PapilioPhylogeny Based on Mitochondrial Cytochrome Oxidase I and II Genes

Molecular Phylogenetics and Evolution Vol. 11, No. 1, February, pp. 122–137, 1999 Article ID mpev.1998.0549, available online at http://www.idealibrary.com on

Papilio Phylogeny Based on Mitochondrial Cytochrome Oxidase I and II Genes Michael S. Caterino and Felix A. H. Sperling Department of Environmental Science, Policy and Management, Division of Insect Biology, 201 Wellman Hall, University of California, Berkeley, Berkeley, California 94720 Received January 26, 1998; revised May 3, 1998

Butterflies of the genus Papilio have served as the basis for numerous studies in insect physiology, genetics, and ecology. However, phylogenetic work on relationships among major lineages in the genus has been limited and inconclusive. We have sequenced 2.3 kb of DNA from the mitochondrial cytochrome oxidase I and II genes (COI and COII) for 23 Papilio taxa and two outgroups, Pachliopta neptunus and Eurytides marcellus, in order to assess the potential of these genes for use in Papilio phylogenetics and to examine patterns of gene evolution across a broad taxonomic range. Nucleotide and amino acid variation is distributed heterogeneously, both within and between genes. Structural features of the proteins are not always reliable predictors of variation. In a combined analysis, these sequences support a nearly fully resolved topology within subgenera and species groups, though higher level relationships among species groups require additional study. The most noteworthy findings are that neither Papilio alexanor nor P. xuthus belongs in the machaon group and that the subgenus Pterourus is paraphyletic with respect to the subgenus Pyrrhosticta. We leave relationships among members of the phorcas species group as a trichotomy. These two protein coding genes, particularly COI, show excellent performance in resolving relationships at the level of species and species groups among Papilionidae. We strongly endorse a similar approach for future studies aimed at these levels. r 1999 Academic Press

INTRODUCTION Butterflies of the genus Papilio are among the best known invertebrate organisms. Several synthetic works on the basic ecology, evolution, genetics, physiology, and conservation status of the species have been published (e.g., Collins and Morris, 1985; Tyler et al., 1994; Scriber et al., 1995b). Worldwide taxonomic, life history, larval, and host plant diversities of the approximately 200 species have been relatively well documented. This strong scientific foundation, in addition to their natural appeal, has led to the use of Papilio 1055-7903/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

species in hundreds of basic studies in a wide variety of biological disciplines. Despite the extensive use of Papilio species in basic research, the phylogenetic framework within which to interpret these studies remains weak. Early work by Ford (1944), Munroe (1961), and others differed on the limits of tribes, species groups, and species of Papilionidae. These studies provided little character support for their hypotheses, and their interpretation in a modern phylogenetic context is therefore ambiguous. Among the earliest attempts to examine relationships in an explicitly cladistic context was the study of Hancock (1983), though his treatment of characters is rather brief. Igarashi (1984) subsequently proposed a phylogeny of most of the major Papilionid lineages on the basis of larval morphology. Miller (1987) expanded on Hancock’s (1983) work with a more extensive discussion of the characters used and their scoring within the taxa studied. Miller also implemented a more appropriate polarization technique through the designation of an outgroup. Brown et al. (1995) have criticized some of Miller’s (1987) homologies and have reexamined his reworked character matrix with the addition of several larval characters in an analysis of combined evidence. The only studies that have comprehensively addressed membership in and relationships among all species groups have been those of Munroe (1961) and Hancock (1983). Numerous studies have attempted to employ hybrid fitness data in evaluating Papilio interrelationships (Clarke and Sheppard, 1953, 1955; Scriber et al., 1995a; Ae, 1995). Ae (1995) used egg viability, sex ratio, F1 fertility, and survivorship on parental hosts to derive a distance measure which he used to construct a phylogenetic hypothesis. However, distances as phylogenetic data, whatever their basis, have the distinct disadvantage of attempting to summarize numerous, independent points of comparison as a single one (Farris, 1981; Albert et al., 1992). Furthermore, distances based on hybridization data, in particular, may be grossly biased by a very small portion of the entire genome (Sperling et al., 1997).

122

Papilio mtDNA PHYLOGENY

Allozyme analysis has been applied to phylogenetic investigation within the machaon, glaucus, and troilus groups (Sperling, 1987; Hagen and Scriber, 1991) but has not been extended to higher taxonomic levels. DNA-based studies on Papilio using mtDNA restriction sites have also focused on relationships within these three species groups (Sperling, 1993a, b; Sperling and Harrison, 1994). While the study of mtDNA in Papilio is of special interest due to its expected concordance with species boundaries (Sperling, 1993b, 1994; Hagen and Scriber, 1995), restriction sites appear to have limited reliability in higher-level phylogenetic problems (Sperling, 1991; Caterino and Sperling, unpublished results). Though our understanding of DNA evolution at the nucleotide level has advanced tremendously in recent years, as have methods of phylogenetic analysis of DNA sequences, no phylogenetic studies employing direct sequencing have yet been published on the Papilionidae. The present study includes 22 species and one subspecies of the approximately 200 recognized species of Papilio. The sampled species include all major lineages in four of the most-studied species groups (the machaon, phorcas, glaucus, and troilus groups). Additional species were chosen to allow examination of some additional longstanding controversies in the classification of the species of Papilio. Though the Papilio machaon group has been the subject of numerous studies, the limits of its membership have been difficult to resolve. Morphological data exhibit numerous ambiguities in resolving the remaining questions. P. alexanor was assigned to the machaon group primarily on the basis of its larval color pattern

123

(Munroe, 1961), which resembles the banded/spotted appearance of most of the rest of the machaon group (see Fig. 1) and possibly facilitates crypsis on its Apiaceae host plants (Brower and Sime, in press). This character is the primary diagnostic feature of the group. Adults of P. alexanor, however, exhibit a striped pattern more like members of the P. glaucus species group, none of which feed on Apiaceae. This adult ‘‘tiger striped’’ pattern is more or less widespread among Papilionidae, known in other species groups (e.g., P. pilumnus in the troilus group) and even other tribes (e.g., some Graphium among the Graphiini). It is thus conceivably plesiomorphic within Papilio. The male genitalia of P. alexanor are somewhat intermediate between that of the machaon group and that of the glaucus group. A restriction site survey across the machaon group revealed a high level of divergence between P. alexanor and the remainder of the group, causing the putative outgroup P. xuthus to appear to be a member of the machaon group (Sperling and Harrison, 1994). The inclusion of both P. alexanor and P. xuthus as well as a full selection of the undisputed species of the machaon group in this study allows us to reexamine the boundaries of the lineage. The relationships among three species of the phorcas group have recently been examined by Vane-Wright and Smith (1991) and Clarke et al. (1991). One of these species, P. dardanus, has served as the foundation of numerous classic studies in complex Batesian mimicry (Ford, 1936; Clarke and Sheppard, 1963). In some subspecies, the dardanus female mimics other sympatric species, including members of the Nymphalidae (Danainae) and some day-flying Lymantriidae (Vane-

FIG. 1. Comparisons of some morphological features of (1) Papilio glaucus, (2) P. alexanor, and (3) P. zelicaon: (a) adult color pattern, (b) larval color pattern, and (c) right genitalic clasper of male.

