*Department of Organismic and Evolutionary Biology, Harvard University; and
Department of Entomology, Oregon State University
Abstract
The sequence evolution of the nuclear gene wingless was investigated among 34 representatives of three lepidopteran families (Riodinidae, Lycaenidae, and Nymphalidae) and four outgroups, and its utility for inferring phylogenetic relationships among these taxa was assessed. Parsimony analysis yielded a well-resolved topology supporting the monophyly of the Riodinidae and Lycaenidae, respectively, and indicating that these two groups are sister lineages, with strong nodal support based on bootstrap and decay indices. Although wingless provides robust support for relationships within and between the riodinids and the lycaenids, it is less informative about nymphalid relationships. Wingless does not consistently recover nymphalid monophyly or traditional subfamilial relationships within the nymphalids, and nodal support for all but the most recent branches in this family is low. Much of the phylogenetic information in this data set is derived from first- and second-position substitutions. However, third positions, despite showing uncorrected pairwise divergences up to 78%, also contain consistent signal at deep nodes within the family Riodinidae and at the node defining the sister relationship between the riodinids and lycaenids. Several hypotheses about how third-position signal has been retained in deep nodes are discussed. These include among-site rate variation, identified as a significant factor by maximum likelihood analyses, and nucleotide bias, a prominent feature of third positions in this data set. Understanding the mechanisms which underlie third-position signal is a first step in applying appropriate models to accommodate the specific evolutionary processes involved in each lineage.
Introduction
The last 40 years have seen considerable activity in the higher systematics and classification of the "true" butterflies (Papilionoidea, including Hesperiidae). However, the multiple hypotheses that have arisen from these efforts do not provide a consistent interpretation of the evolution of the major butterfly lineages. In particular, assessing the monophyly and phylogenetic placement of the large family Riodinidae, which contains over 1,200 species, has been problematic, as can be seen by the conflicting phylogenetic hypotheses derived from morphological data. Although most morphological studies place the riodinids as the sister to the lycaenid butterflies and identify the nymphalids as the closest relatives to this riodinid + lycaenid clade (Ehrlich 1958
; Ehrlich and Ehrlich 1967
; Kristensen 1976
; Scott and Wright 1990
; DeVries 1991, 1997
; Fiedler 1991
; De Jong, Vane-Wright, and Ackery 1996
), few characters support these relationships, and alternate relationships among these groups have been suggested (Robbins 1988a, 1988b;
Martin and Pashley 1992
; for review, see Campbell and Pierce 2000
).
To determine the placement of riodinid butterflies, we examined a new set of molecular characters from wingless (wg), a nuclear gene which has shown utility in reconstructing species level to subfamily level relationships in nymphalids (Brower and DeSalle 1998
). Wingless is a member of the wnt gene family, whose paralogs are easily distinguishable (Sidow 1992
). Primers specific to lepidopteran wingless have been developed from the 3' exon (Carroll et al. 1994
; Brower and DeSalle 1998
). Because this region of wingless showed a rapid rate of substitution in nymphalids, it holds promise for resolving family level relationships in the Lepidoptera (Brower and Egan 1997
; Brower and DeSalle 1998
). Previous molecular studies of the Papilionoidea have used few riodinid representatives (Martin and Pashley 1992
; Weller, Pashley, and Martin 1996
). Our work includes representative taxa from all the main lineages of riodinids, lycaenids, and nymphalids (sensu Harvey [1987
], Eliot [1973
], and Harvey [1991]
, respectively). The aims of this study are (1) to test the hypotheses that the Riodinidae and Lycaenidae are each monophyletic and determine the relationship between these two families and the family Nymphalidae, and (2) to describe patterns of the molecular evolution of wingless and demonstrate the utility of this gene for reconstructing family and subfamily level relationships among the Lepidoptera.
Materials and Methods
Specimens
Taxa were selected to represent each of the main lycaenid, riodinid, and nymphalid lineages (table 1
). Previous studies of butterfly systematics agree on the Hesperiidae as the basal lineage of the Papilionoidea (but see Scoble 1986, 1988
); for this reason, a hesperiid representative was included as an outgroup, as were two representatives of the Pieridae and one species of Papilionidae. Taxa were collected as fresh specimens, and the bodies were stored in 100% ethanol at -80°C. Wings were retained as vouchers in the Harvard Museum of Comparative Zoology (riodinids and lycaenids) and the American Museum of Natural History (nymphalids).
