Phylogeny and Character Evolution in the Empidonax Group of Tyrant Flycatchers (Aves: Tyrannidae): A Test of W. E. Lanyon's Hypothesis Using mtDNA Sequences

Molecular Phylogenetics and Evolution Vol. 22, No. 2, February, pp. 289 –302, 2002 doi:10.1006/mpev.2001.1054, available online at http://www.idealibrary.com on

Phylogeny and Character Evolution in the Empidonax Group of Tyrant Flycatchers (Aves: Tyrannidae): A Test of W. E. Lanyon’s Hypothesis Using mtDNA Sequences Carla Cicero* and Ned K. Johnson* ,† *Museum of Vertebrate Zoology and †Department of Integrative Biology, University of California at Berkeley, Berkeley, California 94720 Received April 9, 2001; revised August 3, 2001

We sequenced mitochondrial DNA from four proteincoding genes for 26 taxa to test W. E. Lanyon’s hypothesis of intergeneric relationships and character evolution in the Empidonax group of tyrant flycatchers. Three genera in this group (Empidonax, Contopus, and Sayornis) successfully occupy north temperate habitats for breeding, while the remaining genera (Mitrephanes, Cnemotriccus, Aphanotriccus, Lathrotriccus, and Xenotriccus) are restricted to neotropical latitudes. Lanyon hypothesized two major clades in the group based on differences in syringeal morphology and proposed relationships among genera using a combination of morphologic, behavioral, and allozymic characters. The mtDNA data strongly support Lanyon’s division of genera into two clades. In addition, the molecular and nonmolecular data sets agree in uniting Aphanotriccus and Lathrotriccus as sister taxa, with Cnemotriccus as basal to these genera. Species of Aphanotriccus, Lathrotriccus, and Cnemotriccus form a clade that exploits a distinctive nesting niche relative to other members of the Empidonax group. Within the second major clade, mtDNA sequences support a reconstruction based on allozymes that places Contopus and Empidonax as sister taxa. This hypothesis contradicts that of Lanyon, who allied Contopus with Mitrephanes on the basis of similarity in foraging mode. Genera in the Empidonax group are members of a larger assemblage that radiated in South America. Occupancy of temperate habitats by certain genera in this group is coincident with their evolution of migratory behavior and with independent diversification in foraging modes that reduces potential competition in sympatry. © 2002 Elsevier Science (USA) Key Words: mitochondrial DNA; molecular phylogeny; Empidonax group; character evolution.

INTRODUCTION The family Tyrannidae, with approximately 100 genera and 400 species (Monroe and Sibley, 1993), comSequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AF447597–AF447700.

prises one of the world’s largest and most diverse avian radiations. Centered biogeographically in the neotropics, this group dominates the avifauna at low latitudes, where the diversity of taxa exhibit a huge range of variation in plumage, morphology, habitat, nesting biology, and foraging behavior. Although classification of some tyrannids has been problematic (Traylor, 1982), most genera fall into one of several well-defined subfamilies (Traylor, 1977; Traylor and Fitzpatrick, 1982). Previous attempts at understanding the evolutionary success of tyrannids have focused on (1) establishing monophyly of the family and its relationship to other groups within the Tyrannoidea (S. M. Lanyon, 1985b; McKitrick, 1985), (2) describing patterns of adaptive radiation in morphology, ecology, life history traits, and behavior (especially foraging mode) at the family level (Keast, 1972; Fitzpatrick, 1980, 1985; McKitrick, 1986; Murphy, 1989), and (3) defining broadscale systematic relationships within particular tyrannid lineages (e.g., W. E. Lanyon, 1984a,b, 1985, 1986, 1988a,b). Additional studies have addressed evolutionary, ecological, or behavioral questions among sympatric and/or sibling species of flycatchers (e.g., Johnson, 1963, 1966, 1980; Verbeek, 1975; Sherry, 1984; Cintra, 1997). To reconstruct intergeneric phylogenies of tyrannids, W. E. Lanyon used cranial characters (Warter, 1965) to separate genera into several major lineages, e.g., kingbirds and allies (Lanyon, 1984a), Myiarchus and relatives (W. E. Lanyon, 1985), an Empidonax assemblage (Lanyon, 1986), a flatbill and tody-tyrant assemblage (Lanyon, 1988a), and an Elaenia assemblage (Lanyon, 1988b). Within each lineage, Lanyon used syringeal morphology (Ames, 1971) to establish the limits of genera or generic groups and postulated relationships among genera by examining similarities in syringeal, cranial, and other traits such as plumage, nest type and location, and foraging mode. The Empidonax assemblage (subfamily Fluvicolinae) contains 33 genera (121 species; Monroe and Sib-

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TABLE 1 Characteristics of Genera in the Empidonax Group of Tyrant Flycatchers Genus

No. species a

Empidonax

15

Contopus

14

Sayornis

3

Mitrephanes

2

Xenotriccus

2

Aphanotriccus

2

Lathrotriccus

2

Cnemotriccus

1

Breeding distribution North and Middle America (lowlands to highlands) North America thru South America, West Indies (lowlands to highlands) North America to northern and western South America (lowlands to midelevations) Middle America, western South America (highlands) Mexico, Middle America (highlands) Middle America, northern South America (lowlands to midelevations) South America, southern Lesser Antilles (lowlands) South America (lowlands)

