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Phylogenetic relationships among the ) inferred from partial sequences of the wingless gene Andrew V. Z. Brower Department of Entomology, Oregon State University, Corvallis, OR 97331-2907, USA [email protected]) A cladistic analysis was performed on a 378 bp region of the wingless gene from 103 nymphalid and three pierid outgroups in order to infer higher level patterns of relationship among nymphalid sub- families and tribes. Although the data are highly homoplastic,in many instances the most parsimonious cladograms corroborate traditionally recognized groups. The results suggest that this short gene region provides a useful source of data for phylogenetic inference,provided that adequate e¡ort is made to sample a diversity of taxa. Keywords: ; butter£y; molecular systematics; taxon sampling

Wing shapes,patterns and colours,which are 1. INTRODUCTION frequently used for the recognition of nymphalid species, The morphologically and ecologically diverse nymphalid are notoriously labile in response to selection for recog- butter£ies have been the subjects of prolonged and intense nition by predators Brower 1984) and mates Silberglied genetic,behavioural and ecological research for over a 1984) or even to di¡erential weather conditions e.g. century. Since the time of Darwin and Bates,workers Brake¢eld 1996). Nijhout 1991) lamented that `The have repeatedly turned to this group for premier exam- freedom with which butter£y species are able to shift, ples of , and realign,hide,unmask and alter the color and shape of Bates 1861; Poulton 1916; Goldschmidt 1945; Fisher 1958; their pattern elements suggests that in principle there is Sheppard 1963; Turner 1977). The study of nymphalids no pattern to which that of another species could not continues to provide fundamental data for the diverse converge' p. 241). The outlook for additional,subtler ¢elds of ecological genetics,chemical ecology,community morphological characters is hardly more sanguine. ecology,development,ethology and conservation biology Indeed,the recent cladistic analysis of adult - Gilbert 1991; Nijhout 1991; Carroll et al. 1994; Wahlberg logical characters by De Jong et al. 1996) o¡ered less et al. 1996; Monteiro et al. 1997; DeVries et al. 1999; see also resolution among nymphalids than Ackery's 1984) tree. the reviews inVane-Wright & Ackery 1984)). Ackery 1984) and DeVries et al. 1985) called for a Yet even after more than a century of taxonomic study, detailed analysis of the larval morphology. Although the phylogenetic relationships among higher groups of Harvey's 1991) classi¢cation was partly based on exami- nymphalids still confound systematic biologists. Unam- nation of the larval ¢liform setae,no resolution of the biguous morphological synapomorphies,such as tricari- relationships among nymphalid subfamilies was implied nate antennae,diagnose the Nymphalidae and there either. Ackery et al.'s 1999) most recent summary further suites of characters permit the relatively uncontro- concluded that `most relationships within and between versial recognition of numerous well-de¢ned subfamilies the various groups remain obscure ...and continuing and tribes Ehrlich 1958; Ackery 1984; Scott 1985; De changes in higher classi¢cation must be anticipated, Jong et al. 1996; Ackery et al. 1999). However,the relative including alterations to limits of many of the subfamilies branching order among these intrafamilial remains themselves' p. 287). uncertain and has been presented by most authors e.g. Given the apparent intractability of the adult Ackery 1984) as a largely unresolved node in the clado- morphology and the current lack of larval specimens for gram of butter£y relationships. It is not clear how existing many taxa,molecular data may provide a new,comple- data may be used to resolve these polytomies: despite mentary approach to deciphering phylogenetic patterns in morphological studies using a variety of data sets and this large and complex group. Martin & Pashley 1992) analytical methods Clark 1947; Ehrlich 1958; Kristensen and Weller et al. 1996) performed the only molecular 1976; Scott & Wright 1990; Harvey 1991; Ackery et al. systematic studies on higher-level relationships among the 1999),the relationships among nymphalid tribes and butter£ies published to date. Martin & Pashley 1992) subfamilies remain poorly understood. Both Harvey's examined the relationships among eight of the 13 recog- 1991) classi¢cation and De Jong et al.'s 1996) cladogram nized nymphalid subfamilies by examining 212 sites in a ¢gure 1) su¡er from a frustrating lack of resolution. region of the 28S ribosomal RNA gene from 15 species, Importantly,the lack of a stable phylogenetic hypothesis and Weller et al. 1996) followed this up with a combined for nymphalids leaves in question the widely cited hypoth- analysis of the 28S data,320 bases of the mitochondrial eses on host use and coevolution Brower & Brower 1964; ND-1 gene and a morphological data matrix of 50 char- Ehrlich & Raven 1965,Ackery 1988),which rely on the acters compiled from Kristensen 1976),Scott 1985) and apparently concordant taxonomic distribution of these Robbins 1988,1989). Although both papers were primarily butter£ies and their food plants. focused on familial and superfamilial relationships,the