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The primers WG1, WG2, and WG2.1, described by Brower and DeSalle (1998)
, were used to amplify the 450-bp wingless fragment from all specimens. The amplification conditions consisted of 1 min denaturation at 94°C, 1 min annealing at 50°C, and 1 min extension at 72°C for 30 cycles. Final concentrations of reagents in a standard 100-µl reaction were 1x Taq buffer (50 mM KCl, 100 mM Tris-HCl, 0.1% Triton X-100), 3 mM MgCl2, 0.5 µM each primer, and 2.5 U Taq polymerase. PCR products were excised from a 1.2% low-melt agarose gel and phenol : chloroform extracted. The purified products were cycle sequenced in both directions using the ABI dye terminator core kit in a Perkin-Elmer 480. Excess nucleotides were removed using CentriSep spin columns (Princeton Separations), and reactions were run out on an ABI 370 automated sequencer. PCR and sequencing of the wingless fragment for the nymphalids was performed as described in Brower and DeSalle (1998)
. Sequence chromatograms were edited and aligned by hand in the program SeqEd (Applied Biosystems, Inc. 1992, SeqEd 1.0.3). Edited nucleotide sequences were translated using GeneJockey (Taylor 1991
). GenBank accession numbers for sequences are listed in table 1
.
Data Analyses and Phylogenetic Methods
We used PAUP*, versions 4.0d54 and 4.0d59 (Swofford 1998
), to calculate pairwise distance matrices, determine likelihood values, and conduct parsimony and LogDet distance analyses. Third-position Euclidean distances based on GC content were calculated as described by Ortí and Meyer (1996)
. Skewness (-g1) scores were calculated from 5,000 random trees generated in PAUP*, version 4.0d59. MacClade, version 3.06 (Maddison and Maddison 1996
), was used to explore character evolution and patterns of nucleotide substitution along tree topologies. MEGA, version 1.01 (Kumar, Tamura, and Nei 1993
) was used to determine proportions of synonymous and nonsynonymous differences. Codon preferences and measures of codon bias (ENC and scaled
2) were calculated using the program Molecular Evolutionary Analysis (MEA; generously provided by the author, Etsuko Moriyama, at Yale University).
Parsimony analyses consisted of heuristic searches with 1050 random-addition replicates using tree bisection-reconnection (TBR) branch swapping. Analysis of amino acid characters employed a transformed Blosum 80 step matrix (see appendix) to weight substitutions among amino acid residues relative to their frequencies of change among observed protein sequences (Henikoff and Henikoff 1992
). In parsimony analyses, nodal support was estimated using bootstrap analyses (100 replications, with 10 random-addition replicates each) and branch support (decay indices; Bremer 1988, 1994
; Donoghue et al. 1992;
Davis 1995
). Distance analyses were carried out using the neighbor-joining algorithm (Saitou and Nei 1987
); estimates of nodal support on distance trees were derived using bootstrap analyses (1,000 replications).
We compared the fit of 12 evolutionary models for this data set by estimating maximum-likelihood (ML) scores for the two trees obtained from the unweighted parsimony search. Likelihood scores for these trees were estimated under four models of evolution: the Jukes-Cantor (JC; Jukes and Cantor 1969
), Kimura (1980)
two-parameter (K2P), HKY85 (Hasegawa, Kishino, and Yano 1985
), and general time reversible (GTR [=REV of Yang 1994
]) models, using ML to estimate nucleotide frequencies for the HKY85 and GTR models. For each of these models, we evaluated likelihood scores under assumptions of (1) equal rates at all sites; (2) a proportion of sites estimated by ML being invariable (I); (3) rates at all sites evolving with rate heterogeneity as approximated by four discrete rate classes of the gamma distribution (
), estimated using ML; and (4) some sites being invariable, with variable sites evolving under a gamma distribution. Likelihood ratio tests were used to determine the model that best fit the data between pairs of nested models (Goldman 1993
; Yang, Goldman, and Friday 1994
; Felsenstein 1995
).