Nest site b

Foraging mode c

Foraging specialization c

Migratory tendency

Cup on open support

Encl. perch-hawk

Generalized

Mixed d

Cup on open support

Aerial hawk

Specialized

Mixed d

Cup on protected vertical surface

Near ground e

Generalized e

Mixed d

Cup on open support

Aerial hawk

Specialized

Nonmigratory

Cup on open support

Unknown

Unknown

Nonmigratory

Cup in cavity or crevice

Encl. perch-hawk

Generalized

Nonmigratory

Cup in cavity or crevice

Encl. perch-hawk

Generalized

Nonmigratory f

Cup in cavity or crevice

Encl. perch-hawk

Generalized

Nonmigratory f

a

Based on Monroe and Sibley (1993) and American Ornithologists’ Union (1998). Construction and placement of nests may vary intragenerically. Sayornis differs from other genera in using mud for nests. See Traylor and Fitzpatrick (1982), Lanyon (1986), Murphy (1989), and Young and Zook (1999). c Categories from Fitzpatrick (1980) and Traylor and Fitzpatrick (1982). d Some species migratory (long distance or short distance), others nonmigratory. Migratory tendency also may vary intraspecifically, e.g., in Sayornis, individuals from different populations may be long-distance migrants, short-distance migrants, or nonmigratory. e Foraging mode is facultative perch-to-ground sallying, which is considered transitional between aerial and terrestrial modes of foraging. Traylor and Fitzpatrick (1982) place Sayornis in a “transitional” group between the Empidonax and the “ground-tyrant” groups. f Mostly nonmigratory, although southern populations may move north during austral winter (Hayes, 1994; Chesser, 1994, 1997). b

ley, 1993; American Ornithologists’ Union, 1998) that Lanyon (1986) classified into eight generic groups. The largest of these lineages, the Empidonax group, is dominated taxonomically by Empidonax and Contopus but also contains Sayornis, Mitrephanes, Cnemotriccus, Aphanotriccus, Lathrotriccus, and Xenotriccus (Table 1). Several other genera (Nesotriccus, Pyrrhomyias, Hirundinea, Myiophobus, and Pyrocephalus) likewise have been considered part of this lineage by other workers (Webster, 1968; Traylor, 1977; Traylor and Fitzpatrick, 1982), but Lanyon (1984a, 1986) did not consider them close relatives. The inclusion of Nesotriccus was based on resemblance to Empidonax and Contopus in juvenile and adult plumage (Traylor, 1977). Lanyon (1984b) reviewed the status of this taxon and concluded that it does not belong even in the broader Empidonax assemblage because it lacks synapomorphies of the nasal septum shared by all other members of the assemblage (also see Sherry, 1985). Pyrrhomyias was placed as a close relative of Mitrephanes and Contopus because it shares a specialized foraging behavior (aerial hawking) and associated morphologic features (relatively long, pointed wings and short tarsi; Traylor, 1977; Traylor and Fitzpatrick, 1982; Murphy, 1989).

Similarly, Hirundinea was regarded as a close relative of Pyrrhomyias because it is even more specialized for aerial hawking and has additional similarities in plumage, bill morphology (“pinched” bill tips), calls, and nesting behavior (Traylor and Fitzpatrick, 1982). Lanyon’s (1986) analysis of various cranial, syringeal, plumage, and nesting characters supported the close relationship between Pyrrhomyias and Hirundinea, but suggested an alliance with Myiophobus in a separate group within the Empidonax assemblage. Finally, Pyrocephalus and Sayornis were treated together by several workers (Traylor, 1977; Fitzpatrick, 1980; Traylor and Fitzpatrick, 1982), either as part of the Empidonax group or as closer to a group with more similar ground-related foraging behaviors. Both of these taxa are near-ground specialists (Fitzpatrick, 1980) and share cranial characters with Empidonax and Contopus (Warter, 1965). Traylor (1977) classified Pyrocephalus with Sayornis because “it bears a close resemblance in general form and in cranial characters.” Syringeal differences, however, prompted Lanyon (1986) to remove Pyrocephalus from the Empidonax group and ally it with Lessonia, Hymenops, and Knipolegus, all of which have sexually dimorphic plum-

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291

TABLE 2 Characters Used by Lanyon (1986) to Reconstruct a Phylogeny of Relationships among Genera in the Empidonax Group of Flycatchers (see Fig. 1) Character Syrinx 1a 2 3 4

Skull 5 Allozymes b 6 7 Plumage 8 9–10 Nesting 11 12 13 Foraging 14

Description

Distribution by taxa

Cartilaginous segments of A2s modified for attachment of internal cartilages. Cartilaginous segments of A2s enlarged caudally but continuous (and in straight line) with calcified A2s. Presence of calcified nodule on lateral surface of each A1 and A2, with cartilaginous connection between them. Cartilaginous segments of A2s modified into broad, transverse cartilages at oblique angle to, and barely if at all connected with, dorsal ends of calcified A2s.

All taxa in Empidonax group (compared to other groups in Empidonax assemblage) Cnemotriccus, Aphanotriccus, Lathrotriccus, and Xenotriccus

Nasal capsule ossified (including alinasal walls and turbinals).

Lathrotriccus

Derived states of six protein-coding loci. Two synapomorphic allozymes.

Cnemotriccus, Aphanotriccus, and Lathrotriccus Aphanotriccus and Lathrotriccus

Adult plumage with rufous wing bars. Prominent, pointed crest (derived independently).

Cnemotriccus, Aphanotriccus, and Lathrotriccus Xenotriccus and Mitrephanes

Nests located in crevices and cavities in trees. Nests typically “saddled” on limb, ledge, beam, or similar substrate. Use of mud in nest foundation (lost in S. saya?).

Cnemotriccus, Aphanotriccus, c and Lathrotriccus Sayornis, Contopus, and Mitrephanes

Principal mode of foraging is aerial hawking and returning to the same perch.