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

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 Figure 2. Strict consensus of the three trees for 16 Morpho MOR nymphalid taxa pruned from the combined cladistic analysis CAL of mtDNA ND-1,28S rDNA and morphological characters for 29 ,redrawn from Weller et al. 1996,their Figure 1. The nymphalid recovered as part of the strict ¢g. 3b). Length ˆ 1058,CI ˆ 0.44 and RI ˆ 0.56. The consensus of the 1580 most parsimonious cladograms from numbers beneath the branches represent unambiguous De Jong et al. 1996,their ¢g. 4) based on 59 exemplar synapomorphies. Subfamily abbreviations as in ¢gure 1. butter£y species and 103 morphological characters. Length ˆ 473,CI ˆ 0.321 and RI ˆ 0.723. The subfamilies, as recognized by Harvey 1991) and Ackery et al. 1999), are indicated as follows: AP,; BRA,Brassolinae are not demonstrably monophyletic and require revision. included in the by Ackery et al. 1999)); CAL, It is currently beyond the scope of any single study to Calinaginae; CHA,; DAN, including incorporate all the species and it is even impractical to the Ithomiinae according to Ackery et al. 1999)); HEL, examine representatives of each putative . However, ; LIB,; LIM,; MOR, as noted by Wheeler 1992) and Judd 1998),appropriate Morphinae; NYM,; SAT,. sampling of taxa is fundamental to the inference of stable phylogenetic hypotheses. I have therefore sampled the diversity of the nymphalid butter£ies rather intensively, more recent paper included 16 species representing 11 obtaining taxa considered to be relevant to higher-level nymphalid subfamilies,which were sampled for at least patterns of hierarchical grouping based on the most one gene ¢ve species were missing one gene or the recent and comprehensive classi¢cation Harvey 1991). other). The strict consensus topology from combined Harvey's 1991) arrangement table 1) serves as the null cladistic analysis of all the data ¢gure 2) is almost fully hypothesis of relationships for comparison with the results resolved and for the most part agrees with traditional of my analyses. hypotheses of grouping,but the taxon sampling is so minimal that the of most subfamilies could not be tested. Of the four subfamilies represented by more 2. MATERIAL AND METHODS than a single exemplar,Satyrinae and Apaturinae appear a) Specimens as monophyletic,while Heliconiinae and Nymphalinae Individual butter£ies were netted in the ¢eld and preserved are paraphyletic. The traditional association of the in cryotubes in liquid nitrogen or in 2^7 ml vials of 100% ethyl `satyrid' subfamilies Brassolinae plus Morphinae plus alcohol EtOH). I found the EtOH preservation technique to be Satyrinae) is recovered,but Ithomiinae plus Danainae is equivalent to liquid nitrogen preservation in the quality and not. Finally,it should be noted that none of the morpho- quantity of DNA obtained and superior from a logistical logical characters employed by Weller et al.1996)is perspective. Specimens collected by colleagues in remote relevant to the resolution of intrafamilial relationships: regions,preserved in EtOH and sent through the mail vastly `Nymphalidae' was a single terminal taxon in Kristenen's expanded the taxonomic and biogeographical scope of the study. 1976),Scott's 1985) and Robbins' 1988,1989) data Locality data on voucher specimens are presented in table 1. matrices. Here I address the problem of nymphalid phylogenetic b) Outgroups relationships at the subfamilial and tribal levels using Because the Nymphalidae and have been consid- DNA sequence data from a coding region of the develop- ered paraphyletic with respect to one another in various ways mental patterning gene wingless wg) see the discussion Ackery 1988; Robbins 1988; De Jong et al. 1996; Campbell et al. in Brower & DeSalle 1998)). The family Nymphalidae 2000),the were selected as the nearest unambiguous currently comprises some 6000^6500 described species outgroup clade. Three pierid genera were included as outgroups, Shields 1989; Ackery et al. 1999) in 350^450 recognized although the cladograms were rooted with Pieris rapae alone. genera Harvey 1991; Ackery et al. 1999),many of which The problem of nymphalid^lycaenid relationships and the

