*Smithsonian Tropical Research Institute, Panamà;
and
Department of Biology, University of Pennsylvania
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Abstract |
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Introduction |
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Surprisingly few studies have employed nucleotide sequences of nuclear exons as phylogenetic markers for avian systematics (e.g., Caspers et al. 1997
; Cooper and Penny 1997
; Groth and Barrowclough 1999
), and these have generally focused on reconstructing relationships among highly divergent avian lineages. Here, we extend the use of c-mos to explore the more recent differentiation within the order Passeriformes, an avian group of particular interest because it is disproportionally diverse, comprising nearly three fifths of extant bird species (Sibley and Monroe 1990
). Our sample of 15 passerine genera (table 1
) provides comparisons across lineages spanning a hierarchical range of differentiation, from representatives of the most ancient passerine clades (e.g., oscines and suboscines) to closely allied genera (e.g., Dendroica and Basileuterus) placed in the same tribe (Sibley and Monroe 1990
) or family (American Ornithologists' Union 1998
) in current classifications. Our choice of particular taxa was influenced primarily by the DNA-DNA hybridizationbased phylogeny of Sibley and Ahlquist (1990)
, which included more than 600 passerine species, and by our own research interests on passerine systematics; figure 1
shows the Sibley and Ahlquist DNA-DNA hybridizationbased reconstruction of relationships among the genera included in the present survey of c-mos differentiation.
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Materials and Methods |
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As part of an ongoing investigation of passerine relationships employing mtDNA markers, we also obtained mitochondrial sequences from 8 oscine representatives of the 15 passerine taxa listed in table 1
using protocols similar to those given above and described elsewhere (e.g., Lovette et al. 1999
). We obtained 2,506 bp of protein-coding mtDNA sequence from each sample, which included the entire coding region of the ATP-synthase 6 (ATPase 6; 684 bp), ATP-synthase 8 (ATPase 8; 168 bp), and NADH dehydrogenase subunit II (NDII; 1,041 bp) genes and a portion of the cytochrome oxidase subunit I gene (COI; 613 bp corresponding to nucleotides 73427954 in the chicken mitochondrial genome sequence; GenBank X52392; Desjardins and Morais 1990
).
c-mos Sequence and Phylogenetic Analysis
Complementary c-mos chromatograms from each individual were aligned with one another, and base calls were confirmed by eye in the program SeqEd to provide 579582 nt of double-stranded sequence per sample. The corresponding c-mos coding regions from two outgroup species (Gallus and Struthio) and one passerine (Acanthisitta) were obtained from GenBank (Schmidt et al. 1988
; Cooper and Penny 1997
) (table 1
). Except where otherwise specified, our characterizations of c-mos variation excluded the two nonpasserine outgroup taxa. Phylogenetic analyses, on the other hand, were rooted using Gallus and Struthio. Sequences were aligned by eye and imported into the program SEQUENCER (B. Kessing, personal communication) to generate nucleotide and amino acid variation statistics. We used three techniques to reconstruct the phylogenetic relationships among sequences. Maximum-likelihood (ML) searches were conducted using the quartet-puzzling method as implemented in PUZZLE, version 4.0 (Strimmer and von Haeseler 1997
). We invoked the "exact" parameter estimation option and estimated the transition : transversion ratio, the nucleotide composition, and the gamma rate parameter from the sequence data. Maximum-parsimony (MP) and neighbor-joining (NJ) searches were conducted using Paup*, version 4.0b2 (Swofford 1999
). Heuristic MP searches were conducted with all nucleotide substitutions weighted equally and with insertions/deletions excluded from analysis, followed by 1,000 heuristic bootstrap replications. Neighbor-joining searches were based on a distance matrix calculated using the Hasegawa-Kishino-Yano (Hasegawa, Kishino, and Yano 1985
) method with rate variation, with the gamma parameter set to the value identified in the ML search.
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Results |
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As expected from our taxonomic sampling strategy, we noted a broad range of c-mos divergence in pairwise comparisons of passerine taxa. The most similar c-mos sequences were those of the two wood-warblers Dendroica and Basileuterus, which differed at two nucleotide sites (0.4% uncorrected divergence). Small distances also separated these warblers and three other nine-primaried oscine taxa (Icterus, Saltator, and Coereba), which differed at 517 nucleotide sites (0.9%2.9%). The greatest distances among passerines were found in pairwise comparisons between oscine and suboscine genera (4462 sites; 7.6%10.7%) and between oscines or suboscines and Acanthisitta (4461 sites; 7.6%10.5%). The Gallus and Struthio outgroup taxa differed from the 15 passerines at 5379 nucleotide sites (9.1%14.8%).
Phylogenetic Reconstructions and Rate Variation Among Taxa
The c-mos trees reconstructed using ML, MP, and NJ techniques were nearly identical (figs. 2 and 3
). Differences among these reconstructions involved only the placement of Acanthisitta relative to the other passerines and the branching order among several closely related nine-primaried oscine passerines. These topological differences and their implications for passerine systematics are discussed below.