Aphanotriccus and Lathrotriccus Sayornis, Contopus, Mitrephanes, and Empidonax

Sayornis Contopus and Mitrephanes

a

Numbers were rearranged from Lanyon (1986) to group suites of characters. Allozyme data are from Lanyon and Lanyon (1986) and support a previous finding by Zink and Johnson (1984). Lanyon (1986) suspected that Aphanotriccus nests in crevices or cavities, but placed a question mark for the genus because data were lacking. Young and Zook (1999) confirmed that the nest site of Aphanotriccus is similar to that of Cnemotriccus and Lathrotriccus. b c

age; he placed these four genera in a separate group within the Empidonax assemblage. As part of a molecular phylogenetic study of the genus Empidonax (Johnson and Cicero, in preparation), we used mitochondrial DNA (mtDNA) to examine relationships among different genera within Lanyon’s (1986) Empidonax group. We limited our analysis to the genera considered by Lanyon (1986) because of the cohesiveness of his group and because of prior explicit hypotheses of relationship based on nonmolecular and other biochemical data. Of the eight genera in Lanyon’s (1986) Empidonax group, five (Mitrephanes, Cnemotriccus, Aphanotriccus, Lathrotriccus, and Xenotriccus) are restricted to neotropical habitats (Table 1). Empidonax, Contopus, and Sayornis likewise occur at lower latitudes, although some representatives have invaded the north temperate zone for breeding. The genus Empidonax has been particularly successful in temperate habitats, where it contains the largest number of species of any tyrannid. Using 14 characters (Table 2), Lanyon (1986) reconstructed a phylogeny for the Empidonax group that divided the genera into two clades (Fig. 1). Simulta-

neously, Lanyon and Lanyon (1986) provided allozyme evidence that supported recognition of these clades, although they excluded two genera (Sayornis and Xenotriccus) from their analysis. These authors also presented evidence for generic status of the Euler’s flycatcher (“Empidonax” euleri), based on differences in syringeal morphology, ossification of the nasal capsule, and alleles at allozyme loci, and proposed a new genus, Lathrotriccus, for this taxon. This finding corroborated a previous allozyme study of Empidonax and Contopus (Zink and Johnson, 1984), which showed that euleri is distinguished from Empidonax by large genetic distances. Mitochondrial DNA has proven a useful marker for elucidating phylogenetic relationships at different taxonomic levels (Avise, 1994). Using this approach, we constructed an independent phylogeny for the Empidonax group and compared our phylogeny with Lanyon’s hypothesis derived from allozymes, morphology, and behavior. Secondarily, we used our molecular data to further explore patterns of character evolution and adaptive radiation in this group. The results of our study provide insight into patterns of ecological, behav-

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137586; D. Dittman, in litt.), was excluded from the study and the remaining sequences were reanalyzed. Our discovery of a misidentified tissue sample underscores the importance of collecting voucher specimens for molecular studies (Remsen, 1995; Winker et al., 1996; Ruedas et al., 2000). The total data set consists of 26 samples (Appendix) sequenced for 3069 nucleotides from four protein-coding mtDNA genes: cytochrome b (cyt b; 1143 bp), NADH2 (ND2; 1041 bp), NADH3 (ND3; 351 bp), and a portion of cytochrome oxidase I (COI; 534 bp). The same genes were sequenced for the mislabeled S. modestus sample. These markers offer different rates of variation and have proved to be informative in other generic-level phylogenetic studies of birds (e.g., Omland et al., 1999; DeFilippis and Moore, 2000; Cicero and Johnson, 2001). DNA Techniques

FIG. 1. Phylogenetic hypothesis of relationships among genera in the Empidonax group based on 14 morphologic, genetic, and behavioral characters (Table 2; Lanyon, 1986). Numbers refer to characters in Table 2.

ioral, and biogeographic diversification in this lineage of tyrant flycatchers. MATERIALS AND METHODS Specimens and Genes Examined We sequenced representatives of seven of the eight genera assigned to the Empidonax group (Lanyon, 1986) (Appendix), including all species analyzed allozymically by Lanyon and Lanyon (1986; Empidonax flaviventris, Contopus virens, Mitrephanes phaeocercus, Aphanotriccus audax, Cnemotriccus fuscatus, and Lathrotriccus euleri). Tissue was unavailable for the monotypic genus Xenotriccus. Although we attempted to sequence at least two samples per genus (of different species, where possible) to verify sequences and minimize potential long-branch problems (Lyons-Weiler and Hoelzer, 1997), one tissue sample of “Cn. fuscatus” was found to be misidentified after completion of all analyses (15.1% different in mtDNA compared to the other sample of Cn. fuscatus included in the study, a distance comparable to the maximum value of 15.4% for intergeneric comparisons). This individual, correctly identified as Sublegatus modestus (subfamily Elaeniinae) from the voucher specimen (LSUMZ

Whole-genomic DNA was extracted from frozen tissue using a DNeasy extraction kit (Qiagen), and different combinations of primers (Cicero and Johnson, 2001) were used to target specific genes or fragments for amplification by polymerase chain reaction (PCR). Double-stranded PCRs were performed in 25-␮l volumes containing 3–5 ␮l of extract and 20 –22 ␮l of a master mix (see Cicero and Johnson, 1995, 2001), with the following conditions: initial denaturation at 93°C for 5 min; 30 –35 cycles of denaturation (93°C for 30 – 60 s), annealing (40 –55°C for 30 – 60 s), and extension (72°C for 45–90 s); and a final extension after the last cycle at 72°C for 3 min. Cycling was performed using a PTC-100 (MJ Research) programmable thermocycler. Extractions and PCRs included at least one negative control, and products were visualized on agarose gels with ethidium bromide staining. Double-stranded products were cleaned using a Qiaquick-Spin PCR Purification Kit (Qiagen) and then cycle sequenced using fluorescent dye-labeled terminators (ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS; Perkin–Elmer). Sequencing reactions were performed in 10-␮l volumes for 24 cycles using the following conditions: 30 s at 96°C, 15 s at 50°C, and 4 min at 60°C. After sequencing, the double-stranded DNA was ethanol precipitated, vacuum dried, and denatured in formamide/blue dextran (5:1) by heating at 90°C for 2 min. Samples were run for 7 h on a 4.8% Page Plus (Amaresco) acrylamide gel using an ABI Prism 377 automated sequencer. Overlapping fragments were sequenced in both directions for verification. Samples that yielded poor quality sequence were reextracted, reamplified, and/or resequenced until unambiguous results were obtained. All final sequences were unambiguous and coded for the functional proteins, indicating that the sequences were mitochondrial vs nuclear in origin (see Collura and Stewart, 1995).