Proc. R. Soc. Lond. B 2000) Phylogenetic relationships among the Nymphalidae A.V.Z. Brower 1203 ) Cont.  places Harvey's `-iti' under ICZN n locks CUIC ¨ ,Lake Bayano,Gatu CUIC ¨ ¨ ,Quatro Barras CUIC ¨ GenBank accession locality voucher NP95-Y177V25ARB319 AF246595 Malaysia: AF246598 ex. Naomi Pierce) AF246538 : Delta Amacuro,Barrancas : Rondonia,Ariquemes AMNH MCZ AMNH RB303RB243 AF246533 AF246534 Brazil: Rondonia,Ariquemes Brazil: Rondonia,Ariquemes AMNH AMNH NY5G-36-5 AF246540 AF246541 USA: New York,Ithaca,Coy French Glen Guiana: Kaw AMNH AMNH NY2NY6VA1PE-10-7NP95-Y314 AF246530RB246 AF246542RB298 AF246609 AF246590 AF246569SA-2-3 USA: New York,UlyssesHBS7 USA: New York,Ithaca,Coy GlenNP95-Y320 AF014142 Malaysia: ex. : Naomi USA: Cuzco,Quebrada Pierce) Washington,DC. ChaupimayoNY1 AF246570 AF246593 AF246594 Brazil: Rondonia,Ariquemes AF246553 Brazil: Rondonia,Ariquemes Malaysia: Sabah,Luasong Malaysia: ex. Naomi AF246529 Pierce) reared in captivity) AMNH AMNH USA: New York,Ulysses AMNH MCZ AMNH AMNH AMNH MCZ AMNH AMNH AMNH VA2 AF169919 USA: Virginia,Blue Ridge AMNH SA-2-2JV3RB248 AF246582 AF014141 AF014140 Malaysia: Sabah,Luasong Indonesia: Brazil: Rondonia,Ariquemes West Java,Bogor AMNH AMNH AMNH E-4-3RB329RB275E-4-5JB1 AF169903 AF169918 AF169920 : AF014135 Pichincha,Tinalandia Brazil: Rondonia,Ariquemes Brazil: Rondonia,Ariquemes AF169883 Ecuador: Pichincha,Tinalandia : Bolivar,Cartagena CUIC AMNH AMNH CUIC CUIC NSW4B-5-1C-17-2SA-2-1RB283 AF246527C-17-4 AF246528P-24-4 AF014139 : AF246585 NSW,Evans Head AF014137 Brazil: Parana Colombia: AF169900 Putumayo,62 km North Mocoa Malaysia: Sabah,Luasong AF169909 Brazil: Rondonia,Ariquemes Colombia: Putumayo,62 km North Mocoa : Panama CUIC AMNH CUIC AMNH AMNH B-10-1P-11-7 AF169921 AF169902 Brazil: Amazonas,Manacapusu Panama: Panama AMNH higher-level classi¢cation of Nymphalidae ) 1991  castelnaui dorcas semistalachtis Phyciodes tharos nauplius bellona interrogationis bella hypatia steneles coenia iphita bolina limborgii phaeton Speyeria cybele emalea erymanthis claudia vibilia Neruda metharme Laparus doris genitrix Actinote stratonice hypsea Philaethrea dido Podotricha telesiphe juno Agraulis vanillae Eurytelina Phyciodina Boloriina uncertain) Biblidini Biblidina Kallimini Euphydryina Heliconiina Exemplar taxa included in this study, listed according to Harvey's Limenitidinae Coloburini Nymphalinae Table 1. Genera within tribesarticle are 29.2 listed International in Commission Harvey's on order. Zoological Nomenclature Genera 1999).) of Satyrinae aresubfamily placed in tribes based on Miller 1968).Heliconiinae The `-ina' tribe ending for subtribes re subtribe genus species sample code

