s a d o l h r a l i l s r o e l a e o . i e i c r o c h n l s e a i c a m a e o h l i n h a h n . n s e m c e t a . h n p a e a i s r s c t t d i g y c a e a b . a m s i l a u o h a e s r r p p l r e r e o . r p n e v m t e l m s f y o s a a e l a c s p c u o h s p p l m s o . i a t a o p e a c i y y a a e i j p a t s e y y s e e e l i o s y a i d c s h c e a s h k m a s s a t m p i s p y n a i n l r a h r n s h c s i a i e i i d p s i i e s a s o r t p i i c a o i a i o a a o i t i r s a o s p u u a i n a h e l s t t n c n a s o h t d i e c h e h e h r m l e a n h n i h r e i o i i m a o n s e h i p c r c o s o c a n c t e c e t p p t i s c n s p s o p o a p u r e r y a n o g a g n t a i e i a e u l r e s e r a r r i c i s o m i l g a y p e c h m e e y s n p x m n t e r l t e t n a e e l s y a l e t o e y e n e a t r m a e y i a y o a i a p r u a d a s u n m i a a i h o r a a e e e g M C E H P B T D D L A T M C P P H M Z A D E M C A A C O H B M L M T M O T C a n i a a a a a n n a m n n a a i i n i a a i a a i a a n e n d n t i i m n n n n i i i n s n n a i i i s i i i a t h t i a i i l e a y l i i e i h d l c n i r n l h l c m i o b r n n i n a y i o g h i t i a a p r o a e e t a a r r i a e h r c n c p h l p t n a t i e i t t p r n t o e y a m i b y y g a n n p p i u a e u i u s E A E D C L E A Z M A B M L M H E M i i n i n i i i i n i i i i i d i s n i n i n n n n i i t d i i i i i u n i i i l x r i n n h t h i n n i a o e s t o r p s e r e t i e a e r s m p y e a a r n t e a b o m y i a i y h n m i l r r i a r t L C C P A M A B H B E S ) . t n o C ( e e e e a a a a . e n y n n n l i a 1 i i i i l x r n h e i a o u m l p r s r t a r s y b f a a t o a a b h p r a u T s C A M B S