Results and Discussion
Alignment and Substitution Patterns
Alignment of the 350-bp wingless fragment for all taxa required a single one-codon insertion in a pierid (Delias). Overall uncorrected sequence divergence percentages among taxa range from 10.3% to 41.6%, greater than those found by Brower and DeSalle (1998)
for comparison of divergences in wingless and the mitochondrial Cytochrome Oxidase I (COI) gene between lepidopteran families (mean uncorrected pairwise difference between Pieridae vs. Nymphalidae reported as 22.8% for wingless, 16.6% for COI). The data set described here yielded 214 parsimony-informative nucleotides when sequences were compared across all taxa (not including outgroup taxa). Of these parsimony-informative nucleotide substitutions, 56 occurred at first-codon-position sites, 43 at second positions, and 115 at third positions (table 2
). When reconstructed on the (two) most-parsimonious trees recovered from unweighted parsimony (trees discussed below), most second positions required smaller numbers of changes across the tree (up to 9 changes per character, averaging 1.3 changes per character) than did first and third positions (first positions made up to 12 changes per character, averaging 2.48; third positions made up to 19 changes per character, averaging 11). This is reflected in higher consistency index (CI) and retention index (RI) values when second positions alone were reconstructed on these trees than when first and third positions were reconstructed (table 3
). The estimated proportion of nonsynonymous differences (±SD) ranged from 1.04 ± 0.73% to 38.08 ± 3.48% over all taxa. When translated, 79% of the 116 amino acid residues in this fragment are variable, showing up to eight character states, and 52% of amino acid sites were phylogenetically informative.
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Although base compositions across taxa are homogeneous at first and second positions, significant chi-square tests of third positions indicate among-taxa compositional heterogeneity (2 = 433.33; df = 111; P < 0.001). When third-position nucleotide content was broken down by family, we found riodinid base composition to be statistically homogeneous among all riodinid taxa examined (
2 = 25.30; df = 33; P = 0.829), with all riodinids showing high AT contents (average AT = 54.7%; fig. 2b
). On the other hand, significantly heterogeneous nucleotide compositions were found among lycaenid species (
2 = 134.17; df = 33; P < 0.001), with four taxa having high AT (%AT > 67%) contents and two having high GC contents (%GC > 73%; average %AT = 48.6%). Nucleotide content among nymphalid taxa was found to be statistically homogeneous (
2 = 36.87; df = 27; P = 0.097). All nymphalids examined have high GC contents (average AT = 42.7%; fig. 2b
). Two different measures of codon bias, ENC and scaled
2, indicate similar (and low) average levels of codon bias in each of the three families, ranging from 0.180 to 0.208 (scaled
2) and from 55.553 to 56.802 (ENC; Shields et al. 1988
; Wright 1990
). However, codon preferences are notably different among the different families, and this may contribute to third-position nucleotide heterogeneity. The significance of nonstationary nucleotide heterogeneity and codon preferences among butterfly families for phylogenetic reconstruction is discussed below.
Maximum-Likelihood Analysis of Nucleotide Evolution
ML analyses were used to assess 16 models of sequence evolution using this data set and the two most-parsimonious trees resulting from the unweighted search. Both trees showed that the single parameter having the greatest effect on improving likelihood scores is among-sites rate heterogeneity (). Table 4
shows scores and parameter estimates for the best (highest likelihood score) of these trees; both trees showed very similar likelihood scores and parameters. Likelihood ratio tests performed on nested models found the GTR+
+I model fit the data significantly better than all of the other models examined (all models shown in table 4 are nested within the GTR+
+I model, Swofford et al. 1996). Estimates of reversible rate parameters among substitution types (A-C, A-G, A-T, C-G, C-T, G-T) are distinct, with high rates between pyrimidines (A-G) and between purines (C-T; R-matrix, see table 4
). A similar increase in rate parameters in these substitution classes was reported by Mason-Gamer, Weil, and Kellogg (1998)
for granule-bound starch synthase in grasses and explains the improvement in likelihood scores with the use of the GTR model as compared with HKY85, which differs from GTR only in allowing different rates for all manners of transitions and transversions.
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A parsimony search using first and second positions alone yielded 111 trees (length = 389; CI = 0.414), and analysis of amino acids recovered 44 most-parsimonious trees (length = 5,119). Although some riodinid and lycaenid relationships were slightly less resolved than in the unweighted analysis, all nodes with bootstrap support >50% in the unweighted search (fig. 3 ) were also recovered with similar support in both analyses. In both weightings, strict consensus resulted in the collapse of all nymphalid nodes. Thus, at least for this sampling of representatives, wingless may not be a reliable marker for basal nymphalid relationships.
On the other hand, relationships within the Riodinidae and the Lycaenidae were remarkably well supported and stable except for two nodes which showed differences in patterns of support in response to the weighting of third-position transitions. One of these is the interpretation of the basal branching patterns of the lycaenids, especially the placement of Curetis. Downweighting or removing third-position transitions placed Curetis as the most basal branch of the lycaenids, with 66% bootstrap support when third position transitions were excluded (fig. 4 ) and 53% support in the analysis of amino acid characters.