Proc. R. Soc. Lond. B 2000) 1204 A.V.Z. Brower Phylogenetic relationships among the Nymphalidae ) Cont.  GenBank accession locality voucher V22RB282RB262RB387RB225 AF246580 AF246536RB221RB348 AF246552 Venezuela: Brazil:RB242 AF246537 Delta Rondonia,Ariquemes Amacuro,BarrancasNY3 AF246535 Brazil:G-36-4 AF246572 Rondonia,Ariquemes Brazil:NP95-Y156 AF246581 Rondonia,Ariquemes Brazil:RB250 AF014143 Rondonia,Ariquemes Brazil:NP95Y191 Rondonia,Ariquemes AF246610 AF246531 Brazil:SA-2-7 AF246574 AMNH Rondonia,Ariquemes Brazil: Rondonia,Ariquemes AF246586RB317 AF246532 Malaysia: ex. Naomi USA: French Pierce) New York,Caroline,Shindagin Guiana: Kaw AMNH HollowRB261 AF246605 Malaysia: ex. Brazil: NaomiF20 Pierce) Rondonia,Ariquemes AMNH AF246557 AMNH AMNH Malaysia:NP95-Y227 Sabah,Luasong AMNH RB295 AF246611 AMNH Brazil:RB318 Rondonia,Ariquemes AF246588 AMNH AF246556 AMNH Brazil:SA-2-4 Rondonia,Ariquemes MCZ RB247 AF014144 Malaysia: ex. Naomi Pierce)RB259 USA: AF246575 Florida,GainesvilleRB353 MCZ AMNH Brazil:RB306 AF246583 Rondonia,Ariquemes AMNH Brazil:QL8 AF246544 Rondonia,AriquemesNP95-Y235 AF246545 AMNH Malaysia:SA-2-8 AF246549 Sabah,Luasong AMNH Brazil:NSW3 AF246568 Rondonia,Ariquemes AF246596 Brazil:F26 Rondonia,Ariquemes AMNH AF246578 Brazil:PE-6-1 Rondonia,Ariquemes MCZ Brazil: AF246600 Malaysia: Rondonia,Ariquemes ex. Naomi Pierce) AF246546 AMNH Australia: Queensland,Brisbane AMNH Malaysia: AF246602 Sabah,Luasong AF246547 AMNH Australia: NSW,Broadwater Peru: USA: Cuzco,Alfamayo AMNH Florida,Gainesville AMNH AMNH AMNH AMNH MCZ AMNH AMNH AMNH AMNH AMNH NY13 AF014145 USA: New York,Ulysses AMNH sp. RB355 AF246550 Brazil: Rondonia,Ariquemes AMNH sp. RB273 AF246555 Brazil: Rondonia,Ariquemes AMNH sp. RB226 AF246539 Brazil: Rondonia,Ariquemes AMNH sp. RB294 AF246548 Brazil: Rondonia,Ariquemes AMNH sp. RB256 AF246554 Brazil: Rondonia,Ariquemes AMNH sp. RB244 AF246573 Brazil: Rondonia,Ariquemes AMNH cyaniris acontius chloe divalis Batesia hypochlora Temenis laothoe maeon clymena cytherea Taenicia julii orsilochus rahria athamas clytemnestra itys Doxocopa Eulaceura osteria chorinaeus Antirrhaea cassina actorion leda mekara fusca abeona sorata pegala Epicaliina Ageroniina Zaretidina Epiphilina Dynaminina Catagrammina Euthaliina Antirrhaeina Melanitina Mycalesina Maniolina Limenitidina Cyrestidini Preponini BiiniElymniiniSatyriini Biina Lethina Hypocystina Anaeina ) Cont. Charaxinae Charaxini Apaturinae Morphinae MorphiniBrassolinae Morphina Satyrinae Table 1.  subfamily tribe subtribe genus species sample code

Proc. R. Soc. Lond. B 2000) Phylogenetic relationships among the Nymphalidae A.V.Z. Brower 1205 GenBank accession locality voucher PE-5-10PE-5-9 AF246587NP95-Y318SA-1-1 AF246597C-3-8 AF246592 Peru: Cuzco,Quebrada SanNSW1 Luis Peru:QL7 Cuzco,Quebrada San LuisCU1 AF246603 Malaysia: ex. Naomi Pierce)RB241 AF246564 AF246565 Malaysia: Sabah,Gum-Gum AF246566 Colombia: Meta,Villavicencio AF246579 Australia: AF246567 Queensland,Brisbane AMNH Australia: Queensland,Brisbane AMNH Cuba: Santiago Brazil: de Rondonia,Ariquemes Cuba MCZ AMNH CUIC AMNH AMNH AMNH AMNH QL1 AF246563 Australia: Queensland,Kirrama State Forest AMNH E-12-1V20RB328RB288 AF246562RB233RB238 AF246561 Ecuador:RB314 AF246584 MacaraRB321 AF014146 Venezuela: AF246560 Delta Amacuro,Barrancas Brazil:RB237 AF246607 Rondonia,Ariquemes Brazil:RB235 AF014147 Rondonia,Ariquemes Brazil:RB305 AF246558 Rondonia,Ariquemes Brazil:RB324 Rondonia,Ariquemes Brazil:PE-10-12 AF246591 Rondonia,Ariquemes Brazil: AF246601 Rondonia,Ariquemes AMNH AF246571 Brazil: AF246559 Rondonia,Ariquemes AF246606 Brazil: Rondonia,Ariquemes Brazil: Rondonia,Ariquemes AMNH Brazil: P: Rondonia,Ariquemes Cuzco,Quebrada AMNH AMNH Chaupimayo AMNH AMNH AMNH AMNH AMNH AMNH AMNH AMNH AMNH RB276 AF246551 Brazil: Rondonia,Ariquemes AMNH NY15NY12 AF246543 AFO14158 USA: Pennsylvannia,Holtwood USA: New York,Ithaca AMNH AMNH sp. PE-5-1 AF246604 Peru: Cuzco,Quebrada San Luis AMNH sp. RB358 AF246576 Brazil: Rondonia,Ariquemes AMNH sp. RB229 AF246599 Brazil: Rondonia,Ariquemes AMNH sp. PE-10-14 AF246589 Peru: Cuzco,Quebrada Chaupimayo AMNH sp. PE-14-3 AF246608 Peru: Cuzco,Rio Lucumayo AMNH sp. NSW2 AF246577 Australia: NSW,Lismore AMNH umbratilis Steroma gaura agleoides Danaus plexippus hamata core briarea cleobaea Corades cistene Tellervo zoilus harmonia maenius Scada theaphia psidii aquata Hyaliris daphnis pheranthes dero nise tucuna pavonii Libytheana carinenta Pieris rapae Delias Anthocharis midea Danaina Itunina Euploeini Euploeina Melinaeini Methonini Mechanitini Oleriini Napeogenini Dircennini new tribe) Godyrini ) Cont. Danainae Amaurina Table 1.  subfamily tribe subtribe genus species sample code Tellervinae Tellervini Ithomiinae Tithoreini Libytheinae Libytheini OUTGROUP Pieridae