124

CATERINO AND SPERLING

Wright and Smith, 1991), while in other areas, the female is an andromorph, resembling the male. P. phorcas is also polymorphic to a lesser degree. One of the major questions in the study of this group has been the direction of evolution of the polymorphism, whether from ancestral andromorphy to Batesian mimicry or the reverse. We included members of all three species of this group in hopes that mtDNA data might help to resolve this trichotomy. There have also been questions regarding the relationships within Pterourus. Hancock (1983) recognized two subgenera in Pterourus: Pterourus s. str. (glaucus and troilus groups) and Pyrrhosticta (zagreus, scamander, and homerus groups). Hybridization studies carried out by Scriber et al. (1991) challenged these groupings. They found higher offspring viabilities when crossing P. glaucus (glaucus group) with members of the scamander group (different subgenus) than with members of the troilus group (same subgenus). The present study examines these relationships with samples from all of these lineages. To address these issues we sequenced the entire cytochrome oxidase I and II genes (COI and COII) plus the intervening gene coding for tRNA-leucine, producing approximately 2.3 kb of sequence. The COI and COII genes code for two of seven polypeptide subunits in the cytochrome c oxidase complex (Capaldi et al., 1983). COI and/or COII sequences have been applied to phylogenetic problems at a wide range of hierarchical levels in insects, from closely related species (Sperling and Hickey, 1994; Beckenbach et al., 1993; Brower, 1996) to genera and subfamilies (Frati et al., 1997), families (Howland and Hewitt, 1995), and even orders (Liu and Beckenbach, 1992). Since we have sequenced both genes, this represents one of the largest sequence data sets generated for phylogenetic study of any group of insects and should put us relatively high on the putative phylogenetic accuracy curve of Hillis (1996). Our goals are to present a mtDNA sequence phylogeny of most of the best-studied lineages of Papilio, to establish the utility of the mitochondrial COI and COII genes for this purpose, and to examine the phylogenetic placements of several lineages which have proven difficult in previous studies. MATERIALS AND METHODS Specimens The specimens used in this study were provided by collaborators or were collected by F. Sperling. A taxomic summary of material examined is provided in Table 1. These represent 10 of the 42 species groups of Hancock (1983). We follow Miller (1987) in using Papilio in the broad sense, encompassing all of these species. Two other Papilioninae, Pachliopta neptunus (Troidini) and Eurytides marcellus (Graphiini), and one member of the Pieridae (Colias eurytheme) were sequenced to

serve as outgroups. Voucher wings and abdomens were deposited in Lot No. 1204 in the insect collection of the Department of Entomology at Cornell University or, for the Colias specimen, in the Essig Museum of Entomology, University of California at Berkeley. Molecular Techniques Total genomic DNA was extracted as in Sperling and Harrison (1994). Most amplified fragments were approximately 4–500 bp long. Amplifications were performed on an Ericomp TwinBlock EasyCycler using a hot start: Taq was added at the end of an initial denaturation at 94°C. This was followed by 35 cycles of 1 min at 94°C, 1 min at 45°C, 1.5 min at 72°C and a subsequent 5-min final extension at 72°C. PCR products were cleaned using Millepore Ultrafree MC filtration tubes or with Qiagen’s PCR Purification Kit. The PCR product was cycle sequenced with Perkin–Elmer/ ABI Dye Terminator Cycle Sequencing Kit with AmpliTaq FS on an MJ Research PTC200 according to Perkin–Elmer’s suggested thermal profile. The sequenced product was filtered through Sephadexpacked columns and dried. This product was resuspended and electrophoresed on an Applied Biosystems International 377 automated sequencer. All fragments were sequenced in both directions. Sequences were aligned manually to the sequence of Drosophila yakuba (Clary and Wolstenholme, 1985). All primers used and their positions relative to D. yakuba are given in Table 2. Phylogenetic Analyses Analyses under parsimony and maximum likelihood were carried out using PAUP 4d(54-55). Sequence alignments were done by eye. In protein-coding regions sequences were translated to amino acids (using GeneJockeyII, version 1.0; Taylor, 1993) prior to alignment. GeneJockeyII was also used to examine amino acid properties [hydrophobicity was calculated using the hydropathy index of Kyte and Doolittle (1982); polarity was calculated using the index of Zimmerman et al. (1968)]. MacClade (version 3.06; Maddison and Maddison, 1992) was used to compute various sequence statistics: nucleotide transformation frequencies, variation among nucleotide positions within the codon, and levels of variation in rate across the entire 2.3 kb. In addition to the core analyses on the nucleotide data, parsimony analyses were carried out on amino acid sequences (all amino acid transformations equally weighted) and on some partitions of the nucleotide data. Starting with all positions equally weighted, we also explored the topological effects of differentially weighting transversions and codon positions (specific schemes discussed under Results). We employed a heuristic search with 50 random taxon addition sequence replicates (all branch-swapping algorithms found the same set of trees). To assess branch support

125

Papilio mtDNA PHYLOGENY

TABLE 1 Species and Specimens Examined Species a Papilionidae Papilionini Papilio (Sinoprinceps) xuthus group xuthus (X) Papilio (Princeps) demoleus group demoleus phorcas group phorcas constantinus dardanus Papilio (Heraclides) thoas group cresphontes anchisiades group anchisiades Papilio (Papilio) machaon group machaon (M1) m. oregonius (M3) zelicaon (Z1) hospiton (H) polyxenes (P1) indra (I) alexanor (A) Papilio (Pterourus) glaucus group glaucus (G1) canadensis (G4) rutulus (R1) multicaudatus (MU1) troilus group troilus (T1) palamedes (PA1) pilumnus (PI) Papilio (Pyrrhosticta) scamander group scamander homerus group garamas Graphiini Eurytides marcellus Troidini Pachliopta neptunus Pieridae Colias eurytheme a

Collection locality

Collector

GenBank Accession no.

Japan: Tokyo

M. Taguchi

AF043999

Malaysia: Penang

D. Goh

AF044000

Kenya Kenya Kenya

I. Gordon I. Gordon I. Gordon

AF044001 AF044002 AF044003

USA: WI: Sauk County

E. Heininger

AF044004

Brazil: Campinas

K. Brown

AF044005

France: Coudoux USA: WA: Palouse Falls USA: CA: Riverside County Sardinia USA: NY: Tompkins County USA: WA: Wawawai France: Dept. Vaucluse

L. Piquemal S. Anderson J. Emmel R. Crnjar M. Carter W. Wehling L. Piquemal

AF044006 AF044007 AF044008 AF044009 AF044010 AF044011 AF044012

USA: MD: Potomac USA: NY: Richford USA: WA: Orcas Island USA: SD: Black Hills

F. Sperling F. Sperling C. Yoon F. Sperling

AF044013 AF044014 AF044015 AF044016

USA: FL: Ocala State Forest USA: FL: Ocala State Forest Mexico: Tamaulipas

F. Sperling F. Sperling R. Lederhouse

AF044017 AF044018 AF044019

Brazil: Campinas

K. Brown

AF044020

Mexico: Tamaulipas

R. Lederhouse

AF044021

USA: FL: Ocala State Forest

F. Sperling

AF044022

Malaysia: Penang

J. Weintraub

AF044023

CAN: ON: Ottawa

F. Sperling

AF044024

mtDNA codes after species names correspond to Sperling (1993b) or Sperling and Harrison (1994).

the equally weighted sequence data set was bootstrapped 1000 times using a heuristic parsimony search with simple taxon addition sequence and NNI branch swapping. All trees were rooted with the sequences of Colias eurytheme, Feltia jaculifera pheromone type A (Lepidoptera: Noctuidae; Sperling et al., 1996), and D. yakuba (Clary and Wolstenholme, 1985). Though these outgroups are rather distant, and faster-evolving positions might be expected to be polarizing randomly (Wheeler, 1990), rootings of trees based only on first-

and second-codon positions were approximately equivalent (the topologies were not identical, making direct comparison difficult). Additionally, unrooted analyses of the Papilionini OTUs alone resulted in near identical topologies. Inclusion of the nonpapilionids allowed us to examine the evolution of these genes over a broad range of divergences. While a multiple gene analysis brings up issues of combinability, adjacent mitochondrial genes are expected to exhibit fully congruent histories; partition homogeneity testing (using PAUP*)

126

CATERINO AND SPERLING

TABLE 2 Primers Used in This Study Primer name k698 k760 Ron RonII RonIII RonIV k699 k808 Jerry JerryIV Nancy k525 BrianII BrianIV BrianXV BrianXVI BrianXVII k741 k807 Mila MilaV MilaVII George GeorgeIII GeorgeVII GeorgeVIII Pat PatII Patrick Pierre Marilyn MarilynII MarilynIII MarilynIV Marlon MarlonII MarlonIII MarlonIV Eva

