The Eastern Arc Mountains of Africa have become one of the focal systems with which to explore the patterns and mechanisms of diversification among montane species and populations. One unresolved question is the extent to which populations inhabiting montane forest interact with those of adjacent lowland forest abutting the coast of eastern Africa. The Tiny Greenbul (Phyllastephus debilis) represents the only described bird species within the Eastern Arc/coastal forest mosaic, which is polytypic across an altitudinal gradient: the subspecies albigula (green head) is distributed in the montane Usambara and Nguru Mountains whereas the subspecies rabai (grey head) is found in Tanzanian lowland and foothill forest. Using a combination of morphological and genetic data, we aim to establish if the pattern of morphological differentiation in the Tiny Greenbul (Phyllastrephus debilis) is the result of disruptive selection along an altitudinal gradient or a consequence of secondary contact following population expansion of two differentiated lineages.

Results:

We found significant biometric differences between the lowland (rabai) and montane (albigula) populations in Tanzania. The differences in shape are coupled with discrete differences in the coloration of the underparts. Using multi-locus data gathered from 124 individuals, we show that lowland and montane birds form two distinct genetic lineages. The divergence between the two forms occurred between 2.4 and 3.1 Myrs ago.

Our coalescent analyses suggest that limited gene flow, mostly from the subspecies rabai to albigula, is taking place at three mid-altitude localities, where lowland and montane rainforest directly abut. The extent of this introgression appears to be limited and is likely a consequence of the recent expansion of rabai further inland.

Conclusion:

The clear altitudinal segregation in morphology found within the Tiny Greenbul is the result of secondary contact of two highly differentiated lineages rather than disruptive selection in plumage pattern across an altitudinal gradient. Based on our results, we recommend albigula be elevated to species rank.

Phenotypic variation within species is an important area of evolutionary biology, for it is this variation which the process of selection directly acts upon. However, the biological factors that contribute to the generation and maintenance of this variability are still not well understood. Part of the ambiguity arises from the occurrence of multiple layers of natural and social selection factors which may be occurring simultaneously, and at different hierarchical levels within and between populations. How these factors interact with each other, and at what level, can generate highly variable traits in which the frequency of diversification differs from the individual to the species level. In addition, in conjunction with selective factors, stochastic genetic variation can impact the evolution and maintenance of phenotypic traits in ways that we do not fully understand, dependent on features such as population size, mutation rate, and the dispersal ability of individuals.

Plumage polymorphism, where multiple forms of a trait are maintained within a species, is a widespread occurrence in many families of birds, but is especially common in birds of prey. The high prevalence of variable plumage color and pattern makes raptors an excellent system in which to study the factors that may contribute to high phenotypic diversity in a species. A large number of polymorphic birds of prey have been identified and examined, but evidence for how multiple selection and stochastic factors interact to maintain these traits has not been well-studied. To explore this question, my research focused on the occurrence and overlap of natural and social selection factors, and how they may interact with genetic stochasticity, in the American Kestrel (Falco sparverius). The American Kestrel is an ideal subject for this study, for it represents one of the most dichromatic raptors with a range of plumage variation found across the New World. In order to investigate the evolutionary factors that originate and maintain this range of plumage variation, I integrated behavioral data collected within California, with museum studies, demographic data, and an analysis of the genetic structure and clinal variation of plumage from across North America.

The American Kestrel has been known to have highly variable plumage, but little work has been conducted to examine specific plumage traits in detail. Part of the difficulty in performing this task lies in the extensive range covered by the species. Found from the highest latitudes of North America to the southern tip of South America, the American Kestrel is almost continuous in its distribution across the New World. In my first chapter I use a combination of museum specimens from multiple collections to examine the full range of the variation found in one highly variable plumage trait of male American Kestrels, the black and white coloration on the tail. By doing so I was able to identify three repeatable morphs and four levels of variation in the color of the tail for males. In my first and subsequent chapters I then use this newly defined polymorphism to examine how the variation present in this trait relates to selective and stochastic factors at both the individual and population level.

Selection through social interactions, such as mate choice and competitive interactions, has often been associated with the evolution of plumage traits in birds. Using both a captive population of American Kestrels maintained in Maryland and free-living individuals from study sites in Northern California, my first chapter examines both the heritability and differences in reproductive fitness associated with the polymorphic tail color of male kestrels. I found that the tail morph appears to change little between molt for adults, and both the morph and level of variation are heritable in the captive population. However, there appeared to be little difference in the reproductive fitness of males with different morphs or variation level in the free-living groups. Males with larger amounts of black and white were shown to nest earlier, but this result was only recovered at one of four study sites. Overall, my research detected no consistent sign of selection on the black and white polymorphic tail of American Kestrels through social interaction for breeding birds.