Proc. R.Soc.Lond. B (2000) Phylogenetic relationships among the Nymphalidae A.V.Z. Brower1 205 r H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H e C h N N N N Z N N N N N N N N N N N N N N N N N N N N N N N N N N I c C u M M M M M U M M M M M M M M M M M M M M M M M M M M M M M M M o v A A A A M A C A A A A A A A A A A A A A A A A A A A A A A A A A t s e r o F s e a t o c a y t n a S a o s s s r m y i i i e e a i r d a u u u n n a o p o m i a a L L L ) B u m o s s s s s s s s s s s s s s a c i b b e e e e e e e e e e e e e e e o r , a m n n n n s s w p c r y i i o h t u e i a a a r m m m m m m m m m m m m m m u l r r r a c e e e e e e e e e e e e e e e e S S S C i o G a u i B B K r a - u u u u u u u u u u u u u u a m v c h a a a , , , a P o b H c q q q q q q q q q q q q q q u a a d d d d d d d i m i i i i i i i i i i i i i i l C , u a c l m r r r r r r r r r r r r r r a a a a n n n u a i m h s u a r r r m r C i i a a a t A A A A A A A A A A A A A A G l l l o d A b b b b V n L I e s s , s , , , , , , , , , , , , , L , e e e a e a , a n , d a , a a a a a a a a a a a a a a o n n n r r h u u u a u i t i i i i i i i i i i i i i i a k N e e e l t o a b a r n n n n n n n n n n n n n n v e e e . W e e Q Q Q R Q e c l g b o o o o o o o o o o o o o o o x u u u S , , , , , a u y a a D d d d d d d d d d d d d d d e i Y s M o o o o o t : ( S Q Q Q N n n n n n n n n n n n n n n Q c c c c : c n M a : : : : : : n w o o o o o o o o o o o o , o o z z z z z a l : n a a a a a a i a e o r u u u u e u e i i i i i i R R R R R R R R R R R R R R l l l l b S c s s o u P N y : : : : : : : : : : : : : : C C C C C a a a z a : y y t l z l l l l l l l l l l l l l d : : m i : : : : : r r i r i i i i i i i i i i i i i r a u a a e l a t t t t l l o z z z z z z z z z z z z z z u u u u u A A s s b s s l n a u C r r r r r a a a a a a a a a a a a a a a a S S e c o u u u u u c e e e e r r r r r r r r e r r r r : r r o l P P P P M M C A A C B A E V B B B B B B B P B B B B P B B U U A 8 7 7 4 8 2 3 4 5 6 9 7 3 2 1 9 4 6 0 7 7 8 9 1 1 1 9 6 6 1 3 7 5 k 8 9 0 0 9 0 6 6 6 7 6 6 6 6 9 8 4 6 0 4 5 8 9 0 7 5 0 7 5 4 7 n 1 5 5 6 6 5 6 5 5 5 5 5 5 5 5 5 5 1 5 6 1 5 5 5 6 5 5 6 5 5 5 5 n o 4 i a 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 4 6 6 4 6 6 6 6 6 6 6 6 6 6 6 1 s 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 4 4 1 4 4 4 4 4 4 4 4 4 4 4 s B e 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 2 2 0 2 2 2 2 2 2 2 2 2 2 O 2 n c e F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F c G a A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A e 8 d 1 o 4 2 3 c 0 3 1 1 Y - - - 1 9 1 1 e - 1 1 9 8 8 3 8 4 1 7 5 5 4 8 6 1 2 l - - - - 4 0 0 - 5 2 8 5 4 2 2 8 3 3 1 2 3 3 0 2 5 7 p 5 5 5 1 1 1 1 - 1 2 7 1 1 1 9 2 2 3 2 2 2 3 3 2 2 3 3 3 2 W W - 0 ------3 1 m L L P S B 2 B B B B B B B B B B B B B Y Y S - U - E E E E E E A a s P P P P N S C N Q C R Q E V R R R R R R R P R R R R P R R N N N s i s l e i a h a t i t t s s a s n a i n n i e r s a a s i t e a a n d b e u m t i n n u r i n i a h i d y n u i a o e i o p i m e i l n s p e i c r a s . a e h p u a n e a a o . m i u m a o l e n d a u a m i h e r p e o p l . b a r p i t r s t e h a c i e d g p a c . i x r s v i s t u . s e e o s r a a s h p e p c s a e i d o m s a s a a e p n a e a p d a s i . l o s p h u i i r i a e i o l z s t a e a n c m n g a p s p a r c p e r c i q a a a i p l a p a p c a e d s m a o e y i a s i s b h s r a s i s a a n h s o a n a e i n a e o i g y i t r v a h n e t i e d c e r r a n i u t s n e o r s a t o i l h s a n i n i m l n m o n o s d o r i r a e t t e h i a a a e h i r l o o p o i l o u t a a u l u y c h i r a o h t l c y d a n y p r r o a p p r p e l t l r t r r e r m c e e e n d z h r e b e a e e a i e i h y y a i y e l u n l t n o y e a y e t i i t c e d g C L P S I P D T E A L T E T M A M M S T O H H N D C P H L A P D a n i l a a i n n a h i i e a r n p e i b n i o u o i a l r a n t n n p o b u a m u r t u s P A D E I i i n n i ) i i i i i i i e i n t n n n n i n i i i i n b i n e i i i i i e n i n n e n e g i r n v a e n a r i o n r h t o i r o n t i o e h a e y h l e i r e y l c t l c w h n p d p l e r t b e e e e b l a a i o i e i u i n r t D E T T M M M O N D ( G L ) . t n o P C U e ( e e a O a a . e n y n i n l a R 1 i e i i e i n v a G i h e r m t d l a m e i T a y l b f o r n l b e U a b h a e i i t u T s D T I L O P