Majority/ minority

Location a (38 end)

J N J J J J N N J J N N J J J J J N N N N N J J J J N N J J N N N N J J J J N

1460 1687 1751 1751 1751 1751 1840 1840 2183 2183 2192 2329 2495 2495 2495 2495 2495 2578 2578 2659 2659 2659 2792 2792 2792 2792 3014 3014 3038 3138 3389 3389 3389 3389 3408 3408 3408 3408 3782

Sequence (58 = 38) TAC CAA GGA GGA GGA GGG AGG TGG CAA CAA CCC ACT CGT CAT CAT CAT CAT TGG TGA GCT GTT GAA ATA ATA ATA ATA TCC TCC CTA AGA TCA TCA TCA TCA CAA CAA CAA CAA GAG

AAT TTA TCG CCT AAA CTT CAG CC TTT CCA AAT CCT CCA ATT AT TCA CCT GAT ATA GCA TTC CC TCC CCT GAT ATA GC(T/C) TTT CC GCA CCT GAC ATA GCT TTC CC GCC CCT GAC ATA GCC TTC CC AGG ATA AAC AGT TCA (C/T)CC AGG GTA TAC TGT TCA ACC CAT TTA TTT TGA TTT TTT GG CAC TTA TTT TGA TTC TTC GG GGT AAA ATT AAA ATA TAA ACT GTA AAT ATA TGA TGA GCT CA CAA TAT TAT GAA GAT TGG G CAA TTT TAT GAA GTT TAG G CAA TT(T/C) TAT GAA GAT TAG G CGA TTC TTT GAA GAT TAA G CTA TTC TTT GAA GAC TAG G AAA TGT GCA ACT ACA TAA TA AAA TGA GCT ACA ACA TAA TA AAT CCA GTG AAT AAT GG AGT CCT GTA AAT AGA GG AGT CCA GTA AAT AAA GG CCT CGA CGT TAT TCA GA CCT CGG CGA TAC TCT GA CCT CGT CGT TAT TCT GA CCT CGT CGA TAT TCC GA AAT GCA CTA ATC TGC CAT ATT A ATT ACA TAT AAT CTG CCA TAT TAG ATA TGG CAG ATT ATA TGT AAT GGA GCC TCT CCT TTA ATA GAA CA TAA GTT CA(A/G) TAT CAT TG TA(T/A) CTT CA(A/G) TAT CAT TG TAT CTT CAG TAT CAC TG TAA CTT CAA TAT CAC CA TGA TAT TGA AGT TAT GA TGA TA(T/C) TGA AG(T/A) TAT GA TGA TAT TGA AGT TAC GA TGA TAT TGA AGA TAT GA ACC ATT ACT TGC TTT CAG TCA TCT

Note. Majority/minority is equivalent to sense/antisense for COI-COII. Position relative to Drosophila yakuba (Clary and Wolstenholme, 1985).

a

revealed no significant difference between genes. We thus base our primary analyses on the full data set. The 50% parsimony bootstrap tree search was used as the starting point for NNI branch swapping under maximum likelihood. The Hasegawa–Kishino–Yano (Hasegawa et al., 1985) model of sequence evolution was implemented using observed nucleotide frequencies, two substitution types (transition/transversion ratio was initially set to 1.5, estimated by PAUP from the parsimony tree), and allowing for rate heterogeneity according to a four-category approximation to a ⌫ distribution (␣ ⫽ 0.3) (see Yang, 1994). This value for ␣ is slightly lower than that implemented by Frati et al. (1997), consistent with an expectation of higher rate heterogeneity among sites (because we have retained third-codon position sites). At the completion of this

search, ␣ and the transition/transversion ratio were estimated (using likelihood ‘‘tree scores’’ in PAUP*) on the obtained topology (giving ␣ ⫽ 0.22; Ti/Tv ⫽ 1.69) and NNI branch swapping was run to completion under these new parameters. We implemented Kishino–Hasegawa (Kishino and Hasegawa, 1989) tests to examine the topological hypotheses described in the Introduction. This method tests for significant differences between two trees under a specified optimality criterion. In the case of parsimony, difference in tree length is tested; under maximum likelihood, difference in likelihood under a specific model is tested. Each hypothesis was generated as a tree in MacClade (version 3.06). Hypotheses of monophyly (for example, of P. alexanor ⫹ machaon group) were programmed as trees with one branch

Papilio mtDNA PHYLOGENY

separating an unresolved test group from an unresolved outgroup containing all other taxa. This tree was then enforced as a PAUP ‘‘constraints’’ statement and the shortest trees under parsimony (using the same weighting scheme described above) which did and did not satisfy this constraint were saved to a file. The tests for significance were carried out in PAUP* under both parsimony and maximum-likelihood criteria. In either case, the same conditions were implemented as during tree searches under those criteria. RESULTS AND DISCUSSION Patterns of Gene Evolution The final aligned sequences yielded 2330 characters (including nucleotides and gaps), extending just into the tRNAs for tyrosine, at the 58 end of the sequence, and lysine at the 38 end. All sequences have been deposited in GenBank; their accession numbers are listed in Table 1. Lunt et al. (1996) and Mitchell et al. (1993) have reviewed the ambiguity in the initiation codon for known insect COI sequences. We likewise found variation in this region, both with respect to other insects and within the study group (see Fig. 2). Alignment is ambiguous until the third amino acid residue (for D. yakuba; Clary and Wolstenholme, 1985). This residue is an arginine, encoded by CGA in all these Lepidoptera as in D. yakuba. However, the Papilio species we

FIG. 2. Manually aligned nucleotide sequences for the junction between tRNA-tyr and the first amino acids of COI. All sequenced Papilio species not listed here are identical to P. machaon. Sequence for Lepidoptera begins immediately after the 38 end of the ‘‘k698’’ primer used to amplify this region. Dots indicate sequence identical to P. machaon.

127

examined exhibit nonsynonymous variation in the preceding codons. Though it would not be surprising if the protein’s terminal amino acids were free to vary, it remains unclear what sequence is recognized for purposes of transcription. Most other length variation is concentrated in the tRNA-leucine gene or the short adjacent spacer sequences, which show several 1-bp indels (mostly phylogenetically uninformative) and a 3-bp insertion immediately following the COI termination codon (in P. zelicaon). A few taxa exhibited length variation in COII as well. Colias exhibits a deletion of 1 codon corresponding to bases 3445–3447 of D. yakuba and P. dardanus showed an insertion of 1 codon between positions 3471 and 3472 of D. yakuba (AAT: arginine) relative to all the other Lepidopteran sequences. This region of COII has revealed length variation in several other insects (Liu and Beckenbach, 1992; Frati et al., 1997). Uncorrected pairwise distances range from 0.2% (m. machaon vs m. oregonius) to ⬃20% (Papilio spp. vs D. yakuba) (see Table 3). Variation within species groups varied from 1–5% among machaon group species to 7–10% among phorcas group species. This latter level is surprisingly high in that divergences between species in different species groups do not range much above this; the highest divergence observed within Papilio is 12.0% (between P. anchisiades and P. phorcas). This plateau indicates the likelihood of third-codon position saturation for some comparisons. Intertribe divergences are similar (10–13%). Only comparisons outside of Papilionidae range over 13%. Nucleotide frequencies across the entire data set are comparably biased to those of other insects, averaging 73.5% A ⫹ T (32.4% A, 40.1% T), 26.5% C ⫹ G (14.3% C, 12.2% G) [see Lunt et al. (1996) for comparison]. A total of 972 sites of the 2330 aligned were variable, with 712 of these within Papilio. We calculated transition/ transversion ratios between all pairs of taxa and plotted this against uncorrected distance between them (see Fig. 3). Transition bias is seen to decline with increasing distance up to about 10%, after which it remains relatively constant at a ratio of less than 1.0. This suggests rapid saturation of transitions for most comparisons. The distribution of variability across codon positions and across genes is shown in Table 4. While total variability is similar for COI and COII (30.8% vs 33.8%), the distribution of variability differs between them, with a higher percentage of first- and secondcodon position substitutions in COII and a resulting amino acid variability approximately 60% higher than that of COI. In order to understand this difference it is necessary to examine amino acid variability from a structural perspective. The cytochrome oxidase I protein comprises 514 amino acids arrayed in a series of hydrophobic membrane-spanning helices separated by extramembrane