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TABLE 3

Phylogenetic Analyses Sequences for each fragment were aligned (Clustal method) and checked against electropherograms using Sequence Navigator (version 1.0.1; Applied Biosystems, Perkin–Elmer) and then joined and translated with MacDNASYSIS Pro (version 1.0; Hitachi Software Engineering America Ltd., 1991). Complete sequences were analyzed with PAUP* (Swofford, 1999) using the beta test version 4.0b4a for Power Macintosh and Windows operating systems. A partition-homogeneity test (Farris et al., 1995) was used to assess intergene heterogeneity because, although the four mtDNA genes work as a single unit, a combined analysis may not be justified if the genes show significant differences. Uncorrected pairwise divergence ( p distance) was plotted against the proportion of transition and transversion substitutions at each codon position to test for evidence of multiple hits (Hillis et al., 1996) in the four genes. In addition, p distances for ND2, ND3, and COI were regressed against those for cyt b to compare rates of divergence between these different genes in the Empidonax group versus other lineages (e.g., Omland et al., 1999; Cicero and Johnson, 2001). Finally, Tamura– Nei (1993) distances were regressed against Nei’s D (Lanyon and Lanyon, 1986) to compare levels of mtDNA versus allozyme divergence among genera within the Empidonax group (see Cicero and Johnson, 1995, p. 551 for justification of using Tamura–Nei). Tree topologies were generated using maximum parsimony and maximum likelihood, with two data partitions: all characters unweighted and third-position transitions excluded (weight ⫽ 0) for some genes. Weighting of characters was based on results of the saturation plots. Parsimony analyses involved 1000 bootstrap replications with full heuristic searches, using the TBR branch-swapping algorithm and random addition of taxa (10 replicates per iteration). Bremer (1988) decay indices for the bootstrapped parsimony tree were calculated with Autodecay version 4.0 (Eriksson, 1998; 100 random-addition replicates per tree). As another measure of robustness, taxa also were sequentially removed (i.e., jackknifed; S. M. Lanyon, 1985a; Cicero and Johnson, 2001) and the resulting data set of n ⫺ 1 taxa was reanalyzed using heuristic parsimony (TBR) with 10 random-addition replicates and strict consensus of trees. PAUP code for the jackknife analysis is unpublished and was provided by S. M. Lanyon (in litt.). Jackknife values were determined manually by comparing the nodes in each consensus tree to those in the bootstrapped parsimony tree with all taxa included. For maximum likelihood, an iterative approach was used in which a starting tree was obtained by parsimony (same criteria as above), and the data and tree topology were subjected to several rounds of likelihood

Properties of Four mtDNA Genes Sequenced for 26 Taxa in the Empidonax Group (Appendix)

% Variable sites % Parsimonyinformative sites % Nonsynonymous sites b

Cyt b

ND2

ND3

COI

Combined a

33.2

37.6

33.9

27.1

33.7

29.4

33.2

30.8

25.3

30.1

4.8

7.0

6.8

0.2

5.0

a

Combined data set for the four genes. Proportion of sites resulting in at least one nonsynonymous substitution. b

analysis until a stable tree and log likelihood value were obtained (see Huelsenbeck, 1998; Huelsenbeck and Crandall, 1997). The HKY85 model (Hasegawa et al., 1985; Hillis et al., 1996) with two substitution types and unequal, empirical base frequencies was used, with three parameters estimated from the data set: transition/transversion ratio, proportion of invariant sites, and shape of the gamma distribution of rate change at different sites. RESULTS Patterns and Levels of Sequence Divergence Of the 3069 bp sequenced for the 26 samples, approximately 34% were variable, 30% were parsimony informative, and 5% had at least one nonsynonymous substitution across all taxa and characters (Table 3). ND2 ranked slightly higher than either cyt b or ND3 in the proportion of variable and parsimony-informative sites, while COI had fewer sites that varied or were parsimony informative. The proportion of nonsynonymous sites showed more extreme variation among genes: substitutions in COI sequences were essentially all synonymous, while approximately 7% of sites in ND2 and ND3 sequences resulted in at least one nonsynonymous substitution. Cyt b also had a moderate level of nonsynonymous substitutions (ca. 5% of sites). Rates of sequence divergence showed a similar pattern comparing genes (Fig. 2), with ND2 evolving slightly faster than cyt b at all levels (slope 1.017), ND3 and cyt b having similar rates for divergences less than approximately 12% (overall slope 1.293), and COI evolving slower than cyt b at levels greater than approximately 10% (overall slope 0.735). Although differences between these slopes were marginally statistically insignificant (F test, see Sokal and Rohlf, 1995, pp. 495– 498; F ⫽ 2.45–3.37, critical F 0.05[1,⬁] ⫽ 3.84), they are meaningful from an evolutionary perspective, especially compared with rate differences between these genes in other groups (e.g., Omland et al., 1999; Cicero and Johnson, 2001).

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although there was a fair amount of scatter (especially in the ND3 plot). In contrast, all genes except for COI showed some evidence of multiple hits in third-position transitions (Fig. 3). Multiple hits in cyt b and ND3 were evident at levels of sequence divergence above 8%, whereas ND2 did not show multiple hits until approximately 13% divergence. The lack of multiple hits in COI is consistent with its lower rate of change relative to the other genes (Fig. 2), suggesting that COI may be more informative at deeper levels of divergence. Based on these plots, phylogenetic analyses were conducted with the following weighting schemes: all characters unweighted and third-position transitions excluded for cyt b, ND2, and ND3. Despite different characteristics of the four genes, a partition-homogeneity test (Farris et al., 1995) revealed significant congruence (P ⫽ 0.150). Therefore, genes were combined to examine overall sequence divergence and to construct phylogenetic hypotheses (see below). With the exception of Mitrephanes olivaceus, for which samples were from widely separate localities (Appendix), intraspecific divergences were less than 0.3% (range 0.1– 0.3%). The two samples of M. olivaceus differed by 6.4%, which falls in the middle of the range for interspecific/intrageneric comparisons (2.7– 9.5%, mean ⫽ 6.6%). The magnitude of difference between these samples was on the order of that found between M. olivaceus and M. phaeocercus (6.5–7.3%) and was greater than the difference between Lathrotriccus and Aphanotriccus (4.7–5.2%). Average divergences between other genera ranged from 9.5 to 15.4% (mean ⫽ 13.3%). Phylogenetic Relationships among Genera: Congruence between Allozymes and mtDNA