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Third-codon-position variability above 20%30% raw pairwise divergence has been considered to be at saturation level in analyses of other nuclear genes (Friedlander, Regier, and Mitter 1994
). Such highly divergent third positions are often downweighted or removed from phylogenetic analyses because they contribute excessive noise and little to no signal (e.g., Friedlander et al. 1996
; Fratí et al. 1997
; but see Brower and DeSalle 1998
). Although we observed divergence levels which, under these guidelines, implicate third-position saturation in wingless (27%78%), a significant -g1 statistic (-0.250) calculated from third positions suggests that these substitutions are not all saturated, and at least some of them contain significant hierarchical signal at the P < 0.01 level (Hillis and Huelsenbeck 1992
; but see Källersjö et al. 1992
). Third positions also show increasing mean pairwise divergences at progressively deeper riodinid and lycaenid phylogenetic levels (fig. 3
). Furthermore, a parsimony search on third positions alone resulted in one most-parsimonious tree (fig. 5
) in which interpretations of riodinid relationships and monophyly were completely concordant with relationships recovered by first and second positions, with the exception of the placement of one riodinid, Eurybia. This search also recovered the sister relationship between the Riodinidae and the Lycaenidae. On the other hand, this tree did not recover lycaenid monophyly or basal lycaenid relationships as reconstructed by parsimony analyses employing first and second positions. Thus, while third positions appear to provide signal consistent for recovery of riodinid relationships and the riodinid + lycaenid node, third positions may contribute to contradictions or noise leading to reduced resolution and bootstrap support for lycaenids (such as that described above for Curetis).
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Another mechanism that might be involved in harboring phylogenetic signal in third positions is among-site rate heterogeneity. If third-position sites evolve at significantly heterogeneous rates, genetic distances between distantly related taxa include multiple hits at more quickly evolving sites which become saturated, as well as changes at more slowly evolving sites which retain phylogenetic signal. In other words, signal persists longer in the data if rate heterogeneity is a prominent feature, such that high third-position divergence creates useful variation in conservatively evolving sites while resulting in saturation of others (Yang 1998
). Wide variation in number of steps per character when nucleotide substitutions were reconstructed onto the topology shown in figure 3
suggests that among-site rate variation exists in this data set (table 3
). Likelihood ratio tests conducted using the unweighted tree topology and the GTR model of evolution to compare hypotheses of among-site rate variation in each nucleotide position individually further identify rate heterogeneity as a significant factor in third positions (
2 = 67.18; P < 0.005;
shape parameter (
) = 3.785), as well as in first and second positions (
2 = 187.66; 38.96,
= 0.443 and 0.865 for first and second positions, respectively; table 5 ).
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Nonstationary base composition across taxa is a third mechanism that may contribute to signal in third positions. At the third position, riodinid, lycaenid, and nymphalid lineages show significantly different nucleotide proportions, with high average AT content in riodinids, intermediate and heterogeneous in lycaenids, and low AT content in nymphalids (fig. 2b
). Unequal nucleotide frequencies among taxa has often been cited as a cause of error in phylogenetic reconstruction because taxa with similar base compositions tend to cluster despite lack of shared ancestry (e.g., Saccone, Pesole, and Preparata 1989
; Sidow and Wilson 1990
; Lockhart et al. 1992, 1994
; Hasegawa and Hashimoto 1993
; Steel, Lockhart, and Penny 1993
; unpublished data). However, if base compositional profiles evolved in a nonhomoplasious manner, they may contain phylogenetic information that reflects historical relationships.
To examine the potential contribution of third-position base composition heterogeneity to phylogenetic signal, trees were reconstructed using third-position distances based on the LogDet correction, which compensates for the effects of biased across-taxa nucleotide compositions in phylogenetic reconstruction (Lockhart et al. 1994
). The resulting tree showed no resemblance to the third-position-only parsimony tree. Specifically, the LogDet tree did not recover riodinid, lycaenid, or riodinid + lycaenid monophyly; rather, taxa from all three lineages were grouped together and randomly distributed across the tree. The complementary experiment, in which neighbor-joining methods were used to reconstruct distances based only on third-position GC content (="GC trees" calculated from Euclidean distances as described by Ortí and Meyer 1996
) recovered a topology far less random than the LogDet tree based on third positions. The GC tree recovered all of the riodinids in one clade (with four lycaenid interlopers which had high GC contents). The Lycaenidae, on the other hand, which show heterogeneous GC contents, were not recovered as monophyletic, and neither were the Nymphalidae. The results of these two analyses implicate the contribution of third-position nucleotide composition in the resolution of riodinid relationships.