Proc. R.Soc.Lond. B (2000) 1206A.V.Z. Brower Phylogenetic relationships among the Nymphalidae placementof Riodininaewill beaddressed elsewhere (Campbell inthe three members ofMelitaeini sampled( Phyciodes, et al. 2000). and ).Thedynamics of the sequence divergencein the wingless geneare addressed inBrower & (c) DNAextraction DeSalle( 1998)and Campbell et al.(2000)and will not DNAwas extracted from individual butter£ ies following the bediscussed further here. Inthis matrix,99 nucleotide protocolpresented in Brower( 1994).Brie£y, specimenswere sites areinvariant, 59 vary in a singletaxon and the maceratedin liquidnitrogen, digested with proteinaseK in a remaining220 are cladistically informative (potential Tris^EDTAlysisbu¡ er and phenol ^chloroformextracted. DNA synapomorphieswithin the Nymphalidae). wasprecipitated with isopropanol,washed with 70%ethanol Theequal-weighted parsimony analysis yielded 336 andresuspended in water.The DNAstocks were stored at equallyparsimonious cladograms of 2760steps. Thestrict 7 208 C. The wingsand abdomens of each specimen were consensus (¢gure 3) implies monophylyof the preservedas vouchers(Brower 1996) and deposited at the Amer- Nymphalidae,the subfamiliesHeliconiinae, Nympha- icanM useumof Natural History ,CornellUniversity linae,Brassolinae, Danainae, I thomiinae,Charaxinae Collectionor the Museum of Comparative Zoology at Harvard andApaturinae and numerous less inclusiveclades identi- (seetable 1 ). ¢edin Harvey’s ( 1991)classi¢cation. The branch support valuesfor many of these cladesare indicated in ¢ gure3. (d) PCR andsequencing TheLimenitidinae and Satyrinae appear as unresolved Polymerasechain reaction (PCR) ampli¢ cations were paraphyleticgroups. Individual will be discussed performedwith theprimers and standard conditions presented further below. in Brower& DeSalle( 1998).The PCR productswere gene SAWresulted ina singlestable topologyafter nine cleaned,cycle sequenced in aPerkin-Elmer9600 and run on an iterations (¢gure 4) .Thiscladogram is 2803steps long ABI 373automated sequencer from sense and anti-sense strands. whenthe characterweights are restored to1 (43steps The automatedsequence outputs were edited manually and longerthan the MP cladogramsunder equal weights) . alignedby eye. The aligneddata matrix is available on the Eighty-oneof the 102nodes are congruent between the WorldWide Web(http:/ /www.ent.orst.edu/browera/datasets/ SAWcladogramand the MP consensus tree (`congruent’ dataset7.htm)and individual sequences have been submitted to meaningthat the SAWtree either exhibitsthe same GenBank(see table 1 forthe accession codes) . cladesas the MP consensus tree orprovides resolution to its polytomies).With the exceptionof twobasal nodes, the (e) Dataanalyses incongruentpatterns ofgrouping are localized within Cladisticanalyses were performed using P AUP3.1(Swo¡ord three clades:Heliconiina, Danainae plus I thomiinaeand 1991).All nucleotidesites were weighted equally and gaps were Satyrinae. codedas missing data. T ree-bisection-reconnection(TBR) branchswapping was performed on ¢ veseparate sets of 1 00 4.DISCUSSION randomaddition sequences. The resultsof these searches were pooledand duplicate trees were culled. The branchsupport (a) Quality (Bremer1988, 1994; Davis 1 995)for selected nodes was Theaverage informative character changes state more calculatedusing anti-constraints with tenrandom additions. T o than1 2times onthe most parsimonioustrees andsome assessthe ability of the data to imply a self-consistenttopology , changeup to 35 times. Theoretically,such rampant successiveapproximations weighting (SA W)(Farris1969) was homoplasysuggests that these dataare not valuable as a performedin PAUPbasedon the maximum rescaled consis- source ofevidence for inferring the patterns ofrelation- tencyindex for each character among the most parsimonious shipamong the groupsrepresented (Sanderson& cladogramsfrom the equal-weighted analyses. U nlessSA W Donoghue1 996).Hillis (1996)concluded from a simula- yieldsa subsetof the MP cladograms,the results of therespect- tionstudy that, although larger sets oftaxa can resolve iveanalyses are not comparable by quantitative means because relationshipswith shorter sequences, approximatelyan thetwo weighting schemes rely on di¡erent philosophical inter- orderof magnitude more sequence per taxonthan was pretationsof the parsimony criterion. The mostparsimonious includedin this matrixis required toinfer the simulated (MP)and SA Wtreesdi¡ ered in thisstudy and are compared branchingpattern accurately.Thesedata o¡ er anempirical qualitativelyhere by assessment of topologicalcongruence ( Judd refutationof those claims. 1998). Thehistorical course ofevolutionary divergence is unknowableand empirical systematists eschewinductive summary measures ofaccuracyor con¢ dence in favourof 3. RESULTS qualitativeassessment ofthe stabilityand corroboration of One hundredand three nymphalidtaxa representing priorhypotheses. The results presented here arestable to 12ofthe 13subfamiliesand 30 ofthe 38tribes recognized the extent that the inferred MP topologyis largelyself- byHarvey ( 1991)andthree pieridoutgroups were consistent underthe impliedweights ( Judd1 998).In compiledfor the wingless region.Nine ofthe sequences general,SA Wreinforcedthe groupsimplied by the datain havebeen published previously in Brower & Egan( 1997), the MP tree. Theincongruence between the MPandSA W 12havebeen published previously in Brower & DeSalle trees withinHeliconiina occurs amongweakly supported (1998)and the remaining85 are new .Theresulting cladesand neither ofthose impliedbranching orders aligneddata matrix of 106 taxa 378characters contains matches the pattern resulting whentaxa are sampled more £ onlythree gapregions: two separate autapomorphic intensivelyat the same locus(Brower & Egan1 997). 3-base deletionsin and ,respectivelyand a Within the Ithomiinaeplus Danainae , three single3-base insertion (aputative synapomorphy) present instances ofincongruence result fromthe instabilityof