FIG. 2. Comparison of uncorrected percentage sequence divergence ( p distances) in cyt b (abscissa) versus three other mtDNA genes (ordinate) for taxa in the Empidonax group. Dotted line in each plot shows equal rates of variation between cyt b and ND2, ND3, and COI, respectively. Dots show pairwise comparisons for samples listed in Appendix. Slopes are represented by the following regression equations: cyt b vs ND2, y ⫽ 1.017x ⫹ 0.019 (r ⫽ 0.896); cyt b vs ND3, y ⫽ 1.293x ⫺ 0.024 (r ⫽ 0.907); cyt b vs COI, y ⫽ 0.735x ⫹ 0.018 (r ⫽ 0.860).

Transition and transversion substitutions at first and second positions increased linearly with percentage sequence divergence in all four genes (not illustrated). Third-position transversions showed a similar pattern, with no evidence of multiple hits (Fig. 3),

Genetic distances based on allozymes (Lanyon and Lanyon, 1986; Sayornis not analyzed) and mtDNA (this study) showed a strong curvilinear correlation (Fig. 4). In both data sets, the lowest distances were found between Empidonax versus Contopus (Nei D ⫽ 0.044, Tamura–Nei D ⫽ 0.088) and Aphanotriccus versus Lathrotriccus (Nei D ⫽ 0.095, Tamura–Nei D ⫽ 0.052). Intermediate values were observed between Empidonax and Contopus versus Mitrephanes (Nei D ⫽ 0.149–0.205, Tamura–Nei D ⫽ 0.119– 0.123) and between Aphanotriccus and Lathrotriccus versus Cneomotriccus (Nei D ⫽ 0.180–0.205, Tamura–Nei D ⫽ 0.154–0.158). Intergeneric distances were highest between Empidonax, Contopus, and Mitrephanes versus Aphanotriccus, Lathrotriccus, and Cnemotriccus (Nei D ⫽ 0.366–0.500, Tamura–Nei D ⫽ 0.165– 0.179), with these two sets of genera clustering together and showing a leveling off of mtDNA divergences relative to allozymes. A comparison of intergeneric relationships using allozymes and mtDNA (parsimony analysis, characters unweighted) revealed identical tree topologies (Fig. 5).

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295

FIG. 3. Proportion of transitions (closed circles) and transversions (open circles) at third positions, versus uncorrected percentage sequence divergence, in four mtDNA genes for the Empidonax group. Symbols show pairwise comparisons for samples listed in Appendix.

Genera fell into two clades that separated Lathrotriccus, Aphanotriccus, and Cnemotriccus from Empidonax, Contopus, Sayornis, and Mitrephanes; these groups were highly supported in all measures. Monophyly of genera based on sampled representatives also was strongly supported. Within the two clades, support was strongest for the sister relationship of Lathrotriccus and Aphanotriccus. Empidonax and Contopus also clustered together with relatively strong support, while Sayornis fell basal to these genera plus Mitrephanes. Exclusion of third-position transitions in the parsimony analysis gave similar results, except that the relationship among Empidonax, Contopus, Mitrephanes, and Sayornis was poorly resolved in bootstrap replicates (values ⬍50%). Maximum likelihood analysis (Fig. 6) also recognized the two major clades and showed long branch lengths leading to major lineages, including the sister pair Aphanotriccus and Lathrotriccus; however, the four genera within the clade containing Empidonax were separated by short basal internodes. Likelihood analyses based on initial

FIG. 4. Relationship between the Nei D (Lanyon and Lanyon, 1986) and the Tamura–Nei D (this study, 3069 bp) for 15 pairwise comparisons (only the taxa analyzed by Lanyon and Lanyon were included). The polynomial equation for the best curve fit is y ⫽ ⫺ 0.797x 2 ⫹ 0.646x ⫹ 0.040.

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FIG. 5. Allozyme and mtDNA trees showing relationships among seven of the eight genera in the Empidonax group. The allozyme data were analyzed using UPGMA with Roger’s D (Lanyon and Lanyon, 1986); values above the branches indicate the number of synapomorphic alleles shared by different genera. The mtDNA data (combined analysis, 3069 bp, characters unweighted) were analyzed using maximum parsimony; sequences were combined because a partition-homogeneity test showed significant congruence among genes (P ⫽ 0.150). Intrageneric branches were collapsed for purposes of illustration (sampled representatives of each genus were monophyletic). Numbers above the branches represent bootstrap values for 1000 replications; numbers below the branches indicate decay indices. All nodes were supported in 100% of jackknifed trees. Sayornis was not included in the allozyme analysis and is represented by a dashed line in that tree. Scores for the mtDNA tree are length ⫽ 2246, CI ⫽ 0.559, RI ⫽ 0.778, RC ⫽ 0.435.