Conclusions
Wingless is a single-copy nuclear gene that shows a level of variation appropriate for phylogenetic reconstruction of butterfly tribes and families. Despite this degree of variation, wingless is easy to PCR-amplify using the primers subscribing the region used here. Because this region is bounded on the 5' end by an intron sequence, amplification of a longer segment has not been possible from genomic DNA to date. However, wingless sequences from other taxa (e.g., for Bombyx) are available from GenBank, and it may be possible to construct primers for PCR amplification of the exon 5' of the region used here. Although the 5' exon sequence is shorter than the fragment collected here (201 bp in Drosophila; Rijsewijk et al. 1987
), the results from this study suggest that this second region of wingless may provide valuable characters for phylogenetic reconstruction.
Wingless has been cited as a useful source of phylogenetic characters for resolving relationships as deep as subfamily level in the Nymphalidae (Brower and DeSalle 1998
). Our results here show that wingless provides an even greater degree of variation in the riodinids and lycaenids than in the nymphalids and resolves relationships in these two families at the tribal level to the among-families level with high nodal support.
In the riodinids, third-position substitutions continue to provide signal consistent with first- and second-position signal well past the 20%30% divergence suggested as the saturation point by Friedlander, Regier, and Mitter (1994)
. In particular, third-position transitions harbor deep signal that supports riodinid monophyly, as well as resolving more recent riodinid relationships. Multiple factors probably contribute to this signal, including preservation of signal as similar nucleotide contents among related taxa and in more slowly evolving sites. However, third positions (especially transitions) also lend support for a paraphyletic Lycaenidae, an interpretation that conflicts with topologies derived from first and second positions. This contributes to unstable lycaenid relationships with decreased bootstrap values when third positions are included. The influence of third positions in defining basal nymphalid nodes appears to be minimal, although third positions do appear to contribute resolution to recent divergences in the nymphalids.
Different processes may be driving the evolution of wingless third positions in different butterfly families. Indications of this come in the form of different nucleotide compositions and codon preferences in different lineages. These phenomena have been documented in other data sets, both mitochondrial (e.g., Fratí et al. 1997
) and nuclear (unpublished data), especially for higher-level phylogenetic problems. In cases such as these, close attention should be paid to any signals suggesting that third positions behave differently in the phylogenetic interpretation of different taxonomic groups. For this data set, excluding third-position transitions from the analysis (fig. 4
) provides a conservative parsimony estimate for this overall analysis of relationships; however, this may exclude useful phylogenetic information, especially in the riodinids. New ML models are just beginning to accommodate nonstationary nucleotide and codon frequencies and may eventually help to solve these problems. To date, though, these models are few in number and rich in parameters (see Yang 1997; Galtier and Guoy 1998
).
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We are especially indebted to Phil DeVries for lending his vast expertise on riodinid biology, for his generous help in collecting and identifying butterflies, and for his improvements in the manuscript. We thank Kelvyn Dunn, Arthur Shapiro, Man-Wah Tan, and David Yeates for assistance in collecting and identifying specimens used in this study. André Mignault and Karen Nutt provided helpful laboratory assistance. Belinda Chang, Brian Farrell, Donald Harvey, Rodney Honeycutt, Jeff Jensen, Toby Kellogg, Carla Penz, Bob Reed, Chris Simon, and two anonymous reviewers provided helpful discussions and comments. Thanks to David Swofford for allowing us to publish results from test versions of PAUP*. This work was supported by a National Science Foundation Doctoral Dissertation Improvement Grant (DEB-9520824) and a National Institute of Health training grant to D.L.C. and by NSF DEB-9615760 to N.E.P. Grants from the Putnam Expedition Fund of the MCZ (to D.L.C. and N.E.P.), the Harvard Department of Organismic and Evolutionary Biology, and the Harvard Graduate Student Council, and support from the La Selva Lodge in Ecuador enabled us to collect many of these specimens. A.V.Z.B.'s contributions to this research were supported by NSF BSR-9220317 and DEB-9303251 and by a postdoctoral fellowship from the Smithsonian Institution and the American Museum of Natural History.
Footnotes
Rodney Honeycutt, Reviewing Editor
1 Present address: University Park, Maryland.
2 Keywords: Riodinidae
Lycaenidae
Nymphalidae
molecular phylogenetics
wingless,
gene utility
third codon positions
maximum likelihood
3 Address for correspondence and reprints: Dana L. Campbell, Department of Biology, Biology/Psychology Building, University of Maryland, College Park, Maryland 20742. E-mail: dcampbell{at}oeb.harvard.edu
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