AbstractSong Diversification and Speciation in the Empidonax difficilis-occidentalis-flavescens Complexby Andrew Christopher RushDoctor of Philosophy in Integrative BiologyUniversity of California, BerkeleyProfessor Rauri Bowie, ChairSpeciation can occur when barriers to gene flow form between populations. Phenotypic differences resulting from divergent selection can act as strong pre-zygotic barriers to gene flow. Birds provide excellent opportunities to examine the effectiveness of pre-zygotic barriers, because phenotypic traits, such as song and plumage, which affect species recognition and mate choice, are often conspicuous and relatively easy to observe and quantify. In the research outlined in this dissertation, I analyze the effects of divergence in song on lineage diversification in the six principal taxa that comprise the Empidonax-difficilis-occidentalis-flavescens complex, a clade of suboscine passerines. This dissertation is one of the broadest examinations of the interaction between genotype and innate song type yet conducted, and provides insights relevant to the role of divergent signals in speciation in a wide range of organisms.In Chapter 1, I examine an area of geographic contact in southwestern Canada between two of these taxa, the Pacific-slope Flycatcher (E. d. difficilis) and the Cordilleran Flycatcher (E. o. hellmayri). Contact zones between recently diverged taxa offer unique opportunities to test whether the forms are reproductively isolated and therefore distinct species. I present the first analysis of genetic variation across this region, in order to determine whether hybridization and gene flow occurs between these taxa. I determine that parental populations of Pacific-slope and Cordilleran Flycatchers have distinct mitochondrial haplotypes, and that all of the individuals sampled in interior southwestern Canada have the Pacific-slope haplotype. In contrast, analysis of nuclear DNA (AFLPs) indicates a high level of population admixture between Pacific-slope and Cordilleran Flycatchers in this region, although I find no evidence of nuclear gene flow into core parental populations. I suggest that the discordance between the mitochondrial and nuclear markers most likely results from stochastic loss of Cordilleran mitochondrial haplotype lineages facilitated by asymmetries in mating due to earlier arrival and greater abundance of Pacific-slope Flycatchers in the contact zone. Although the discovery of hybridization between Pacific-slope and Cordilleran Flycatchers in southwestern Canada calls into question their status as distinct species, the lack of evidence of gene flow into parental populations indicated a need for genetic analyses of populations representing a broader sampling of the geographic range of each species. In the remaining two chapters of the dissertation, I address two major questions:1. Does innate song function as a particularly strong isolating mechanism between incipient suboscine species?2. How do patterns of divergence in innate song differ from those of learned song, and how has song divergence affected lineage diversification across the E. difficilis-occidentalis-flavescens clade?In Chapter 2, I perform an extensive study of song variation in Pacific-slope and Cordilleran Flycatchers to determine its effects on hybridization and gene flow. Suboscines offer interesting opportunities to investigate the effects of song divergence on lineage diversification, because songs develop without learning. When songs diverge between populations, they can create behavioral barriers to gene flow. Divergence in innate song in suboscine passerines could result in particularly strong behavioral barriers to gene flow because song type is more closely correlated with genotype, and thus a more direct marker of lineage affiliation than in passerine species with learned song. In this chapter, I demonstrate high levels of introgression in both mitochondrial and nuclear genetic markers, although the pattern of introgression is asymmetrical, with introgression limited into core Pacific-slope populations. Moreover, I highlight extensive geographic discordance between the frequencies of mitochondrial and nuclear markers. I demonstrate that the songs of the two taxa are distinct, and highly correlated with nuclear genotype, and that the songs of admixed individuals exhibit spectral characteristics intermediate to the parental species. Song playback experiments demonstrate that both species show some level of discrimination based on song, and highlight lineage-specific behavioral differences that have likely affected the outcome of secondary contact. Pacific-slope Flycatchers seem to rely more on song in territorial interactions, and may discriminate more among song types. Cordilleran Flycatchers exhibit higher levels of aggressiveness in response to playback. Based on the pattern of geographic variation in genetic and song characters, and on the results of the playback experiments, I propose a historical scenario of secondary contact in which asymmetrical introgression of nuclear alleles was facilitated by the social dominance of more aggressive Cordilleran Flycatchers. Finally, I predict that Cordilleran populations will become increasingly introgressed, while introgression into Pacific-slope populations may be limited by a combination of ecological and behavioral factors. In Chapter 3, I extend the examination of genetic and song variation to include the six principal taxa that comprise the Empidonax difficilis-occidentalis-flavescens clade, to examine whether song divergence has affected lineage divergence by acting as a species discrimination trait. Numerous studies have shown that the level of complexity can affect the efficacy of song in birds, but these studies have focused mainly on birds with learned song. In this study, I offer a novel approach for examining patterns of vocal repertoire evolution, by comparing within repertoire complexity (syllable diversity) across homologous vocalization types present in all six taxa. I find varying rates of song divergence across taxa and across latitude. Songs are distinct between some taxa, but not others. Song divergence is not correlated with mtDNA distance, but it is correlated with latitudinal distance between taxa. Song complexity seems to be higher in higher latitude migratory taxa, but this is due the extremely divergent song of one taxon (E. d. difficilis). Moreover, a high level of divergence in one particular vocalization (Song 2) is most responsible for the overall divergence of E. difficilis song. Song playback experiments show varying levels of discrimination among song types, and there is at least preliminary evidence that lower latitude species are able to use more subtle vocal cues in taxon recognition than higher latitude migratory taxa. This study provides a unique view into how vocal repertoires can evolve in birds, and how this relates to lineage diversification. Moreover, this study is consistent with other studies that have found elevated rates of signal diversification and song complexity in higher latitude migratory species.Thus, innate song does seem to be able to function as a strong isolating mechanism, but this depends on ecological and behavioral contexts. That is, the abbreviated breeding seasons at higher latitudes might drive more extreme reproductive behaviors such as singing and territorial defense compared to lower latitude taxa. This can affect the effectiveness as song as a taxon discrimination trait, by affecting both the rate of evolution of song and the behavioral context in which the song is performed. Morevoer, I found that patterns of song divergence correlate closely to patterns of genetic divergence in the comparison of song divergence and admixture between Pacific-slope and Cordilleran Flycatchers, but that varying rates of song divergence relative to genetic divergence in the broader phylogenetic comparison make this correlation weaker. Song seems capable of a high level of consistency across relatively large genetic distances or a high level of divergence over relatively small genetic distances, indicating that in at least some of these taxa, song divergence has been driven by selection. Claiming that I have evidence that song differences drove lineage diversification would be premature, but song differences do seem to have important roles in maintaining taxon boundaries. Despite the high level of admixture between Pacific-slope and Cordilleran Flycatchers at interior sites, gene flow does not occur at any significant level into core Pacific-slope populations. Multiple lines of evidence suggest that Pacific-slope song has become highly derived, relative to the songs of other taxa in this clade and that it has a greater, or at least more varied, role in reproduction. Thus, attributing an important role to song differences in decreasing gene flow from Cordilleran populations into core Pacific-slope populations seems reasonable. Moreover, the high level of discrimination in lower latitude taxa among very similar song types suggests that song could be an effective cue for assortative mating in these taxa. This comes with the caveat that the context that often exists in secondary contact zones, such as low population density, can promote hybridization in taxa that would likely mate assortatively if the cost of mate searching were lower.