the clade containing Lathrotriccus, Aphanotriccus, and Cnemotriccus. To further explore patterns of character evolution in the Empidonax group, we used MacClade version 3.06 (Maddison and Maddison, 1996) to map foraging and nesting behavior as well as breeding distribution and migratory tendency onto the mtDNA phylogeny (Tables 1 and 2, Figs. 7 and 8). On the basis of these results, Contopus and Mitrephanes either (1) inherited their specialized foraging behavior (aerial hawking) from a common ancestor, as suggested by Lanyon (1986; see Fig. 1 and Table 2), with subsequent change to a more generalized perch-hawking behavior in Empidonax, or (2) evolved this specialized behavior independently. The mtDNA data are unable to distinguish between these competing hypotheses, although the observation that generalized foraging is more widespread in the group (all taxa except Contopus and Mitrephanes, Fig. 7) lends support to the latter hypothesis. With regard to nesting behavior, the distinctive nest sites of Lathrotriccus, Aphanotriccus, and Cnemotriccus (Table 1) relative to other members of

parsimony topologies constructed using the two different weighting schemes (see Materials and Methods) gave identical trees. Phylogenetic Reconstructions and Character Evolution Lanyon’s (1986) reconstruction (Fig. 1) based mostly on morphologic and behavioral characters agrees with the phylogeny derived from mtDNA sequences (Fig. 5) in also recognizing two major clades with the same groups of genera. Comparing the two trees, the only apparent difference is in the putative relationship among Empidonax, Contopus, Mitrephanes, and Sayornis. Whereas Lanyon allied Contopus and Mitrephanes together on the basis of similarity in foraging behavior, the mtDNA data placed Contopus as the sister taxon to Empidonax in most analyses. The allozyme data (Fig. 5) also suggested this relationship, although only one synapomorphic allele united these genera. The basal position of Empidonax in Lanyon’s (1986) phylogeny (Fig. 1) apparently is attributed to differences in nesting biology (Johnson and Cicero, in preparation) compared to Sayornis, Contopus, and Mitrephanes. In general, fewer nonmolecular (Fig. 1) and molecular (Fig. 5) characters supported resolution of relationships within this clade compared to

FIG. 6. Maximum likelihood topology for samples of the Empidonax group, based on 3069 bp of mtDNA analyzed using an initial, unweighted parsimony tree (see Materials and Methods). Numbers above the branches represent branch lengths. The log likelihood value of the optimized tree is 14,274.49.

PHYLOGENY AND CHARACTER EVOLUTION IN Empidonax GROUP

297

DISCUSSION Intergeneric Relationships in the Empidonax Group: Evidence from Molecular and Nonmolecular Characters A major result of this study is the strong congruence between mtDNA sequences and other characters (allozymes, morphology, behavior) used by Lanyon (1986; also see Lanyon and Lanyon, 1986) to hypothesize intergeneric relationships within the Empidonax group. These varied data sets all support a distinct clade comprising Lathrotriccus, Aphanotriccus, and Cnemotriccus, which share a number of molecular and nonmolecular characters that distinguish them from

FIG. 7. Phylogenetic reconstructions for the evolution of foraging behavior and specialization in species sequenced in this study (Appendix). Tree is based on 3069 bp of mtDNA analyzed using unweighted maximum parsimony (same tree as in Fig. 5, but with individual species depicted).

the group are congruent with their phylogenetic alliance based on mtDNA (not illustrated) and Lanyon’s (1986) characters (Table 2, Fig. 1). A more complex pattern emerged when analyzing migratory behavior (Table 1, Fig. 8), which was not considered by Lanyon (1986). Migration within Empidonax and Contopus appears to have originated from a common ancestor, while migratory behavior in Sayornis—whose phylogenetic placement was not well resolved (Figs. 5 and 6)—may share either the same or an independent origin relative to Empidonax–Contopus. Despite this uncertainty, the evolution of migratory behavior is closely tied to occupancy of north temperate environments by species in these genera. Because intrageneric and intraspecific (i.e., interpopulation) differences in migratory tendency occur within the Empidonax group, a more complete sampling of taxa or population variants is necessary for a thorough phylogenetic reconstruction of the evolution of migration in this lineage.

FIG. 8. Phylogenetic reconstructions for primary breeding distribution (temperate versus neotropical) and the evolution of migratory behavior in species sequenced in this study (Appendix). Tree is based on 3069 bp of mtDNA analyzed using unweighted maximum parsimony (same tree as in Fig. 5, but with individual species depicted). Cnemotriccus and Lathrotriccus are nonmigratory through most of their range, although southern populations may exhibit austral movements (see text). Populations of Sayornis vary in migratory tendency: some exhibit long- or short-distance movements, others are nonmigratory.

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Empidonax, Contopus, Mitrephanes, and Sayornis. The putative position of Xenotriccus as the sister group to Lathrotriccus, Aphanotriccus, and Cnemotriccus (Lanyon, 1986), all of which share a similar syrinx, cannot be corroborated because of the lack of either allozyme (Lanyon and Lanyon, 1986) or sequence data. Although L. euleri has historically been placed in the genus Empidonax, Zink and Johnson (1984) reported large genetic distances separating this taxon from all other Empidonax. They further suggested that euleri may be closely related to Cnemotriccus because of observed similarities in external morphology, ecology, and behavior (“specimens of these forms are often confused in collections”). However, they did not examine the proteins of Cnemotriccus and thus were unable to confirm this relationship. Lanyon and Lanyon (1986), in a follow-up study, verified that Lathrotriccus and Cnemotriccus are closely related but showed that they are not sister taxa. Rather, these authors presented evidence for a sister taxon relationship between Lathrotriccus and Aphanotriccus on the basis of external morphology, syringeal morphology, synapomorphic alleles, and relatively low genetic distance. The close relationship of these two genera is strongly supported by mtDNA sequences, which show a level of divergence (mean ⫽ 5%) less than that found between many cryptic or congeneric species of neotropical birds [e.g., M. olivaceus (6.4%) and M. olivaceus vs. M. phaeocercus (6.9%), this study; Hackett, 1995, 1996; Garcı´a-Moreno and Arctander, 1998; Bates et al., 1999]. While such a low mtDNA divergence might argue for congeneric status, genetic distances alone should not be used as a taxonomic yardstick (Cicero and Johnson, 1995; Johnson et al., 1999). Accordingly, continued recognition of the genus Lathrotriccus is justified on the basis of its distinctive cranial morphology (ossified nasal capsule; Lanyon, 1986) and the presence of several synapomorphic alleles in Aphanotriccus (Lanyon and Lanyon, 1986; Fig. 5). Ecological, Behavioral, and Biogeographic Diversification in the Empidonax Group Tyrant flycatcher species comprise nearly 20 –30% of the avifaunas in neotropical and south temperate environments (Keast, 1972), while only 35 of the approximately 400 species (10 of 100 genera) in the family have invaded the north temperate zone for breeding. The tremendous success of tyrannids in the neotropics is attributed to an unparalleled capacity to occupy diverse habitats over broad elevational and latitudinal zones. Furthermore, species of tyrannids have evolved highly diverse morphologies, foraging modes, and reproductive and nesting strategies (Keast, 1972; Fitzpatrick, 1980, 1985; Murphy, 1989). This diversification is evident not only at the family or subfamily level, but also within different lineages. In Lanyon’s (1986) Empidonax group, representatives restricted to the