Proc. R. Soc. Lond. B 2000) 1206 A.V.Z. Brower Phylogenetic relationships among the Nymphalidae placement of Riodininae will be addressed elsewhere Campbell in the three members of Melitaeini sampled Phyciodes, et al. 2000). Eresia and Euphydryas). The dynamics of the sequence divergence in the wingless gene are addressed in Brower & c) DNA extraction DeSalle 1998) and Campbell et al. 2000) and will not DNA was extracted from individual butter£ies following the be discussed further here. In this matrix,99 nucleotide protocol presented in Brower 1994). Brie£y,specimens were sites are invariant,59 vary in a single taxon and the macerated in liquid nitrogen,digested with proteinase K in a remaining 220 are cladistically informative potential Tr i s ^EDTA lysis bu¡er and phenol^chloroform extracted. DNA synapomorphies within the Nymphalidae). was precipitated with isopropanol,washed with 70% ethanol The equal-weighted parsimony analysis yielded 336 and resuspended in water. The DNA stocks were stored at equally parsimonious cladograms of 2760 steps. The strict 720 8C. The wings and abdomens of each specimen were consensus ¢gure 3) implies monophyly of the family preserved as vouchers Brower 1996) and deposited at the Amer- Nymphalidae,the subfamilies Heliconiinae,Nympha- ican Museum of Natural History,Cornell University linae,Brassolinae,Danainae,Ithomiinae,Charaxinae Collection or the Museum of Comparative Zoology at Harvard and Apaturinae and numerous less inclusive clades identi- see table 1). ¢ed in Harvey's 1991) classi¢cation. The branch support values for many of these clades are indicated in ¢gure 3. d) PCR and sequencing The Limenitidinae and Satyrinae appear as unresolved Polymerase chain reaction PCR) ampli¢cations were paraphyletic groups. Individual clades will be discussed performed with the primers and standard conditions presented further below. in Brower & DeSalle 1998). The PCR products were gene SAW resulted in a single stable topology after nine cleaned,cycle sequenced in a Perkin-Elmer 9600 and run on an iterations ¢gure 4). This cladogram is 2803 steps long ABI 373 automated sequencer from sense and anti-sense strands. when the character weights are restored to 1 43 steps The automated sequence outputs were edited manually and longer than the MP cladograms under equal weights). aligned by eye. The aligned data matrix is available on the Eighty-one of the 102 nodes are congruent between the World Wide Web http://www.ent.orst.edu/browera/datasets/ SAW cladogram and the MP consensus tree `congruent' dataset7.htm) and individual sequences have been submitted to meaning that the SAW tree either exhibits the same GenBank see table 1 for the accession codes). clades as the MP consensus tree or provides resolution to its polytomies).With the exception of two basal nodes,the e) Data analyses incongruent patterns of grouping are localized within Cladistic analyses were performed using PAUP 3.1 Swo¡ord three clades: Heliconiina,Danainae plus Ithomiinae and 1991). All nucleotide sites were weighted equally and gaps were Satyrinae. coded as missing data. Tree-bisection-reconnection TBR) branch swapping was performed on ¢ve separate sets of 100 random addition sequences. The results of these searches were 4. DISCUSSION pooled and duplicate trees were culled. The branch support a) Quality Bremer 1988,1994; Davis 1995) for selected nodes was The average informative character changes state more calculated using anti-constraints with ten random additions. To than 12 times on the most parsimonious trees and some assess the ability of the data to imply a self-consistent topology, change up to 35 times. Theoretically,such rampant successive approximations weighting SAW) Farris 1969) was homoplasy suggests that these data are not valuable as a performed in PAUP based on the maximum rescaled consis- source of evidence for inferring the patterns of relation- tency index for each character among the most parsimonious ship among the groups represented Sanderson & cladograms from the equal-weighted analyses. Unless SAW Donoghue 1996). Hillis 1996) concluded from a simula- yields a subset of the MP cladograms,the results of the respect- tion study that,although larger sets of taxa can resolve ive analyses are not comparable by quantitative means because relationships with shorter sequences,approximately an the two weighting schemes rely on di¡erent philosophical inter- order of magnitude more sequence per taxon than was pretations of the parsimony criterion. The most parsimonious included in this matrix is required to infer the simulated MP) and SAW trees di¡ered in this study and are compared branching pattern accurately.These data o¡er an empirical qualitatively here by assessment of topological congruence Judd refutation of those claims. 1998). The historical course of evolutionary divergence is unknowable and empirical systematists eschew inductive summary measures of accuracy or con¢dence in favour of 3. RESULTS qualitative assessment of the stability and corroboration of One hundred and three nymphalid taxa representing prior hypotheses. The results presented here are stable to 12 of the 13 subfamilies and 30 of the 38 tribes recognized the extent that the inferred MP topology is largely self- by Harvey 1991) and three pierid outgroups were consistent under the implied weights Judd 1998). In compiled for the wingless region. Nine of the sequences general,SAWreinforced the groups implied by the data in have been published previously in Brower & Egan 1997), the MP tree. The incongruence between the MP and SAW 12 have been published previously in Brower & DeSalle trees within Heliconiina occurs among weakly supported 1998) and the remaining 85 are new. The resulting clades and neither of those implied branching orders aligned data matrix of 106 taxaÂ378 characters contains matches the pattern resulting when taxa are sampled more only three gap regions: two separate autapomorphic intensively at the same locus Brower & Egan 1997). 3-base deletions in Euptoieta and Lethe,respectively and a Within the Ithomiinae plus Danainae clade,three single 3-base insertion a putative synapomorphy) present instances of incongruence result from the instability of