Proc. R.Soc.Lond. B (2000) Phylogenetic relationships among the Nymphalidae A.V.Z. Brower1 207

Heliconius Laparus Heliconiinae Neruda Agraulis Heliconiina 6 Podotricha Dryadula 2 Dryas 8 Acraea Actinote 3 Euptoieta Speyeria 22 Euphydryas Phyciodes Nymphalinae Eresia Colobura Vanessa 1 3 Limenitis 3 Biblis Batesia Charaxinae 3 1 Caligo Brassolinae Megisto 5 Corades Steroma Haetera Amathusia Morpho Libytheana 2 Doxocopa Asterocampa Apaturinae Eulaceura Ithomiinae Hyaliris 2 2 Scada Mechanitini 4 Melinaeini 7 2 Tithoreini Tellervo 6 Danaus Danainae Danaina 6 10 Itunina 9 Amaurina Temenis Lethe Anthocharis Pieris Delias

Figure3. Strict consensus of 336equally parsimonious cladograms for the wingless datamatrix with equalweights (length 2760, CI 0.189 and RI 0.531).Traditionally recognized subfamilies and tribes (or subtribes) appearing as clades ˆ ˆ ˆ hereare indicated by black and white bars, respectively. The branchsupport values are indicated for these groups.

Proc. R.Soc.Lond. B (2000) 1208A.V.Z. Brower Phylogenetic relationships among the Nymphalidae

Heliconius Heliconiinae Laparus Neruda Eueides Philaethria Podotricha Heliconiina Dryadula Dryas Dione Agraulis Acraea Acraeini Actinote Actinote (paraphyletic) Cethosia Cirrochroa Euptoieta Boloria Speyeria Cupha Limenitis Limenitidini Adelpha (paraphyletic) Tanaecia Euphydryas Phyciodes Nymphalinae Eresia Hypolimnas Melitaeini Precis Junonia Colobura Vanessa Polygonia Hypanartia Nymphalini Symbrenthia Kallima Siproeta Biblis Vila Biblidini Mestra (paraphyletic) Laringa Diaethria Batesia Panacea Temenis Dynamine Hamadryas Doxocopa Asterocampa Apaturinae Eulaceura Eunica Catonephele Myscelia Marpesia Memphis Zaretis Charaxinae Hypna Polyura Prepona Oleria Ceratinia Ithomiinae Hypoleria (paraphyletic) Dircenna Hyaliris Hypothyris Pteronymia Napeogenes Mechanitis Mechanitini Scada Thyridia Tithorea Tithoreini Elzunia Danaus Danainae Tirumala Ideopsis Danaina Parantica Amaurina Lycorea Anetia Itunina Euploea Methona Melinaea Melinaeini Athyrtis Tellervo Caligo Bia Brassolinae Opsiphanes Antirrhea Caerois Morpho Tisiphone Oressinoma Satyrinae Cercyonis (+ ) Pedaliodes Corades Taygetis Lymanopoda Steroma Megisto Haetera Lethe Melanitis Mycalesis Amathusia Libytheana Chersonesia Anthocharis Delias Pieris

Figure4. The mostparsimonious cladogram resulting from SAW (withweights restored to unity)(length 2803, CI 0.18 and ˆ ˆ RI 0.521).Traditionally recognized subfamilies and tribes (or subtribes) appearing as cladesare indicated by black and white ˆ bars,respectively. Three paraphyletic grades are marked as well: archeslink disjunct sections of theBiblidini andIthomiinae. Crossesmark the nodes incongruent with themost parsimonious trees discovered with equalweighting.