A lack of evidence for the use of the tail color in behavioral interactions does not mean that the trait is not under other forms of selection. A wide-ranging species could encounter a large scope of different microhabitats and ecological factors. Natural selection, such as specialization in habitat type or differing fitness under divergent climatic variables, could potentially drive the maintenance of multiple morphs if different types were specialized for different ecological areas. Such a pattern is often thought to result in clinal distribution of forms across large areas. In my fourth chapter, I used plumage data from 633 male kestrels collected during the breeding season from across North America to construct a map of the frequencies of the three morphs and four variation levels for the continent. A geographic cline was discovered, where birds along the eastern Atlantic Coast showed higher frequencies of both the pattern and level of variation with the greatest degree of black and white. Extending to the west and ending at the Pacific Coast, birds showed a much higher frequency of morphs and patterns with smaller amounts of the two colors. The geographic division in morph frequencies suggested that broad scale climatic or ecological variables might play a role in the distribution of American Kestrels with different tail morphs. However, no correlation was found between a range of bioclimatic variables and tail color. There was also found to be no relationship between morph and variation level, and broad habitat type. Thus, it appears that the highly variable tail pattern of male American Kestrels is not strongly affected by either natural or social selection factors.

The inability to discover any strong relationship between selection factors and the polymorphic tail color of American Kestrels suggests that the trait may be under weak or no selection at this point in time. In such cases, the major influences on the tail color may instead be from stochastic genetic variation, and changes in population size, genetic population structure, and the movement of birds across the landscape. In chapter three, I used demographic and genetic data from a long-term study program of American Kestrels in Saskatchewan, Canada to show that kestrels do experience rapid changes in population size. Findings also suggested that the species appears to be very fluid with its movement into local breeding areas, and is readily affected by changes in land-use and habitat. In chapter four, I turn back to the continent wide data set and compared the different morph frequencies seen with population structure constructed from 375 genetic samples of American Kestrels collected during the breeding season. The genetic structure detected separated out three populations, one in the southeast corresponding with the subspecies F. s. paulus, one along the Atlantic Coast, and a third that appeared to encompass the rest of the continent. This does not correspond exactly with the geographic cline of morph frequencies, but both the genetic and plumage data highlight that the Atlantic Coast is a unique area for the American Kestrel.

Broadly, my dissertation used a combination of research at both the individual and population level to identify a new polymorphic plumage trait in American Kestrels and to determine that the trait appears to exist in different frequencies among populations on the North American continent. While no direct association was found between tail color and natural selection factors at the broad geographical level, or social selection factors at the individual level, the findings do not rule out these areas completely. Overall, the work conducted here suggests that the factors influencing plumage polymorphism in birds of prey are complex, and that stochastic genetic variation may have a larger impact on the maintenance of plumage polymorphism then originally recognized.

Identifying the biological processes that drive phenotypic evolution is a central challenge in evolutionary biology. Achieving this goal is particularly difficult when examining complex phenotypes that are likely to be impacted by both genotype and environmental factors as well as potential interactions between these parameters. While experimental studies of reaction norms in inbred lines of lab animals can elucidate the effects of genes versus environment, such results do not encompass the breadth and complexity of naturally occurring variation in phenotypes. As a result, the implications of such studies for natural populations may be limited. An alternative approach employs a phylogeographic framework to investigate phenotypic variation among populations distributed across a range of environments. Under such a framework, the impacts of both phylogenetic relationships and environmental factors are considered, allowing researchers to explore the critical interactions between genotype and environment that generate patterns of phenotypic diversity in natural populations.

Within this phylogeographic framework, contact zones provide a natural laboratory for exploring the interplay between genotypes and environments because they place distinct genotypes in common environments. To date, complex traits remain relatively unexplored in this context. To understand the roles of genotypic and environmental factors in shaping complex phenotypes, I examined two traits - gut microbiome diversity and olfactory cues in urine - across a California vole (Microtus californicus) contact zone. Previous research suggested that California voles have diverged into two phylogenetic lineages that were thought to come into contact in Santa Barbara County. Additionally, lab studies indicated that F1 males generated by crossing these two lineages were sterile, providing potential evidence of Bateson-Dobzhansky- Muller incompatibility. This dissertation builds upon these earlier studies to examine the differentiation of complex phenotypic traits during divergence of closely related phylogenetic groups.