neotropics include Lathrotriccus, Aphanotriccus, Cnemotriccus, Mitrephanes, and Xenotriccus. The former three genera form a clade that exploits a distinct nesting niche of cavities or crevices in trees (Lanyon and Lanyon, 1986; Young and Zook, 1999). Interestingly, the putative sister taxon to these genera (Xenotriccus; Lanyon, 1986) reportedly constructs an open-cup nest (Rowley, 1962, 1963; Alvarez del Toro, 1964) similar to that of Empidonax, Contopus, and Mitrephanes. Sayornis also constructs a cup nest, although members of this genus have adapted the use of mud to adhere nests to protected vertical surfaces (Murphy, 1989). The evolution of differences in nesting behavior apparently has occurred repeatedly within the Tyrannidae. For example, in the Empidonax assemblage, four of the five generic groups with multiple taxa contain genera that show distinctive nesting behavior. Such variation also is apparent in other generic assemblages (e.g., kingbirds and allies; Lanyon, 1984), as well as among species within genera (e.g., Empidonax; Murphy, 1989; Johnson and Cicero, in preparation). Because differences in construction or placement of nests often are associated with variation in egg color and spotting patterns (Oniki, 1985), exploitation of diverse nesting opportunities can be an important factor in reproductive isolation, especially in tropical habitats where such opportunities are probably greatest. In addition to nesting biology, tyrant flycatchers also exhibit remarkable flexibility in foraging behavior (Fitzpatrick, 1980, 1981, 1985). The evolution of diverse foraging strategies, from generalized to highly specialized modes, has occurred repeatedly in unrelated taxa. This is evident at the level of subfamily as well as within generic lineages, whose members include both generalized and more specialized foragers (Fitzpatrick, 1980). In the Empidonax group, foraging strategy does not appear to be phylogenetically informative. For example, two genera (Contopus and Mitrephanes) exhibit specialized aerial-hawking behavior, whereas all other genera except Sayornis are generalist enclosed-perch hawkers. Although Mitrephanes has been described as “a miniature Contopus in habits” (Webster, 1968; Traylor, 1977), these genera do not emerge as sister taxa in either allozyme (Lanyon and Lanyon, 1986) or mtDNA (this study) analyses. Interestingly, the only genera that have invaded the north temperate zone (Empidonax, Contopus, and Sayornis) all exhibit different foraging strategies, although they are members of the same phylogenetic clade. Such differences seemingly reduce potential competition among these taxa in sympatry (Verbeek, 1975). Likewise, species of Empidonax flycatchers are able to coexist in local sympatry because of divergences in habitat use and foraging spheres that apparently evolved allopatrically (Johnson, 1966; Johnson and Cicero, in preparation). In the neotropics, seasonally sympatric species of flycatchers show differences in habitat use

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and foraging breadth as well as feeding rate and prey size (Sherry, 1984). Because divergence in foraging behavior is correlated with variation in morphologic as well as life history traits in tyrant flycatchers (Fitzpatrick, 1985; Murphy, 1989), these factors, combined with differences in nesting ecology, would promote speciation and ecological diversification into the wide array of niches and habitats occupied by lineages within this family (Keast, 1972; Traylor and Fitzpatrick, 1982). Although the sister lineage to the Empidonax group is unresolved based on morphologic characters (see Lanyon, 1986), and has not been studied using molecular data, early adaptive radiation of this group is most likely traced to South America. The main evidence for this hypothesis is the distribution of species in the broader Empidonax assemblage (Lanyon, 1986), over 75% of which breed in South American tropical or temperate latitudes. Within the Empidonax group, 2 primarily tropical species (Cn. fuscatus and L. euleri) have extended their range into the south temperate zone, while 16 species (39%, dominated by the genus Empidonax) breed primarily in north temperate habitats. Occupancy of north temperate latitudes is correlated with the evolution of long-distance or short-distance migratory behavior (this study; Johnson and Cicero, in preparation; also see Cicero and Johnson, 1998). Likewise, populations of neotropical flycatchers that inhabit south temperate regions—including Cnemotriccus and Lathrotriccus— have evolved at least short-distance, austral migratory tendencies (Hayes et al., 1994; Chesser, 1994, 1997). The evolution of migratory behavior in birds appears to represent a relatively rapid, genetically driven response to changing environmental conditions, most likely from an ancestral state of partial migration (Berthold, 1998).