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The passerine c-mos trees are largely congruent with those based on DNA-DNA hybridization (fig. 1
), and thus the c-mos phylogenetic results fit well with and contribute additional support for prevailing views regarding the evolutionary history of these taxa. Although containing several unresolved polytomies, the consensus c-mos tree generated from the results of the ML, MP, and NJ searches (which is identical to the MP consensus tree shown in fig. 3
) differs from the DNA-DNA hybridizationbased tree of Sibley and Ahlquist (1990
, fig. 1
) only in the relative branching order in the suboscine clade comprising Conopophaga, Myrmotherula, and Formicarius. Similarly, the c-mos and mtDNA trees for oscine passerines are highly congruent (figs. 24
). Despite the relatively short length (582 nt) of the c-mos fragment sequenced, many nodes in the c-mos reconstructions received strong reliability (fig. 2
) or bootstrap (fig. 3
) support.
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Discussion |
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The c-mos reconstructions provide further evidence that the Acanthisittidae are representatives of an ancient lineage but are ambiguous regarding the placement of the acanthisittid lineage relative to the suboscine or the oscine clades. Instead, the c-mos data suggest that the Acanthisitta lineage became evolutionarily independent at approximately the same time that the suboscine and oscine clades diverged from one another, producing the three-branch polytomy seen in our reconstructions. The uncertainty of relationship resulting from this early passeriform radiation is reflected in the topological differences between the ML, MP, and NJ reconstructions: in the ML tree (fig. 2
), Acanthisitta is basal to a clade comprising both the oscines and the suboscines; in the MP consensus tree (fig. 3
), Acanthisitta, the suboscines, and the oscines form a three-way polytomy; and in the NJ tree (fig. 3
,) Acanthisitta is the sister lineage of the suboscines, with a very short internode separating it from the basal passerine bifurcation. In no reconstruction was the placement of Acanthisitta supported by high bootstrap or reliability scores. Likelihood ratio tests (Kishino and Hasegawa 1989
) conducted in Paup* indicated no significant difference between the observed ML tree (fig 2
; -Ln = 2,457.4), in which Acanthisitta is basal to the combined suboscine/oscine clade and trees in which Acanthisitta was constrained to be the basal lineage within either the suboscines or the oscines (for both alternatives, -Ln = 2,458.9; -
Ln = 1.5, T = 0.6, P > 0.5). Similarly, parsimony criteria identified negligible tree length differences between these three alternative topologies: Acanthisitta (subocines, oscines) = 324 steps; oscines (Acanthisitta, suboscines) = 323 steps; suboscines (Acanthisitta, oscines) = 323 steps.
Although the c-mos reconstructions are inconclusive regarding the sister taxon of the Acanthisittidae, they provide a molecular perspective on the origin of the Acanthisittidae lineage relative to further diversification within the suboscine and oscine clades. In all reconstructions, moderately long internodes separated the root of the Acanthisitta lineage from the earliest sampled bifurcation within either the suboscine or the oscine clade, suggesting that the Acanthisittidae lineage originated before the subsequent and extensive radiations of lineages within these two groups. Although additional sequences representing several groups of enigmatic "Old World suboscines" (taxa in Sibley and Ahlquist's [1990]
Infraorder Eurylaimides) are needed to test the hypothesis that the derivation of the Acanthisittidae preceded the further diversification of extant passerines, the available c-mos evidence is consistent with previous suggestions that the Acanthisittidae represent an ancient lineage relative to other extant passerines. If this hypothesis holds, it would help explain why different morphological characters have provided conflicting results regarding acanthisittid affinities.
Phylogenetic Relationships of Suboscine and Oscine Passerines
The c-mosbased topology for the other 14 passerine taxa is highly congruent both with the traditional classification of these species and with the DNA-DNA hybridizationbased reconstruction of Sibley and Ahlquist (1990)
. These 14 passerine taxa are divided between two well-defined clades that correspond to their traditional separation into suboscine and oscine groups.
Suboscines
In the c-mos reconstructions, the five suboscine taxa fell into two clades: the tyrannid flycatchers Elaenia and Mionectes, and the antbirds Myrmotherula and Formicarius and the gnatcatcher Conopophaga. This basal suboscine bifurcation is congruent with the phylogeny of Sibley and Ahlquist (1990)
, which placed the tyrannids as the sister taxon to all other New World suboscines. The placement of Conopophaga as the sister taxon to Myrmotherula, however, conflicts with the Sibley and Ahlquist tree, in which the clade containing Myrmotherula (their parvorder Thamnophilidae) is basal to a clade (their superfamily Formicariodea) containing both Conopophaga and Formicarius. Short internodes separate these three lineages in both the c-mos (figs. 2 and 3
) and the Sibley and Ahlquist (fig. 1
) trees. The c-mos topology relating these three lineages was supported by a high reliability score in the ML analysis (fig. 2
) but not by high bootstrap values in the MP or NJ reconstructions (fig. 3
).