Towards this goal, in Chapter One I characterize the contact zone between the northern and southern lineages of M. californicus. I use a mitochondrial locus and genome-wide nuclear markers acquired from ddRAD sequencing to determine the degree of genetic divergence between these lineages and to test for evidence of hybridization. My results indicate that while both northern and southern mitochondrial genetic lineages are present in contact zone

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populations in Santa Barbara County, there is no evidence for admixture of nuclear genetic markers within this zone. This result is unexpected given the relatively recent divergence between the two lineages (8,500 - 54,000 years ago) and suggests that despite their recent origin, these lineages are not exchanging genetic materials.

In Chapter Two, I explore variation in a complex phenotypic trait – gut microbial diversity - to assess the relative contributions of phylogenetic history and current environmental variation in shaping this trait. Previous studies using lab animals have demonstrated the importance of the gut microbiome on host well being but the impact of host divergence on gut microbial diversity remains unclear. To explore this theme, I use sequence data from the bacterial 16S rRNA (V4 region) to examine patterns of gut microbial community composition across the M. californicus contact zone. Patterns of microbial diversity are assessed relative to predictions based on shared host ancestry (phylosymbiosis hypothesis), changes in gut microbial composition in cybrid (mismatched mitochondrial and nuclear genomes) hosts (hologenome speciation hypothesis), and increased microbial dissimilarity with distance (dispersal limitation hypothesis). My analyses reveal that gut microbial community structure in M. californicus is best explained by the geographic distances among host populations, as predicted by the dispersal limitation hypothesis, and by climatic factors. The relative abundance of several genera of bacteria, however, differs between voles with pure parental genotypes versus cybrids, as predicted by the hologenome speciation hypothesis. Thus, the factors contributing to overall gut microbial diversity may differ from those impacting specific baterial taxa.

Finally, in Chapter Three, I examine geographic variation in olfactory cues in vole urine across the same contact zone characterized in Chapters 1 and 2. Previous research has revealed selection against mating by individuals from different lineages of M. californicus, suggesting that cues facilitating mate choice differ between lineages. How olfactory cues vary among natural populations of voles or other rodents remains poorly understood and thus it is not known if cues in the urine of voles allow individuals to discriminate between genetic lineages. I use gas chromatography-mass spectrometry to examine the chemical profiles of urine from male voles. My data suggest that overall chemical profiles differ between populations within versus outside the contact zone, with sampling localities, and with month of collection. My analyses of individual components of urine reveal two compounds that differ between populations in the contact zone versus those within the northern lineage (outside the contact zone). This pattern is consistent with predictions based on phylogeographic divergence of vole lineages. In contrast, variation in climatic factors across the contact zone does not explain variation in urinary chemical compounds. These analyses suggest that phylogenetic dynamics between lineages may be a key determinant of variation in reproductive important olfactory cues in this species of rodent.

In summary, while variation in both phenotypic traits examined appears to be influenced by environmental conditions (e.g., geographic distance, sampling locality), evidence of genotypic (lineage) impacts on specific elements of these traits is evident. These findings underscore the importance of considering multiple aspects of phenotypic variability when assessing the interplay of genetic and environmental factors in shaping complex phenotypes.