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

Dros. yakuba Feltia Colias Pach. neptunus Eury. marcellus P. xuthus P. demoleus P. constantinus P. phorcas P. dardanus P. indra P. hospiton P. zelicaon P. polyxenes P. machaon P. m. oregonius P. cresphontes P. anchisiades P. troilus P. palamedes P. pilumnus P. alexanor P. glaucus P. canadensis P. multicaudatus P. rutulus P. garamas P. scamander

0.180 0.199 0.200 0.200 0.204 0.196 0.200 0.208 0.209 0.197 0.194 0.199 0.199 0.196 0.195 0.204 0.203 0.216 0.219 0.211 0.211 0.197 0.197 0.204 0.197 0.209 0.209

1

0.137 0.131 0.127 0.129 0.124 0.130 0.142 0.137 0.128 0.124 0.129 0.129 0.128 0.129 0.134 0.129 0.132 0.137 0.141 0.137 0.123 0.122 0.125 0.125 0.134 0.134

0.59

2

0.133 0.131 0.139 0.137 0.143 0.151 0.145 0.132 0.130 0.132 0.132 0.131 0.132 0.147 0.143 0.145 0.147 0.148 0.151 0.139 0.135 0.136 0.140 0.143 0.145

0.63 0.75

3

0.117 0.115 0.112 0.120 0.135 0.124 0.114 0.113 0.116 0.118 0.117 0.118 0.121 0.114 0.126 0.129 0.129 0.125 0.108 0.108 0.110 0.111 0.125 0.121

0.57 0.68 0.75

4

0.113 0.116 0.116 0.132 0.127 0.112 0.104 0.111 0.111 0.108 0.109 0.114 0.117 0.127 0.125 0.128 0.119 0.105 0.105 0.112 0.113 0.119 0.118

0.53 0.67 0.64 0.78

5

0.083 0.081 0.092 0.092 0.074 0.072 0.076 0.078 0.074 0.075 0.095 0.090 0.093 0.095 0.098 0.101 0.086 0.085 0.087 0.088 0.108 0.096

0.64 0.93 0.65 0.70 0.67

6

0.080 0.097 0.092 0.075 0.076 0.079 0.079 0.079 0.080 0.095 0.102 0.107 0.106 0.114 0.096 0.091 0.089 0.092 0.092 0.111 0.098

0.53 0.80 0.58 0.76 0.56 0.89

7

0.081 0.073 0.077 0.085 0.084 0.085 0.080 0.081 0.102 0.097 0.104 0.098 0.102 0.101 0.091 0.090 0.092 0.094 0.105 0.098

0.59 0.80 0.65 0.74 0.56 0.77 1.17

8

0.094 0.095 0.097 0.092 0.094 0.092 0.093 0.111 0.120 0.107 0.110 0.113 0.116 0.102 0.101 0.107 0.104 0.114 0.109

0.63 0.96 0.73 0.86 0.79 1.15 1.25 2.57

9

0.095 0.092 0.095 0.095 0.092 0.093 0.107 0.108 0.110 0.111 0.115 0.111 0.101 0.097 0.102 0.104 0.112 0.105

0.64 0.86 0.71 1.07 0.59 1.06 1.43 1.58 1.56

10

0.048 0.049 0.054 0.047 0.048 0.093 0.091 0.096 0.101 0.098 0.094 0.079 0.080 0.079 0.084 0.102 0.090

0.54 0.67 0.65 0.67 0.57 0.90 0.74 0.76 1.10 0.85

11

0.034 0.038 0.034 0.035 0.091 0.095 0.101 0.099 0.105 0.100 0.084 0.082 0.085 0.085 0.104 0.096

0.60 0.80 0.74 0.87 0.73 1.35 0.93 0.93 1.34 1.06 1.19

12

0.026 0.038 0.039 0.095 0.098 0.101 0.101 0.106 0.099 0.086 0.082 0.084 0.085 0.103 0.095

0.62 0.78 0.61 0.78 0.69 1.45 0.90 1.11 1.58 1.12 1.12 3.00

13

0.042 0.042 0.096 0.099 0.102 0.104 0.105 0.102 0.087 0.085 0.086 0.088 0.103 0.099

0.67 0.81 0.64 0.74 0.65 1.29 0.74 1.03 1.56 1.00 0.94 2.25 16.00

14

0.002 0.095 0.099 0.096 0.093 0.101 0.096 0.084 0.082 0.085 0.088 0.099 0.093

0.56 0.88 0.78 0.78 0.59 1.37 1.04 0.93 1.64 1.13 1.50 4.25 2.71 2.13

15

0.096 0.100 0.098 0.094 0.102 0.097 0.085 0.082 0.085 0.088 0.100 0.094

0.55 0.90 0.80 0.80 0.61 1.42 1.07 0.96 1.68 1.17 1.58 4.50 2.86 2.25 1.50

16

0.081 0.099 0.101 0.104 0.102 0.084 0.085 0.092 0.085 0.102 0.087

0.60 0.82 0.65 0.74 0.86 0.97 0.80 0.97 1.33 0.93 0.84 1.07 1.03 0.94 1.03 1.06

17

0.093 0.099 0.101 0.105 0.083 0.083 0.091 0.089 0.105 0.093

0.69 0.85 0.61 0.64 0.67 0.81 0.67 0.90 0.91 1.10 0.73 0.80 0.76 0.79 0.83 0.85 1.37

18

0.064 0.083 0.099 0.077 0.074 0.079 0.081 0.092 0.087

0.76 1.27 0.98 1.04 1.20 1.31 1.29 1.52 1.62 1.33 1.32 1.28 1.42 1.41 1.57 1.61 1.53 1.23

19

0.088 0.100 0.086 0.085 0.087 0.087 0.099 0.092

0.75 1.24 1.06 1.04 1.29 1.58 1.75 1.67 2.31 1.79 1.72 1.72 1.69 1.70 2.04 2.09 2.12 1.38 4.44

20

0.104 0.085 0.083 0.092 0.087 0.102 0.096

0.67 0.92 0.93 0.67 0.86 0.82 0.90 0.84 1.16 0.96 1.21 0.97 1.11 0.95 1.09 1.12 1.18 0.90 2.37 2.67

21

0.085 0.083 0.089 0.088 0.107 0.099

0.60 0.96 0.66 0.77 0.85 0.83 0.78 0.93 1.31 1.17 0.75 1.17 0.97 0.93 1.12 1.15 1.29 0.86 1.91 2.59 0.88

22

0.013 0.033 0.026 0.077 0.068

0.50 0.96 0.69 0.76 0.84 1.00 0.78 0.97 1.19 1.20 0.63 0.78 0.77 0.69 0.83 0.87 1.07 0.70 1.90 2.53 1.48 1.13

23

0.032 0.022 0.077 0.070

0.53 1.04 0.70 0.80 0.94 1.08 0.89 1.10 1.33 1.34 0.68 0.90 0.88 0.80 0.97 1.00 1.14 0.76 2.05 2.72 1.67 1.30 3.00

24

0.037 0.079 0.072

0.58 0.94 0.79 0.74 0.90 1.11 0.80 0.91 1.41 1.09 0.79 1.06 0.97 0.89 1.10 1.14 1.23 0.89 2.00 2.23 1.54 1.36 4.20 3.67