Traylor (1977) hypothesized that Aphanotriccus and Xenotriccus each contain two relict species that “may be remnants of an earlier stock from which the currently successful Central and North American genera Contopus, Empidonax, and Sayornis were derived.” Furthermore, because Contopus is more evenly distributed (i.e., in neotropical and temperate latitudes) than the other genera, it “may well have been the primitive stock from which the others were derived.” Traylor (1977) does not speculate on the biogeographic origin of Mitrephanes. Whether Aphanotriccus or Xenotriccus represents the ancestral stock cannot be determined with present data sets. However, mtDNA sequences do not support Traylor’s (1977) hypothesis that Contopus is basal to Empidonax and Sayornis. Although short internodes between Empidonax, Contopus, Sayornis, and Mitrephanes suggest relatively rapid diversification of these four lineages, there was some support for Empidonax and Contopus as sister taxa (e.g., Figs. 5 and 6). Furthermore, report of an intergeneric hybrid between Empidonax trailli and Contopus sordidulus (Short and Burleigh, 1965) provides additional evidence that these genera are closest living relatives. The relationship of Sayornis, on the other hand, was poorly resolved in all analyses. While a molecular phylogenetic study of the entire genus Empidonax has been completed (Johnson and Cicero, in preparation), sampling of the remaining species of Contopus, as well as of additional populations of Sayornis and Mitrephanes, would shed further light on the biogeographic history of this diverse lineage. A phylogeographic analysis of S. nigricans would be especially interesting in light of its widespread distribution, which extends from western North America into South America along the length of the Andes, where reportedly it has invaded relatively recently (Traylor, 1977).

APPENDIX Tissue Samples (n ⴝ 26) of Taxa in the Empidonax Group That Were Amplified and Sequenced for Four mtDNA Genes (cyt b, ND2, ND3, COI) Taxon Cnemotriccus fuscatus fuscatior Lathrotriccus euleri euleri L. euleri euleri Aphanotriccus audax A. audax Mitrephanes phaeocercus eminulus Mitrephanes olivaceus M. olivaceus Contopus cooperi

Locality

Specimen number a

Rio Amazonas opposite Aysana, 80 m, ca. 80 km NE Iquitosca, Depto. Loreto, Peru El Tirol, 19.5 km by road NNE Encarnacion, Depto. Itapua, Paraguay El Tirol, 19.5 km by road NNE Encarnacion, Depto. Itapua, Paraguay Cana on E slope Cerro Pirre´, 650 m, Darie´n Prov., Panama Cana on E slope Cerro Pirre´, 650 m, Darie´n Prov., Panama Ca. 6 km NW Cana, 1200 m, Darie´n Prov., Panama Cerro Asunta Pata, 82 km by road E Charzani, Prov. Bautista Saavedra, Depto. La Paz, Bolivia Cushi, 1800 m, Depto. Pasco, Peru Finch Creek, 2500 ft, 1.5 mi N and 2 mi E Chews Ridge Ranger Station, Monterey Co., California

LSUMZ 120152 MVZ 168850 MVZ 168855 LSUMZ 108927 LSUMZ 108499 LSUMZ 108479 LSUMZ 163168 LSUMZ 128762 MVZ 169193

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APPENDIX—Continued Locality

Specimen number a

Sawmill Creek, 4400 ft, 1 mi SE San Benito Mountain, San Benito Co., California Wells Range, 2400 ft, N foothills Caliente Range, San Luis Obispo Co., California Shovel Creek, 4450 ft, 4 mi N and 7 mi W Macdoel, Siskiyou Co., California 1.5 mi N and 1.5 mi W La Salle Creek Bog, 1450 ft, Hubbard Co., Minnesota 1 mi N and 1 mi E La Salle Creek Bog, 1450 ft, Hubbard Co., Minnesota 3 mi E Waskish, 1180 ft, Beltrami Co., Minnesota 3 mi E Waskish, 1180 ft, Beltrami Co., Minnesota Finch Creek, 2500 ft, 1.5 mi N and 2 mi E Chews Ridge Ranger Station, Monterey Co., California Finch Creek, 2500 ft, 1.5 mi N and 2 mi E Chews Ridge Ranger Station, Monterey Co., California 5 km SE Porrosati, 1800 m, Prov. Heredia, Costa Rica 5 km SE Porrosati, 1800 m, Prov. Heredia, Costa Rica 6 mi N and 1 mi W McCurtain, 480 ft, Haskell Co., Oklahoma 6 mi N and 1 mi W McCurtain, 480 ft, Haskell Co., Oklahoma Smoke Creek, 4200 ft, Washoe Co., Nevada Smoke Creek, 4200 ft, Washoe Co., Nevada Deer Creek, 2300 ft, 3.5 mi S and 5 mi W Crockett Peak, Lake Co., California Site of Troy, Troy Canyon, W slope Grant Range, Nye Co., Nevada

MVZ 170139

Taxon Co. cooperi Contopus sordidulus veliei Co. sordidulus veliei Contopus virens Co. virens Empidonax flaviventris E. flaviventris Empidonax difficilis difficilis E. difficilis difficilis Empidonax flavescens flavescens E. flavescens flavescens Sayornis phoebe S. phoebe Sayornis saya saya S. saya saya Sayornis nigricans semiatra S. nigricans semiatra

MVZ 169024 MVZ 168549 MVZ 168652 MVZ 168655 MVZ 168659 MVZ 168660 MVZ 169269 MVZ 169273 MVZ 178156 b MVZ 178158 b MVZ 178208 c MVZ 178209 c MVZ 170117 MVZ 170118 MVZ 169295 MVZ 177922

a LSUMZ, Louisiana State University Museum of Natural Science, Baton Rouge, LA. MVZ, Museum of Vertebrate Zoology, University of California, Berkeley. b MVZ catalog number for tissue sample only. Voucher at American Museum of Natural History (MVZ 178156 ⫽ AMNH skeleton No. 12789; MVZ 178518 ⫽ AMNH skeleton No. 12788). c MVZ catalog number for tissue sample only. Voucher at Oklahoma Museum of Natural History (MVZ 178208 ⫽ OMNH 18210; MVZ 178209 ⫽ OMNH 18209).

ACKNOWLEDGMENTS We especially thank Frederick H. Sheldon and Donna L. Dittman for generously providing tissue samples from the Museum of Natural Science, Louisiana State University. J. Van Remsen and Donna L. Dittman verified the identifications and localities of voucher specimens for these samples. Ka Yin (Harold) Fong performed the laboratory work. Karen Klitz prepared the final figures. Jaime Garcı´aMoreno and one anonymous reviewer provided helpful comments that greatly improved the manuscript. Laboratory expenses were funded by a grant from the Lulu Von Hagen Foundation.

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