Nectar-feeding birds have enchanted scientists for centuries due to the remarkable morphological similarities between geographically and phylogenetically distant lineages, extreme life histories, and adaptations to a sugary diet. Though many characters have been attributed to the success of this ecological niche, no singular adaptation is shared across all radiations. Nectarivores range greatly in terms of body size, bill size and shape, tongue morphology, ecology, feeding behavior, and diet composition. Here, I assess nectarivory in birds as a syndrome through the lenses of feeding morphology, genetics, and nutritional ecology. I sought to better understand the evolution of nectarivory by investigating three different aspects of nectar-feeding adaptations: (1) tongues for transporting nectar into the throat; (2) the evolution of genes underlying bill morphology; and (3) the ecology of ethanol in the diets of birds. In chapters 1 and 2, I explore the skeletal morphology of the feeding apparatus and the genetic underpinnings of bill size and shape in Sunbirds (Passeriformes: Nectariniidae). In Chapter 3, I measure the prevalence of ethanol, a by-product of the fermentation of the sugars in nectar, with a focus on birds that feed on nectar and fruit. Specifically, I compare the prevalence of dietary ethanol within several species of hummingbirds and between hummingbirds and other bird species that serve as exemplars of other diet categories.In Chapter 1, I describe the tongue apparatus in sunbirds and explore the patterns of size and shape variation in the morphology of the skeletal elements using several phylogenetic comparative methods. When compared to their non-nectarivorous relatives, sunbirds have evolved a highly modified tongue apparatus to feed on nectar, yet sunbird species vary in the degree to which they specialize on nectar. To better understand the evolution of sunbird tongues, I first described the sunbird tongue apparatus and the function of key components by reconstructing the skeletal elements and associated soft tissues in situ of an Olive Sunbird (Cyanomitra olivacea). The functional significance of sunbird tongue morphology in nectar-feeding was hypothesized by comparing it to previous studies of avian tongue anatomy and function. Then I investigate patterns in morphological diversity across sunbirds and the relationship between size and shape, phylogeny, and feeding ecology by reconstructing the tongue skeleton from 41 CT scans representing sunbirds (Nectariniidae), flowerpeckers (Dicaeidae), and their relatives (Irenidae, Chloropseidae, Modulatricidae, Promeropidae). Two-dimensional and three-dimensional morphometrics were used to quantify size of the individual skeletal elements and the shape of the basihyal-urohyal. Two-dimensional data were supplemented with previously published bill measurements and body masses in the AVONET dataset. The morphology of the Olive Sunbird tongue apparatus supports previous hypotheses that sunbirds feed on nectar by generating a negative-pressure space (hermetic suction) with the tongue body and upper palate. I also describe a structure on the paraglossalia unique to sunbirds that likely provides structural support to the tongue body. The morphometric results suggest a complex interplay between bill size, tongue size and shape, and diet (as a proxy for feeding behavior) that is partially driven by phylogenetic history. Diet predicts shape depending on how body mass is included as a co-variable. Lastly, I compare the tongue apparatus between other nectarivorous radiations - hummingbirds and Australian honeyeaters - and discuss the differences in form and function of nectar-feeding adaptations across birds. In Chapter 2, I explore whether known candidate genes for bill morphology are implicated in the evolution of sunbird bills. Selection is expected to have occurred at the ancestral branch of sunbirds after the split from flowerpeckers (Dicaeidae) which coincides with an increase in bill length. I surveyed genes previously implicated in studies of bill size and shape. I generated sequence data of 11 candidate genes and phylogenetically informative sites for 139 taxa using a target capture method, including sunbirds (Nectariniidae), flowerpeckers, and several outgroups. Using a multilocus phylogeny constructed from the same individuals, exonic regions of these genes were tested for positive selection at the site level and at the branch level. I found evidence of episodic positive selection at a total of 28 sites across the protein ALX1, BMP4, LPP, RNASEH2B, and SHH. Fourteen sites were detected in LPP alone, six of which occurred in one exon, and seven were detected in RNASEH2B. No statistically significant, elevated rates of evolution was detected for the ancestral branch of sunbirds, nor for any other clades with elongated beaks. Although positive selection was not detected where I hypothesized, the results do not preclude these genes potentially being implicated in phenotypic changes in other ways (e.g., changes in gene expression). Chapter 3 focuses on the nutritional ecology of feeding on nectar. Ethanol is a naturally occurring molecule produced via fermentation of sugar-rich foods by yeast. Birds that regularly consume fruit and nectar may accordingly be ingesting ethanol on a chronic basis, although this possibility has not been documented in the wild. However, ethanol catabolism is followed by production of secondary metabolites such as ethyl glucuronide (EtG), which can be measured to provide a retrospective view of dietary exposure. We hypothesized that avian species with diets rich in fermentable sugars consume non-trivial amounts of ethanol, and therefore accumulate detectable levels of EtG. Assays for the presence of EtG in avian feathers yielded positive results for 10 of 17 avian species; EtG was also present at substantial levels in the livers of two of five study species. We found that EtG was present in nectarivorous hummingbirds, but also in species in other trophic niches (3 granivores, 1 omnivore, 3 invertivores, and 1 vertivore). Dietary exposure to ethanol may thus be much more widespread than has previously been recognized, and diverse features of avian nutritional ecology (e.g., secondary consumption via ingestion of prey items) may contribute to its accumulation in tissues.

Researchers have sought to understand the link between the avian skull and dietary preferences for centuries by studying vision, brain size, beak shape, and the complex evolutionary relationship of the beak relative to the cranium. One group of birds of recurring interest to researchers are the raptors. The term ‘raptor’ colloquially encompasses three orders: Accipitriformes, Falconiformes, and Strigiformes. This study provides a comprehensive comparison of species among the diurnal raptors: Accipitriformes and Falconiformes, including the skulls of 150 species. It is one of the largest studies of the overall morphology of these two orders to date and lays the groundwork for further research into the diet, function, anatomy, taxonomy, and morphological diversity of living and extinct raptors. All three chapters rely on the use of 2D geometric morphometrics to draw conclusions about shape variation in the skulls of raptors.