25

0.077 0.072

0.54 0.88 0.71 0.77 0.92 1.15 0.82 0.97 1.24 1.22 0.73 0.97 0.94 0.79 1.11 1.14 1.11 0.85 1.91 2.24 1.60 1.25 7.00 5.00 4.80

26

Uncorrected p-Distances for All Pairwise Comparisons (below Diagonal) and Transition/Transversion Ratio (above Diagonal)

TABLE 3

0.081

0.76 1.08 1.00 0.91 1.18 1.38 1.31 1.35 1.65 1.37 1.19 1.47 1.39 1.32 1.31 1.34 2.14 1.49 1.73 1.90 1.60 1.47 1.86 1.91 1.84 1.73

27

0.67 1.17 1.07 1.00 1.26 1.52 1.11 1.42 1.65 1.32 1.35 1.91 1.48 1.46 2.00 2.05 1.67 1.17 1.77 2.17 1.39 1.46 2.43 2.47 2.35 2.71 3.14

28

128 CATERINO AND SPERLING

Papilio mtDNA PHYLOGENY

129

FIG. 3. Uncorrected (p) distances for pairwise comparisons between complete 2.3-kb nucleotide sequences (x-axis) against corresponding transition/transversion ratios (y-axis). The point not shown (indicated by the arrow) is at (0.013, 8.7). The majority of comparisons are at or near saturation of transitions relative to transversions.

loops. Variation among functional regions of the COI gene (following Lunt et al., 1996) is shown in Fig. 4. In general our results agree with those of Lunt et al. (1996). The most conserved residues are found in the metal-binding transmembrane helices M2, M6, M7, TABLE 4 Distribution of Variability among Genes and Codon Positions Based on Papilio Species Only COI Total sites 1542 Total sites variable/ percent of total sites 475/30.8% 1st position variable/% of 1st positions 88/17.1% 2nd position variable/% of 2nd positions 17/3.3% 3rd position variable/% of 3rd positions 374/72.8% Amino acids variable/% of amino acids 51/9.9%

tRNAleucine

COII

All sequence

78

681

2330

7/9.0% —

—

—

—

230/33.8% 712/30.6 51/22.5% 139/18.6%

13/5.7%

30/4.0%

166/73.1% 540/72.1%

37/16.3%

88/11.8%

and M10 and in the putative proton-conducting M8 helix. The internal loops are generally less conserved in amino acid sequence while no consistent pattern appears for external loops. In contrast to Lunt et al. (1996), our most variable region was found in external loop 4 (E4), though this region spans only 6 amino acids. In further contrast to Lunt et al., the protein’s carboxy terminus is relatively well conserved in these butterflies and is substantially less variable than the preceding loops and helices. Thus, the constraints operating on the COI protein appear to vary across phylogenetic space. The structure of COII is fundamentally different from that of COI. The COII protein spans 227 amino acids and comprises two distinct regions (Capaldi et al., 1983; Saraste, 1990). Two membrane-spanning helices anchor the N-terminal portion in the inner mitochondrial membrane. The majority of the protein resides outside of the membrane and is involved in copper binding. Amino acid residues 25–43 and 57–70 form distinctly hydrophobic regions and are postulated as membrane-spanning regions 1 and 2 (Fig. 4; M1 and M2) with internal loop 1 (I1) separating them. Saraste (1990) assigned residues 121–193 to the putative copperbinding region (Cu), which is followed by the carboxy

130

CATERINO AND SPERLING

FIG. 4. Mean nucleotide (white bar) and amino acid (black bar) variability across structural regions of COI and COII for all Papilio species. Variability is calculated as total number of nucleotides or amino acids observed averaged over all sites. See text for description of protein regions.

terminus (COOH). The region spanning residues 71– 120 is of uncertain function and is termed the linking region (Lnk). These limits, particularly for the membrane-spanning regions, differ slightly from those recognized in Capaldi et al. (1983) but are supported by patterns of hydrophobicity and polarity in our data. Variation among these regions is shown in Fig. 4. At the amino acid level the most conserved regions are the N-terminal region and the linking region, while the membrane-spanning and copper-binding regions themselves are the least conserved. Examining this pattern at a finer scale (Fig. 5) reveals that defined regions do not accord very well with actual patterns of variation. Three well-conserved regions are seen: one spans half each of the N-terminal and M1 regions; another spans approximately two-thirds of the linking region; and the third spans the downstream portion of the copperbinding region and part of the carboxy terminus. An interesting region of variability occurs over the first 10 residues of the copper-binding region and is responsible for its overall high variability. In general structural regions seem to be poor predictors of variability for this gene. The constraints on COII may be more related to its interactions with other polypeptides, particularly subunit I and cytochrome c (Saraste, 1990).

The reconstructed tRNA-leucine for Papilio machaon is shown in Fig. 6. There was little variation observed in this region within Papilionidae. However, there is an interesting difference from homologous tRNAs of other insects. The aminoacyl stem has been shortened from the standard seven nucleotide pairs of other insects (Clary and Wolstenholme, 1985) to six. This was found to be the case in the outgroup Lepidoptera as well. While most of the additional variation in this molecule occurs in nonpairing loop nucleotides, there is variation in the innermost nucleotide pair in the D-stem. Phylogenetic Analyses The unweighted parsimony search resulted in two trees of 3074 steps (CI ⫽ 0.426; RI ⫽ 0.432). A strict consensus of these is presented in Fig. 7a with corresponding bootstrap percentages where these are 50% or greater. The maximum-likelihood phylogram is shown in Fig. 7b. The following discussion contrasts these results and addresses variation among alternative analyses. In general, the previously recognized subgenera and species groups were recovered with good support (with the exceptions of the machaon group, into which P. alexanor could not be parsimoniously placed, and the

Papilio mtDNA PHYLOGENY

131

FIG. 5. Amino acid variability over all sites for COII (among Papilio species) calculated as number of amino acids observed per site. Structural regions are indicated along the x-axis.

FIG. 6. The reconstructed tRNA-leucine for Papilio machaon. Boxed nucleotides vary among Papilio species. The starred nucleotide indicates an additional variable site when the papilionid outgroups are considered. Stem nucleotides joined by circles would be paired according to known insect tRNA structures, but would constitute non-Watson–Crick pairings.

subgenus Pterourus, which is apparently paraphyletic with respect to Pyrrhosticta). The papilionid outgroups (Pachliopta neptunus and Eurytides marcellus) are not clearly resolved. Additional sampling is clearly needed, as well as sequence from more slowly evolving loci, in order to address intertribal relationships. Basal relationships within the Papilionini proved difficult to resolve with these data. The strict consensus tree in Fig. 7a reveals five major lineages: (machaon group ⫹ phorcas group ⫹ demoleus ⫹ xuthus; hereafter MPDX), (cresphontes ⫹ anchisiades; CA), (glaucus group ⫹ garamas ⫹ scamander; GGS), (troilus group), and P. alexanor. These are found in all nucleotide analyses (though not in all partitions or amino acid analyses) and have majority bootstrap support. However, resolutions among these vary substantially under different analytical conditions. Table 5 summarizes the results with respect to these major clades. Most parsimony analyses concur in placing the CA clade (corresponding to Heraclides) as the sister group to the remainder of the tribe. However, this resolution is not supported in the maximum-likelihood analysis nor by stepmatrix weighting of third-position changes. Maximum likelihood favors a basal dichotomy between MPDX and (alexanor ⫹ CA ⫹ troilus group ⫹ GGS), though this resolution was not significantly more likely. Amino acids place both cresphontes and anchisiades basally, but not as each other’s closest relative. A clade composed of Papilio alexanor and the troilus group is found under most parsimony schemes as well as under