Proc. R. Soc. Lond. B 2000) Phylogenetic relationships among the Nymphalidae A.V.Z. Brower 1207

Heliconius Laparus Heliconiinae Neruda Eueides Dione Agraulis Heliconiina 6 Podotricha Dryadula 2 Dryas 8 Acraea Actinote Actinote Acraeini 3 Cethosia Euptoieta Boloria Speyeria Cupha Cirrochroa 22 Euphydryas Nymphalinae Phyciodes Eresia Hypolimnas Melitaeini Colobura Vanessa 1 Polygonia 3 Hypanartia Nymphalini Symbrenthia Kallima Siproeta Precis Junonia Limenitis Limenitidini 3 Adelpha Marpesia Biblis Vila Laringa Mestra Batesia Panacea Catonephele Myscelia Memphis Zaretis Charaxinae 3 Prepona Hypna Polyura 1 Caligo Opsiphanes Brassolinae Bia Tisiphone Cercyonis Pedaliodes Megisto Taygetis 5 Corades Lymanopoda Steroma Haetera Amathusia Morpho Libytheana Chersonesia Hamadryas 2 Doxocopa Asterocampa Apaturinae Eulaceura Diaethria Oleria Ceratinia Ithomiinae Dircenna Hypoleria Pteronymia Hyaliris Hypothyris 2 Napeogenes Mechanitis 2 Scada Mechanitini Thyridia 4 Melinaea Athyrtis Melinaeini 7 2 Tithorea Elzunia Tithoreini Methona Tellervo 6 Danaus Danainae Tirumala Danaina 6 Euploea 10 Lycorea Anetia Itunina 9 Ideopsis Parantica Amaurina Temenis Dynamine Eunica Caerois Melanitis Mycalesis Lethe Oressinoma Anthocharis Pieris Delias

Figure 3. Strict consensus of 336 equally parsimonious cladograms for the wingless data matrix with equal weights length ˆ 2760,CI ˆ 0.189 and RI ˆ 0.531). Traditionally recognized subfamilies and tribes or subtribes) appearing as clades here are indicated by black and white bars,respectively. The branch support values are indicated for these groups.