Oscines
One initially surprising but now widely accepted systematic revision that followed from Sibley and Ahlquist's (1990)
DNA-DNA hybridization studies was the separation of the oscine passerines into two sister groups, the parvorders Corvida and Passerida. The Corvida include a number of morphologically variable taxa endemic to Australia, New Guinea, and Southeast Asia that had previously been grouped into disparate passerine families, plus the New World vireos and the panglobally distributed corvines (Sibley and Ahlquist 1990
). In the c-mos reconstructions, Vireo and Cyanocorax together form the sister clade to the remaining oscines, thereby supporting Sibley and Ahlquist's Corvida hypothesis. The internode separating the Corvida lineage from the basal bifurcation within the Passerida, however, is short, suggesting that the Corvida lineage separated from the remaining oscines not long before further cladogenesis occurred in the Passerida.
Within the Passerida, the c-mos reconstructions identify a highly supported clade composed of Dendroica, Basileuterus, Icterus, Saltator, and Coereba. These five taxa are all representatives of the so-called "New World nine-primaried oscines" (Sibley and Ahlquist's family Fringillidae), a highly diverse radiation that includes eight tribes in their classification (Sibley and Ahlquist 1990
). Phylogenetic relationships within the nine-primaried oscines have long been considered particularly problematic because of the recent and explosive diversification of this group (Hellmayr 1935, 1936
; Mayr and Amadon 1951
; Beecher 1953
; Storer 1969
; Sibley 1970
; Paynter and Storer 1970
; Lovette and Bermingham 1999
). Both the c-mos and mtDNA trees provided high levels of support for two pairs of sister taxa, Dendroica/Basileuterus and Coereba/Saltator.
Comparative Nuclear and Mitochondrial Sequence Divergence
In comparing c-mos and mitochondrial sequences, we were primarily interested in determining relative rates of nucleotide substitution. A second goal was to use c-mos differentiation as a standard to evaluate the rate at which these mitochondrial loci approach saturation. We therefore included in our c-mos survey a set of eight oscine passerine taxa (Vireo, Cinclocerthia, Myadestes, Dendroica, Basileuterus, Saltator, Coereba, and Icterus) from which we had previously obtained long mitochondrial sequences.
The absence of significant c-mos rate variation among passerine lineages supports our underlying assumption that the c-mos gene has accumulated substitutions at an approximately constant rate in these lineages. We therefore used c-mos divergence as a standard against which to measure the relative frequencies of various classes of mitochondrial nucleotide substitution. These comparisons were valuable because they provided a calibration of mtDNA differentiation against an independent locus with a relatively low intrinsic rate of saturation. In figure 5
, we show uncorrected pairwise mitochondrial distances plotted against the corresponding gamma-corrected c-mos divergence values. A striking but expected feature of this graphic analysis is the lack of linearity in the plot of mitochondrial transitions. A plateau at approximately 10% transition distance is reached at a very low level of c-mos differentiation, suggesting that mitochondrial transitions approach complete saturation at a distance that corresponds to one or two c-mos nucleotide substitutions. On the other hand, uncorrected distances based on mitochondrial transversions appear to accumulate at an approximately constant rate for a much longer period before reaching a saturation plateau. The slope of this initial mtDNA transversion : c-mos relationship is approximately 0.81.0, suggesting that c-mos nucleotide substitutions accumulate at a rate similar to that of mitochondrial transversion substitutions. These differences in mtDNA transition and transversion saturation rates mirror long-standing assumptions about the molecular evolution of vertebrate mtDNA derived from comparisons of different classes of mitochondrial substitution (e.g., Kocher et al. 1995
). As has also long been recognized, the rapid saturation of mitochondrial transitions may cause biases in genetic distances calculated by methods that do not account for these saturation effects (e.g., Yang 1997
). These comparisons suggest that nucleotide saturation effects may cause large underestimates of sequence divergence even between avian taxa that differ by less than 10% uncorrected mitochondrial divergence, a degree of differentiation frequently observed between congeneric avian species and closely related avian genera (e.g., Helbig et al. 1995
; Klicka and Zink 1997
; Lovette and Bermingham 1999
).
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Acknowledgements |
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Footnotes |
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1 Present address: Center for Tropical Research, Department of Biology, San Francisco State University.
2 Keywords: c-mos,
mitochondrial DNA
Passeriformes
phylogeny
substitution rates
3 Address for correspondence and reprints: Irby J. Lovette, Center for Tropical Research, Department of Biology, San Francisco State University, 1600 Holloway Avenue, San Francisco, California 94132. E-mail: ilovette{at}sas.upenn.edu
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