132

CATERINO AND SPERLING

FIG. 7. (A). The strict consensus of two unweighted parsimony trees (3074 steps; CI ⫽ 0.426, RI ⫽ 0.432). Subgenera are shown to illustrate taxonomic congruence. Bootstrap percentages are shown above branches supported in at least 50% of 1000 replicates. Search conditions: heuristic search with 50 random taxon addition replicates (single replicates under bootstrapping); TBR branch-swapping. (b) Maximum-likelihood hypothesis of relationships. ML branch-swapping was performed on the 50% majority rule consensus from parsimony bootstrapping. Model: HKY85 with observed nucleotide frequencies, transition/transversion ratio estimated (1.69), and ⌫-distributed rate heterogeneity (estimated ␣ ⫽ 0.22).

maximum likelihood. This group is, in turn, most frequently resolved as the sister lineage to the GGS lineage though this is not strongly supported. Otherwise there is little that is consistent among analyses with regard to relationships among these main lineages. Analysis of these basal relationships using a more slowly evolving, nuclear locus has proven more effective (Reed and Sperling, in press). The monophyly of the MPDX lineage is supported in 83% of bootstrap replicates, as well as in maximumlikelihood analysis. Relationships within MPDX are

relatively well supported. Though not strongly supported by bootstrapping, P. demoleus consistently resolved as sister to the phorcas group. Resolution among the species of the phorcas group itself proved as difficult to determine with mtDNA sequence data as with other data generated for this problem. Both unweighted parsimony trees group constantinus and dardanus while Vane-Wright and Smith’s (1991) favored hypothesis of a sister relationship between phorcas and dardanus to the exclusion of constantinus is favored by maximum likelihood. Examining the sequence in

133

Papilio mtDNA PHYLOGENY

TABLE 5 Resolutions among Major Lineages Reconstructed by Different Analyses Resolution

UNW

SAW

W1

(CA(((AL ⫹ TR)GGS)MPDX)) (CA((AL ⫹ TR)(GGS ⫹ MPDX))) ((CA(GGS(AL ⫹ TR)))MPDX) ((AL ⫹ GGS)((CA ⫹ TR)MPDX)) (C(A(TR ⫹ GGS)(AL ⫹ MPDX))) a

● ●

●

●

W2

W3

ML

AA

● ●

● ●

Note. Bullets indicate which resolutions were found by the particular search. Analyses: UNW, all positions included, equally weighted (two most parsimonious trees found); SAW, all positions reweighted once by rescaled consistency indices; W1, nt1 (⫻1.5), nt2 (⫻2); W2, Tv (⫻2); nt1 & nt2 (⫻2); W3, nt3 changes weighted as inverse to minimum observed transformation frequencies; ML, maximum likelihood (HKY85; ⌫: ␣ ⫽ 0.22; Ti/Tv ⫽ 1.69); AA, equally weighted amino acids. Taxa: MPDX, machaon group ⫹ phorcas group ⫹ demoleus ⫹ xuthus; CA, cresphontes ⫹ anchisiades; GGS, glaucus group ⫹ garamas ⫹ scamander; AL, alexanor; TR, troilus group. a Heraclides (CA) paraphyletic.

greater detail (see Table 6) reveals a high level of homoplasy, with nearly equivalent numbers of substitutions supporting all possible arrangements. Kishino– Hasegawa testing, likewise, does not significantly support any one resolution over another. Maximum likelihood and unweighted parsimony concur in resolving xuthus as basal within the MPDX lineage (as in Fig. 9). However, this varies in the weighted trees; xuthus is found as the sister to the Princeps species alone by the W2 scheme and as the sister to the machaon group alone by the W3 scheme. None of these resolutions is significantly more likely. The relationships among the species of the machaon group largely accord with those suggested by Sperling and Harrison (1994); the monophyly of zelicaon ⫹ polyxenes is strongly supported in all analyses (100% of bootstrap replicates) as is the basal placement of indra with respect to the remainder of the group. The placement of alexanor in the machaon group is not supported by any of our analyses and is strongly rejected by Kishino–Hasegawa tests [P (likelihood) ⫽ 0.0043]. Otherwise, our results differ primarily from those of previous studies in the placement of hospiton, though its placement as the sister lineage to zelicaon ⫹ polyxenes is not strongly supported (63% of boostrap replicates). A more thorough sampling of TABLE 6 Characters Unambiguously Supporting Possible phorcas Group Resolutions Resolution (const(dard, ((const,dard) ((const,phorc) phorc)) phorc) dard) First codon position Second codon position Third codon position Noncoding positions Total synapomorphies

0 0 14 2 16

2 1 13 0 16

4 0 11 0 15

sequences among machaon subspecies will be necessary to conclusively place this species. Our analyses strongly support the close relationship among the scamander, homerus (represented by garamas), and glaucus groups asserted by Scriber et al. (1991) on the basis of hybrid surviviorship. The monophyly of these to the exclusion of the troilus group is found by all parsimony schemes examined, by maximum likelihood, and in 81% of unweighted bootstrap replicates. The resolution within the glaucus group is unambiguous and supported by all analyses, but it differs somewhat from previous mtDNA results. Sperling (1993b) (using mtDNA restriction site data) and Hagen and Scriber (1991) (on the basis of allozymes) found rutulus and multicaudatus to be sister species, a resolution that finds no support in our data. The rooting of both earlier studies using the troilus group, which appears to be less closely related than previously thought, may be partly responsible for the disagreement. Our data agree with Hagen and Scriber’s (1991) allozyme results with regard to relationships among species of the troilus group. A (troilus, palamedes) pilumnus)) resolution is supported in 97% of bootstrap replicates based on the entire COI ⫹ COII. Additional sampling will be necessary to address the issue of troilus paraphyly with respect to palamedes (as suggested by Hagen and Scriber, 1991). Given the popularity of these genes for phylogeny reconstruction in insects, we felt it worthwhile to examine the performance of each gene alone relative to the entire data set. These trees are presented in Fig. 8. The bootstrapping results, while consistent with those based on the full data set, are each substantially less resolved. Both genes recover most of the species groups with good support. COII alone, however, does not strongly support the monophyly of the phorcas group. Surprisingly, COII alone does not provide strong support for Papilionini monophyly, though this is supported by 93% of boostrap replicates based on the full

134

CATERINO AND SPERLING

FIG. 8. Parsimony topologies based on individual genes. Bootstrap percentages are shown above branches supported in at least 50% of 500 replicates. Search conditions: all positions equally weighted; heuristic search with 50 random taxon addition replicates (single replicates under bootstrapping); NNI branch-swapping. Left tree: COI; single most parsimonious tree. Right tree: COII; strict consensus of four equally parsimonious trees.

data set. COI alone does not strongly support monophyly of the garamas/scamander lineage and resolved only scamander as closely related to the glaucus group. Only COI provides strong support for the MPDX lineage, but this is invariant among analyses based on the full data set. Overall, it is clear that the two genes together provide information that is only weakly present in either one alone. CONCLUSIONS This study clearly demonstrates the utility of the COI and COII sequence for the study of species-grouplevel relationships in Papilionini. Most of the shallower nodes are strongly supported and we predict that relationships within species groups which we have not thoroughly sampled are likely to be well resolved using a similar approach. However, it remains to be seen how well relationships among species groups will be resolved. It seems likely that these genes are nearing saturation at third-codon positions for the deeper comparisons. More extensive sampling of taxa may improve phylogenetic accuracy as demonstrated by Hillis (1996). Nuclear genes currently under development also show greater promise for resolving deeper phylogenetic divergences (Cho et al., 1995; Friedlander et al., 1996; Fang et al., 1997; Reed and Sperling, in preparation). We suggest the tree in Fig. 9 as our working hypothesis. It is one of the two most parsimonious (unweighted) trees and is also found by weighting scheme