Proc. R.Soc.Lond. B (2000) Phylogenetic relationships among the Nymphalidae A.V.Z. Brower1 209

Hypoleria andthe remainingfour result fromthe nestingof subfamily.TheMP tree implies reciprocalmonophyly of the Danainaebetween the basaland derived I thomiinaein the Ithomiinaes. s. andDanainae s. s., butthe Itho- the SAWtree. Theincongruence in the Satyrinaeis not miinaebecome paraphyletic in the SAWtree. Itis intri- attributableto the instabilityof speci¢c taxaor cladesand guingthat the -feeding Tithorea and Elzunia mayinstead result frominsu¤ cient samplingof this extre- arebasal to the Apocynaceaeand Asclepiadaceae-feeding melydiverse group (which represents nearlyone-third of `Danainae’in the latter hypothesis.Both cladograms allN ymphalidae)( Ackery et al. 1999). supportthe hypothesisthat Apocynaceae-feeding,apose- Thecorroboration of prior systematic hypothesesalso matic larvaeare primitive characteristics ofthe Danainae confers some degreeof plausibility to the current results. s. l. (Edgar et al. 1974;Brown 1 987). TheMP consensus implies monophylyin seven out of ten Ackery et al. (1999)resurrected Ehrlich’s (1958)in- subfamilies(T ellervinaeand Libytheinae with a single clusiveMorphinae, encompassing both the Palaeotropical representative eachincluded are also trivially monophy- Amathusiinaeand the NeotropicalBrassolinae and Mor- letic).Thetraditional subfamilies not supported here have phinaeof various authors. Among the morphineand longbeen considered arti¢ cial (e.g. Limenitidinae) or are satyrinelineages sampled, only the monophylyof the notdiagnosed by morphological synapomorphies (e.g. Brassolinae(which clearly includes the formerlyenigmatic Satyrinaeto the exclusionof Brassolinae) ( Ackery et al. genus Bia) (vide Miller 1968;De Vries et al. 1985) is 1999).Thus,without the `aid’of special process models supportedby the MP cladogram.However ,the SAWtree andmathematical corrections, this short generegion supports the monophylyof numerous traditionally recog- generallycorroborates higher taxa supported by evidence nizedclades, including the Brassolinae,Morphini and the fromother character systems andfails to corroborate sister relationshipof the Morphinaes. l.and Satyrinae, those whichare not so supported. but Amathusia (the onlyamathusiine sampled) joinsa basalclade with Old Worldsatyrines. Thisresult implies (b) Taxonomic implications the parallelevolution of features associatedwith large Thesister relationshipbetween the Heliconiinaand size inthe Neotropicaland Palaeotropical realms. Acraeiniimplied here necessitates reinterpretation ofthe However,given the diversityof this clade,these patterns femaleabdominal `stink clubs’ (Mu « ller 1886)from the arebest viewedmerely asintriguing hypotheses to synapomorphyuniting the sensu Harvey( 1991) stimulate further research. toa synapomorphyuniting the Heliconiinaebut lost inthe Thesnout butter£ ies (Libytheinaeor Libytheidae) are Acraeini.D .Harvey(personal communication) suggested oftenviewed as the sister taxonof all other Nymphalidae that the loss maybe associated with the simpli¢ed court- dueto the plesiomorphicpresence offunctional forelegs shipperformed byacraeines, which exhibit sperm priority inthe females (Ehrlich 1958;Scott &Wright1 990; enforcedby a sphragis(Smith 1984).The ad hoc invocation Ackery et al.1999).TheSA Wtree implies abasalposition ofstink club homoplasy is compensatedfor by the more for Libytheana, with Chersonesia (Cyrestidini Marpesiini) ˆ parsimoniousoccurrence ofthe capacityto sequester or asits immediate outgroupand the sister taxonto the synthesize cyanoglucosides(N ahrstedt &Davis1981 ), Libytheinaeplus all other nymphalids. The other cyresti- which,according to this hypothesis,is uniquelyderived dine, Marpesia,is sister tothe Nymphalinaeplus Biblidini, amongthe Heliconiinaplus Acraeini. suggestingthat the tribe mayrepresent apolyphyletic Thelarge and diverse `subfamilyLimenitidinae’ has basalassemblage. Both Libytheana and Chersonesia exhibit beenlong and almost unanimously recognized as an enlargedlabial palpi (see Ackery et al. (1999) for unnaturalassemblage (Mu « ller 1886;Scott &Wright comments onthe rangeof variation in this character 1990;Harvey 1991 ;Ackery et al. 1999),aviewcorro- amongnymphalid taxa) . boratedby the wingless data.The SA Wtree suggests a¤nities betweenthe Limenitidiniand Heliconiinae, (c) Prospects andbetween the NeotropicalBiblidini and Nymphalinae. Thisstudy supports the hypothesis(Brower & DeSalle Oddly,the apaturinessampled nest wellinside the 1998;Campbell et al. 2000)that the small fragmentof the Biblidini,although morphological features implytheir wingless geneexamined provides a usefulsource ofcharac- closer a¤nity to the Charaxinae(Ackery et al. 1999). ters forcladistic inference amongbutter£ y subfamilies, Thisis the least orthodoxresult fromthe wingless data tribes andgenera. Given the relativelysmall number of andsuggests thatmore detailedsampling of Old World generegions that havebeen employed with success inthe representatives ofboth the Apaturinaeand `Limeniti- molecularsystematics ofinsects todate (Caterino et al. dinae’is necessary. 2000),the empiricalsuccess ofthe wingless generepresents Althoughno unique morphological synapomorphy has asigni¢cant addition to the batteryof markers currently beenfound to unite the Danainaes. l.( Danainaeplus available.Although phylogenetic hypotheses based on ˆ Ithomiinaeplus T ellervinae),most authorshave consid- datafrom single sources arepotentially subject tobias or ered them toform a monophyleticgroup (Bates 1861; `inaccuracy’,the dense taxonomicsampling employed Clark1 947;Ehrlich1958; Ackery 1 984,1 988).Ackery et al. here appearsto permit synapomorphyto overcome high (1999)argued that the use ofpyrrolizidine alkaloids for levelsof homoplasyand corroborate clades well supported courtshippheromone synthesis (andpossibly for chemical bydata from morphology .Theinitial phylogenetic struc- defence) (Brower1 984)is amore promisingshared trait ture presented here providesa frameworkfor subsequent thananything yet described frommorphology .The investigationof particular nymphalid clades in greater Danainaes.l. is the most stronglysupported major clade detail.The taxa in particular need of more intensive inthis study,corroboratingAckery et al.’s(1999)ranking samplingor improved resolution are the Limenitidini, ofthe Ithomiinaeplus Tellervinaeplus Danainae as asingle Satyrinae,Amathusiini and I thomiini.