Proc. R. Soc. Lond. B 2000) 1208 A.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 Brassolini Antirrhea Caerois Morpho Tisiphone Oressinoma Satyrinae Cercyonis (+ Amathusiini) Pedaliodes Corades Taygetis Lymanopoda Steroma Megisto Haetera Lethe Melanitis Mycalesis Amathusia Libytheana Chersonesia Anthocharis Delias Pieris

Figure 4. The most parsimonious cladogram resulting from SAW with weights restored to unity) length ˆ 2803,CI ˆ 0.18 and RI ˆ 0.521). Traditionally recognized subfamilies and tribes or subtribes) appearing as clades are indicated by black and white bars,respectively. Three paraphyletic grades are marked as well: arches link disjunct sections of the Biblidini and Ithomiinae. Crosses mark the nodes incongruent with the most parsimonious trees discovered with equal weighting.

Proc. R. Soc. Lond. B 2000) Phylogenetic relationships among the Nymphalidae A.V.Z. Brower 1209

Hypoleria and the remaining four result from the nesting of subfamily. The MP tree implies reciprocal monophyly of the Danainae between the basal and derived Ithomiinae in the Ithomiinae s. s. and Danainae s. s.,but the Itho- the SAW tree. The incongruence in the Satyrinae is not miinae become paraphyletic in the SAW tree. It is intri- attributable to the instability of speci¢c taxa or clades and guing that the -feeding Tithorea and Elzunia may instead result from insu¤cient sampling of this extre- are basal to the Apocynaceae and Asclepiadaceae-feeding mely diverse group which represents nearly one-third of `Danainae' in the latter hypothesis. Both cladograms all Nymphalidae) Ackery et al. 1999). support the hypothesis that Apocynaceae-feeding,apose- The corroboration of prior systematic hypotheses also matic larvae are primitive characteristics of the Danainae confers some degree of plausibility to the current results. s. l. Edgar et al. 1974; Brown 1987). The MP consensus implies monophyly in seven out of ten Ackery et al. 1999) resurrected Ehrlich's 1958) in- subfamilies Tellervinae and Libytheinae with a single clusive Morphinae,encompassing both the Palaeotropical representative each included are also trivially monophy- Amathusiinae and the Neotropical Brassolinae and Mor- letic). The traditional subfamilies not supported here have phinae of various authors. Among the morphine and long been considered arti¢cial e.g. Limenitidinae) or are satyrine lineages sampled,only the monophyly of the not diagnosed by morphological synapomorphies e.g. Brassolinae which clearly includes the formerly enigmatic Satyrinae to the exclusion of Brassolinae) Ackery et al. genus Bia)vide Miller 1968; DeVries et al. 1985) is 1999). Thus,without the `aid' of special process models supported by the MP cladogram. However,the SAW tree and mathematical corrections,this short gene region supports the monophyly of numerous traditionally recog- generally corroborates higher taxa supported by evidence nized clades,including the Brassolinae,Morphini and the from other character systems and fails to corroborate sister relationship of the Morphinae s. l. and Satyrinae, those which are not so supported. but Amathusia the only amathusiine sampled) joins a basal clade with Old World satyrines. This result implies b) Taxonomic implications the parallel of features associated with large The sister relationship between the Heliconiina and size in the Neotropical and Palaeotropical realms. Acraeini implied here necessitates reinterpretation of the However,given the diversity of this clade,these patterns female abdominal `stink clubs' Mu«ller 1886) from the are best viewed merely as intriguing hypotheses to synapomorphy uniting the Heliconiini sensu Harvey 1991) stimulate further research. to a synapomorphy uniting the Heliconiinae but lost in the The snout butter£ies Libytheinae or Libytheidae) are Acraeini. D. Harvey personal communication) suggested often viewed as the sister taxon of all other Nymphalidae that the loss may be associated with the simpli¢ed court- due to the plesiomorphic presence of functional forelegs ship performed by acraeines,which exhibit sperm priority in the females Ehrlich 1958; Scott & Wright 1990; enforced by a sphragis Smith 1984). The ad hoc invocation Ackery et al. 1999). The SAW tree implies a basal position of stink club homoplasy is compensated for by the more for Libytheana,with Chersonesia Cyrestidini ˆ Marpesiini) parsimonious occurrence of the capacity to sequester or as its immediate outgroup and the sister taxon to the synthesize cyanoglucosides Nahrstedt & Davis 1981), Libytheinae plus all other nymphalids. The other cyresti- which,according to this hypothesis,is uniquely derived dine, Marpesia,is sister to the Nymphalinae plus Biblidini, among the Heliconiina plus Acraeini. suggesting that the tribe may represent a polyphyletic The large and diverse `subfamily Limenitidinae' has basal assemblage. Both Libytheana and Chersonesia exhibit been long and almost unanimously recognized as an enlarged labial palpi see Ackery et al. 1999) for unnatural assemblage Mu«ller 1886; Scott & Wright comments on the range of variation in this character 1990; Harvey 1991; Ackery et al. 1999),a view corro- among nymphalid taxa). borated by the wingless data. The SAW tree suggests a¤nities between the Limenitidini and Heliconiinae, c) Prospects and between the Neotropical Biblidini and Nymphalinae. This study supports the hypothesis Brower & DeSalle Oddly,the apaturines sampled nest well inside the 1998; Campbell et al. 2000) that the small fragment of the Biblidini,although morphological features imply their wingless gene examined provides a useful source of charac- closer a¤nity to the Charaxinae Ackery et al. 1999). ters for cladistic inference among butter£y subfamilies, This is the least orthodox result from the wingless data tribes and genera. Given the relatively small number of and suggests that more detailed sampling of Old World gene regions that have been employed with success in the representatives of both the Apaturinae and `Limeniti- molecular systematics of to date Caterino et al. dinae' is necessary. 2000),the empirical success of the wingless gene represents Although no unique morphological synapomorphy has a signi¢cant addition to the battery of markers currently been found to unite the Danainae s. l.  ˆ Danainae plus available. Although phylogenetic hypotheses based on Ithomiinae plus Tellervinae),most authors have consid- data from single sources are potentially subject to bias or ered them to form a monophyletic group Bates 1861; `inaccuracy',the dense taxonomic sampling employed Clark 1947; Ehrlich 1958; Ackery 1984,1988). Ackery et al. here appears to permit synapomorphy to overcome high 1999) argued that the use of pyrrolizidine alkaloids for levels of homoplasy and corroborate clades well supported courtship pheromone synthesis and possibly for chemical by data from morphology. The initial phylogenetic struc- defence) Brower 1984) is a more promising shared trait ture presented here provides a framework for subsequent than anything yet described from morphology. The investigation of particular nymphalid clades in greater Danainae s.l. is the most strongly supported major clade detail. The taxa in particular need of more intensive in this study,corroborating Ackery et al.'s 1999) ranking sampling or improved resolution are the Limenitidini, of the Ithomiinae plusTellervinae plus Danainae as a single Satyrinae,Amathusiini and .