W2 (Tv[x2]; n1 & n2 [x2]). In general, our findings concur with previous morphological work. The species groups of Munroe (1961) were all recovered apart from the exclusion of alexanor from the machaon group. Hancock’s (1983) suggested phylogeny of the species groups is generally supported by our results. We similarly propose a lineage composed of the machaon group, the phorcas group, P. xuthus, and P. demoleus (Hancock’s Papilio and Princeps). We also support paraphyly of Pterourus with respect to Pyrrhosticta as suggested by Scriber et al. (1991). This finding has significant ramifications for the understanding of the evolution of host plant preference and particularly the origin of polyphagy in Pterourus (Scriber et al., 1995a). The exclusion of alexanor from the machaon group seems necessary based on these data; support for machaon group monophyly without alexanor is very strong. It would appear, then, that Apiaceae feeding has arisen independently in alexanor and that its larval coloration is convergent with that of the machaon group. Such a convergence has precedent: some populations of P. demodocus (demoleus group) that have switched from Rutaceae to Apiaceae in South Africa have independently arrived at a pattern very similar to that of P. hospiton, another machaon group Apiaceae feeder (Clarke et al., 1963). The actual affinities of alexanor remain unclear. We suggest a close relationship to the troilus group. However, several alternative placements are approximately equally likely. Mitochondrial DNA has not provided any resolution for the question of phorcas group relationships. It is

135

Papilio mtDNA PHYLOGENY

FIG. 9. Our suggested hypothesis of relationships. This is one of two most parsimonious trees from the unweighted search. See text for justification of selection of this particular topology.

possible that introgression of mtDNA haplotypes through hybridization, such as that demonstrated by Clarke et al. (1991), may have obscured phylogenetic patterns in this marker. It is also likely that substantial intraspecific variation that we have failed to recognize exists. It also must be considered that a trichotomy may be an accurate representation of the relationships of these taxa (Hoelzer and Melnick, 1994). In any case, representatives from additional areas within the range of each, particularly from andromorphic dardanus populations, would likely provide useful information. While 2.3 kb of mitochondrial sequence data has enabled us to shed light on some longstanding problems in Papilio systematics, many remain. Mitochondrial DNA data can be strictly interpreted only in terms of maternal lineages. Sequence studies using nuclear, biparentally inherited genes need to be completed for a balanced molecular evaluation of these problems. Future studies should also focus on balanced taxon sampling. Particularly interesting species to include would be additional representatives of the P. (Heraclides) lineage, especially P. esperanza, an enigmatic, highly localized species from Oaxaca, Mexico. Data on Meandrusa, an East Asian genus of two species which has variously been placed as a Papilionine, a Graphiine, or with Teinopalpus, as a fourth tribe in the Papilioninae (see Igarashi, 1979, 1984; Miller, 1987; Sperling, 1991; Brown et al., 1995) would also likely help resolve the base of the tree of Papilionini. Given the broad interest in Papilio and ongoing, rapid developments in methods of phylogenetic analysis, we are confident that a robust understanding of the relationships in this prominent group is near at hand.

ACKNOWLEDGMENTS In addition to the collectors listed in Table 1, we thank M. Berenbaum, C. Clarke, C. Hauser, M. Peterson, M. Scriber, and R. Vane-Wright for facilitating specimen acquisition. Many thanks also are due to D. Swofford for permission to use test versions of PAUP* and to J. Huelsenbeck, D. Kain, and R. Reed for much useful advice. We thank A. Brower, T. Friedlander, R. DeSalle, and one anonymous reviewer for useful comments on an earlier version of the manuscript. This work was supported in part by a California Agricultural Experimentation Grant to F. Sperling and by the Margaret C. Walker Fund for Systematic Entomology to M. Caterino.

REFERENCES Ae, S. A. (1995). Ecological and evolutionary aspects of hybridization in some Papilio butterflies. In ‘‘Swallowtail Butterflies: Their Ecology and Evolutionary Biology’’ (J. M. Scriber, Y. Tsubaki, and R. C. Lederhouse, Eds.), pp. 229–236, Scientific Publishers, Gainesville, FL. Albert, V. A., Mishler, B. D., and Chase, M. W. (1992). Character-state weighting for restriction site data in phylogenetic reconstruction, with an example from chloroplast DNA. In ‘‘Molecular Systematics of Plants’’ (P. S. Soltis, D. S. Soltis, and J. J. Doyle, Eds.), pp. 369–403, Chapman and Hall, London. Beckenbach, A. T., Wei, Y. W., and Liu, H. (1993). Relationships in the Drosophila obscura species group, inferred from mitochondrial cytochrome oxidase II sequences. Mol. Biol. Evol. 10: 619–634. Brower, A. V. Z. (1996). Parallel race formation and the evolution of mimicry in Heliconius butterflies: A phylogenetic hypothesis from mitochondrial DNA sequences. Evolution 50: 195–221. Brower, A. V. Z., and Sime, K. R. (in press). Mimicry and aposematism in the caterpillars of the Papilio machaon group: A reconsideration. J. Lepidopt. Soc. Brown, K. S., Klitzke, C. F., Berlingeri, C., and Euze´bio Rubbo dos Santos, P. (1995). Neotropical swallowtails: Chemistry of food plant relationships, population ecology and biosystematics. In ‘‘Swallow-

136

CATERINO AND SPERLING

tail Butterflies: Their Ecology and Evolutionary Biology’’ (J. M. Scriber, Y. Tsubaki, and R. C. Lederhouse, Eds.), pp. 405–446, Scientific Publishers, Gainesville, FL. Capaldi, R. A., Malatesta, F., and Darley-Usmar, V. M. (1983). Structure of cytochrome c oxidase. Biochim. Biophys. Acta 726: 135–148. Cho, S., Mitchell, A., Regier, J. C., Mitter, C., Poole, R. W., Friedlander, T. P., and Zhao, S. (1995). A highly conserved gene for low level phylogenetics: Elongation factor-1 alpha recovers morphologybased tree for heliothine moths. Mol. Biol. Evol. 12: 650–656. Clarke, C. A., Dickson, C. G. C., and Sheppard, P. M. (1963). Larval color pattern in Papilio demodocus. Evolution 17: 130–137. Clarke, C. A., Gordon, I. J., Smith, C. R., and Vane-Wright, R. I. (1991). Phylogenetic relationships of three African swallowtail butterflies, Papilio dardanus, Papilio phorcas, and Papilio constantinus: New data from hybrids. Syst. Entomol. 16: 257–274. Clarke, C. A., and Sheppard, P. M. (1953). Further observations on hybrid swallowtails. Entomol. Rec. Suppl. 65: 1–12. Clarke, C. A., and Sheppard, P. M. (1955). The breeding in captivity of the hybrid swallowtail Papilio machaon gorganus Fru¨hstorfer (F) ⫻ Papilio hospiton Guene´e (M). The Entomologist 88: 265–288. Clarke, C. A., and Sheppard, P. M. (1963). Interactions between major genes and polygenes in the determination of the mimetic patterns of Papilio dardanus. Evolution 17: 404–413. Clary, D. O., and Wolstenholme, D. R. (1985). The mitochondrial DNA molecule of Drosophila yakuba: Nucleotide sequence, gene organization and genetic code. J. Mol. Evol. 22: 252–271. Collins, N. M., and Morris, M. G. (1985). ‘‘Threatened Swallowtail Butterflies of the World,’’ IUCN, Gland and Cambridge. Fang, Q. Q., Cho, S., Regier, J. C., Mitter, C., Matthews, M., Poole, R. W., Friedlander, T. P., and Zhao, S. (1997). A new nuclear gene for insect phylogenetics: Dopa decarboxylase is informative of relationships within Heliothinae (Lepidoptera: Noctuidae). Syst. Biol. 46: 269–283. Farris, J. S. (1981). Distance data in phylogenetic analysis. In ‘‘Advances in Cladistics: Proceedings of the First Meeting of the Willi Hennig Society’’ (V. A. Funk and D. R. Brooks, Eds.), pp. 3–23, New York Botanical Garden, New York. Ford, E. B. (1936). The genetics of Papilio dardanus Brown. Trans. R. Entomol. Soc. London 85: 435–466. Ford, E. B. (1944). Studies in the chemistry of pigments in the Lepidoptera, with reference to their bearing on systematics. 4. The classification of the Papilionidae. Trans. R. Entomol. Soc. London 94: 201–223. Frati, F., Simon, C., Sullivan, J., and Swofford, D. L. (1997). Evolution of the mitochondrial Cytochrome oxidase II gene in Collembola. J. Mol. Evol. 44: 145–158. Friedlander, T. P., Regier, J. C., Mitter, C., and Wagner, D. L. (1996). A nuclear gene for higher level phylogenetics: Phosphoenolpyruvate carboxykinase tracks Mesozoic-age divergences within Lepidoptera. Mol. Biol. Evol. 13: 594–604. Hagen, R. H., and Scriber, J. M. (1991). Systematics of the Papilio glaucus and Papilio troilus species groups (Lepidoptera: Papilionidae): Inferences from allozymes. Ann. Entomol. Soc. Am. 84: 380–395. Hagen, R. H., and Scriber, J. M. (1995). Sex chromosomes and speciation in tiger swallowtails. In ‘‘Swallowtail Butterflies: Their Ecology and Evolutionary Biology’’ (J. M. Scriber, Y. Tsubaki, and R. C. Lederhouse, Eds.), pp. 211–227, Scientific Publishers, Gainesville, FL. Hancock, D. L. (1983). Classification of the Papilionidae: A phylogenetic approach. Smithersia 2: 1–48. Hasegawa, M., Kishino, M., and Yano, T. (1985). Dating the human–