Proc. R.Soc.Lond. B (2000) 1210A. V.Z. Brower Phylogenetic relationships among the Nymphalidae

Foremost,I thankRob DeSalle and Jim Miller forsponsorship Patternformation and eyespot determination in butter£y andsupport during my work at the American Museum of wings. Science 265, 109^114. NaturalHistory ( 1993^1997).IthankG. Austin, F .Beniluz, Caterino,M. S., Cho,S. &Sperling,F .A.H.2000The current A.Berry,C. Bridges,T .Collins,J .Hall,B. Hermier,G. Lamas, stateof insect molecular systematics: a thrivingtower of K.Matsumoto,N. Pierce, D. Otero, K. Sime,C. Snyder,M.-W . Babel. A.Rev .Entomol. 45.(Inthe press. ) Tan,A. Warren,K. Willmott,M. Whitingand D .Yeatesfor Clark,A. H.1 947The interrelationshipsof the several groups assistancein the¢ eldand/ orproviding specimens. I also within thebutter£ y superfamilyNymphaloidea. Proc.Entomol. acknowledgethe dedicated laboratory work of E. Bonwich, Soc. Wash. 49,148^149and192. M. Egan,G. Grills and F .Vital.I amindebted to P .Ackery, Davis,J. I. 1995 A phylogeneticstructure for the monocotyle- P.DeVries,D. Harvey ,J.Rawlinsand R. 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