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

Foremost,I thank Rob DeSalle and Jim Miller for sponsorship Pattern formation and eyespot determination in butter£y and support during my work at the American Museum of wings. Science 265,109^114. Natural History 1993^1997). I thank G. Austin,F. Beniluz, Caterino,M. S.,Cho,S. & Sperling,F. A. H. 2000 The current A. Berry,C. Bridges,T. Collins,J. Hall,B. Hermier,G. Lamas, state of insect molecular systematics: a thriving tower of K. Matsumoto,N. Pierce,D. Otero,K. Sime,C. Snyder,M.-W. Babel. A. Rev. Entomol. 45.Inthepress.) Tan,A. Warren,K. Willmott,M. Whiting and D. Yeates for Clark,A. H. 1947 The interrelationships of the several groups assistance in the ¢eld and/or providing specimens. I also within the butter£y superfamily Nymphaloidea. Proc. Entomol. acknowledge the dedicated laboratory work of E. Bonwich, Soc.Wash. 49,148^149 and 192. M. Egan,G. Grills and F. Vital. I am indebted to P. Ackery, Davis,J. I. 1995 A phylogenetic structure for the monocotyle- P. DeVries,D. Harvey,J. Rawlins and R. Vane-Wright for dons,as inferred from chloroplast DNA restriction site discussion of nymphalid systematics. Finally,I thank D. Judd for variation,and a comparison of measures of clade support. critical insights in the data analysis. This research was supported by National Science Foundation NSF) DEB-9220317 Syst. Bot. 20,503^527. to Rob DeSalle and Jim Miller and by NSF DEB-9303251 and De Jong,R.,Vane-Wright,R. I. & Ackery,P. R. 1996 The an American Museum of Natural History postdoctoral fellow- higher classi¢cation of butter£ies Lepidoptera): problems ship to the author. and prospects. Entomol. Scand. 27,65^101. DeVries,P. J.,Kitching,I. J. & Vane-Wright,R. I. 1985 The systematic position of Antirhaea and Caerois,with comments on REFERENCES the classi¢cation of the Nymphalidae Lepidoptera). Syst. Ackery,P. R. 1984 Systematic and faunistic studies on butter£ies. Entomol. 10,11^32. In The biology of butter£ies ed. R. I. Vane-Wright & P. R. DeVries,P. 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