ape split by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22: 160–174. Hillis, D. M. (1996). Inferring complex phylogenies. Nature 383: 130–131. Hoelzer, G. A., and Melnick, D. J. (1994). Patterns of speciation and limits to phylogenetic resolution. Trends Ecol. Evol. 9: 104–107. Howland, D. E., and Hewitt, G. M. (1995). Phylogeny of the Coleoptera based on mitochondrial cytochrome oxidase I sequence data. Insect Mol. Biol. 4: 203–215. Igarashi, S. (1979). ‘‘Papilionidae in Their Early Stages,’’ Vols. 1 and 2, Tokyo. [In Japanese]. Igarashi, S. (1984). The classification of the Papilionidae mainly based on the morphology of their immature stages. Tyo To Ga 34: 41–96. Kishino, H., and Hasegawa, M. (1989). Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea. J. Mol. Evol. 29: 170–179. Kyte, J., and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105–132. Liu, H., and Beckenbach, A. T. (1992). Evolution of the mitochondrial cytochrome oxidase II gene among 10 orders of insects. Mol. Phylogenet. Evol. 1: 41–52. Lunt, D. H., Zhang, D.-X., Szymura, J. M., and Hewitt, G. M. (1996). The insect cytochrome oxidase I gene: Evolutionary patterns and conserved primers for phylogenetic studies. Insect Mol. Biol. 5: 153–165. Maddison, W. P., and Maddison, D. R. (1992). ‘‘MacClade, Version 3.06,’’ Sinauer, Sunderland, MA. Miller, J. S. (1987). Phylogenetic studies in the Papilioninae (Lepidoptera: Papilionidae). Bull. Amer. Museum Nat. Hist. 186: 365–512. Mitchell, S. E., Cockburn, A. F., and Seawright, J. A. (1993). The mitochondrial genome of Anopheles quadrimaculatus species A: Complete nucleotide sequence and gene organization. Genome 36: 1058–1073. Munroe, E. (1961). The classification of the Papilionidae. Can. Entomologist Suppl. 17: 1–51. Reed, R. D., and Sperling, F. A. H. Sources of error in the phylogenetic analysis of multiple loci: An example from the swallotail butterfly genus Papilio. Mol. Biol. Evol., in press. Saraste, M. (1990). Structural features of cytochrome oxidase. Quart. Rev. Biophys. 23: 331–366. Scriber, J. M., Lederhouse, R. C., and Brown, K. S. (1991). Hybridization of the Brazilian Papilio (Pyrrhosticta) (section V) with the North American Papilio (Pterourus) (section III). J. Res. Lepidoptera 29: 21–32. Scriber, J. M., Lederhouse, R. C., and Dowell, R. V. (1995a). Hybridization studies with North American swallowtails. In ‘‘Swallowtail Butterflies: Their Ecology and Evolutionary Biology’’ (J. M. Scriber, Y. Tsubaki, and R. C. Lederhouse, Eds.), pp. 269–282, Scientific Publishers, Gainesville, FL. Scriber, J. M., Tsubaki, Y., and Lederhouse, R. C. (1995b). ‘‘Swallowtail Butterflies: Their Ecology and Evolutionary Biology,’’ Scientific Publishers, Gainesville, FL. Sperling, F. A. H. (1987). Evolution of the Papilio machaon species group in western Canada. Quaestiones Entomol. 23: 198–315. Sperling, F. A. H. (1990). Interspecific hybrids in Papilio butterflies: Poor taxonomy or interesting evolutionary problem? Can. J. Zool. 68: 1790–1799. Sperling, F. A. H. (1991). ‘‘Mitochondrial DNA Phylogeny, Speciation, and Hostplant Coevolution of Papilio Butterflies,’’ Ph.D. dissertation. Cornell University. Sperling, F. A. H. (1993a). Mitochondrial DNA phylogeny of the

Papilio mtDNA PHYLOGENY Papilio machaon species group (Lepidoptera: Papilionidae). Mem. Entomol. Soc. Can. 165: 233–242. Sperling, F. A. H. (1993b). Mitochondrial DNA variation and Haldane’s rule in the Papilio glaucus and P. troilus species groups. Heredity 71: 227–233. Sperling, F. A. H. (1994). Sex-linked genes and species differences in the Lepidoptera. Can. Entomologist 126: 807–818. Sperling, F. A. H., Byers, R., and Hickey, D. (1996). Mitochondrial DNA sequence variation among pheromotypes of the dingy cutworm, Feltia jaculifera (Gn.) (Lepidoptera: Noctuidae). Can. J. Zool. 74: 2109–2117. Sperling, F. A. H., and Feeny, P. (1995). Umbellifer and composite feeding in Papilio: Phylogenetic frameworks and constraints on caterpillars. In ‘‘Swallowtail Butterflies: Their Ecology and Evolutionary Biology’’ (J. M. Scriber, Y. Tsubaki, and R. C. Lederhouse, Eds.), pp. 299–306, Scientific Publishers, Gainesville, FL. Sperling, F. A. H., and Harrison, R. G. (1994). Mitochondrial DNA variation within and between species of the Papilio machaon group of swallowtail butterflies. Evolution 48: 408–422. Sperling, F. A. H., and Hickey, D. (1994). Amplified mitochondrial DNA as a diagnostic marker for species of conifer-feeding Choristoneura. Can. Entomologist 127: 277–288.

137

Sperling, F. A. H., Spence, J. R., and Andersen, N. M. (1997). Mitochondrial DNA, allozymes, morphology, and hybrid compatibility in Limnoporus water striders (Heteroptera: Gerridae): Do they all track species boundaries? Ann. Entomol. Soc. Am. 90: 401–415. Taylor, P. L. (1993). ‘‘GenejockeyII, Version 1.0,’’ Biosoft, Cambridge, UK. Tyler, H., Brown, K. S., and Wilson, K. 1994. ‘‘Swallowtail Butterflies of the Americas: A Study in Biological Dynamics, Ecological Diversity, Biosystematics and Conservation,’’ Scientific Publishers, Gainesville, FL. Vane-Wright, R. I., and Smith, C. R. (1991). Phylogenetic relationships of three African swallowtail butterflies, Papilio dardanus, Papilio phorcas, and Papilio constantinus: A cladistic analysis. Syst. Entomol. 16: 275–292. Wheeler, W. C. (1990). Nucleic acid sequence phylogeny and random outgroups. Cladistics 6: 363–367. Yang, Z. (1994). Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: Approximate methods. J. Mol. Evol. 39: 306–314. Zimmerman, J. M., Eliezer, N., and Simha, R. (1968). The characterization of amino acid sequences in proteins by statistical methods. J. Theor. Biol. 21: 170–201.