In Chapter 1, I investigated the effect of the rhamphotheca on the shape of the bird beak in raptors by comparing the shape of the beak with and without a rhamphotheca in the same species. This chapter provides answers to the question of whether a combination of skulls with and without the rhamphotheca can be used in geometric morphometric analyses of skull shape. The rhamphotheca adds length and creates a sharper point in the beaks of raptors, but the extent to which the shape of the beak changes compared to the bone underneath had not been investigated. A geometric morphometric analysis of five different species of raptors from different orders and families found that the rhamphotheca does differ significantly from the shape of the underlying bony beak in every case. This has implications for the study of bird beaks when the rhamphotheca isn’t available, as is often the case when looking at fossil birds. These results also resulted in the exclusion of rhamphotheca from the analyses of skulls in chapters two and three.

In Chapter 2, I explored the general morphology of the raptor skull using 2D landmarks of skulls from the Accipitriformes and Falconiformes. This chapter details the morphology of the raptor skull, what features scavenging members of each order have in common, and how scavengers and non-scavengers differ morphologically. Scavenging has a significant effect on the overall shape of the skull, driving the evolutionary difference between the families and subfamilies of the Accipitriformes and Falconiformes. The results of this chapter strongly suggest the presence of a feeding guild structure within the accipitrid and cathartid vultures and point to the progressive evolution of ‘vulture-like’ traits in the Polyborinae subfamily of the Falconiformes.

In Chapter 3, I analyzed the scavenging members of each order in greater detail, to discuss and reevaluate the guild structures proposed by Kruuk (1967) and Hertel (1994). I also evaluate the inclusion of the caracaras into this guild structure, based on geographical distribution and known overlap with the vultures of the Accipitriformes. Chapter 3 also discusses the shape of the vulture skull through time by including fossil specimens from the La Brea and McKittrick tar pits. Using the knowledge about existing scavenger guild structures, I apply this knowledge to fossil and extinct species of raptors and scavengers of the Pleistocene. The statistical results from this study support the existence of the guild structures proposed by Hertel (1994), with only minor differences with respect to which species fit within each guild. The caracaras may comprise their own guild structure independent of cathartid and accipitrid vultures, but more observational data needs to be collected in order to draw firm conclusions.

The island of Madagascar and surrounding volcanic and coralline islands are considered to form a biodiversity hotspot with large numbers of unique taxa. The origin of this endemic fauna can be explained by two different factors: vicariance or over-water-dispersal. Deciphering which factor explains the current distributional pattern of a given taxonomic group requires robust phylogenies as well as estimates of divergence times. The lineage of Indian Ocean scops-owls (Otus: Strigidae) includes six or seven species that are endemic to Madagascar and portions of the Comoros and Seychelles archipelagos; little is known about the species limits, biogeographic affinities and relationships to each other. In the present study, using DNA sequence data gathered from six loci, we examine the biogeographic history of the Indian Ocean scops-owls. We also compare the pattern and timing of colonization of the Indian Ocean islands by scops-owls with divergence times already proposed for other bird taxa.

Results:

Our analyses revealed that Indian Ocean islands scops-owls do not form a monophyletic assemblage: the Seychelles Otus insularis is genetically closer to the South-East Asian endemic O. sunia than to species from the Comoros and Madagascar. The Pemba Scops-owls O. pembaensis, often considered closely related to, if not conspecific with O. rutilus of Madagascar, is instead closely related to the African mainland O. senegalensis. Relationships among the Indian Ocean taxa from the Comoros and Madagascar are unresolved, despite the analysis of over 4000 bp, suggesting a diversification burst after the initial colonization event. We also highlight one case of putative back-colonization to the Asian mainland from an island ancestor (O. sunia). Our divergence date estimates, using a Bayesian relaxed clock method, suggest that all these events occurred during the last 3.6 myr; albeit colonization of the Indian Ocean islands were not synchronous, O. pembaensis diverged from O. senegalensis about 1.7 mya while species from Madagascar and the Comoro diverged from their continental sister-group about 3.6 mya. We highlight that our estimates coincide with estimates of diversification from other bird lineages.

Conclusion:

Our analyses revealed the occurrence of multiple synchronous colonization events of the Indian Ocean islands by scops-owls, at a time when faunistic exchanges involving Madagascar was common as a result of lowered sea-level that would have allowed the formation of stepping-stone islands. Patterns of diversification that emerged from the scops-owls data are: 1) a star-like pattern concerning the order of colonization of the Indian Ocean islands and 2) the high genetic distinctiveness among all Indian Ocean taxa, reinforcing their recognition as distinct species.