Monkeypox virus (MPXV), a zoonotic DNA virus, is now considered the most important orthopoxvirus infection in human populations since the eradication of smallpox. The role of reservoir host species is highly important in this zoonotic disease due to the large percentage of human infections attributed to wildlife origins. As a disease that infects multiple hosts, limited knowledge of MPXV prevalence across host species and the relative importance of each host species to disease maintenance makes it difficult to predict human risk and target disease management efforts. In this study, we screened over 1000 museum skin specimens collected from 1899 to 1993 for viral DNA to understand historical MPXV infection in various Funisciurus species across the Congo Basin. MPX viral DNA was found in 93 of 1038 (9.0%) specimens from as early as 1899 and in five different Funisciurus species: Funisciurus anerythrus, Funisciurus carruthersi, Funisciurus congicus, Funisciurus lemniscatus, and Funisciurus pyrropus. We identify two new potential host species, gain insight into the relative prevalence rates of infection within museum skins between various Funisciurus species, and examine the spatial and temporal distribution of monkeypox virus in these possible host species.

Unraveling the unique ways anthropogenic effects impact the genomes of populations, and applying such genomic knowledge to improve conservation policy, is paramount for effective preservation and management of biodiversity in our changing world. The central African country of Cameroon, widely recognized as a global conservation priority, is a prime landscape to study these issues. Here, I explore genetic and epigenetic variation between natural and human-altered habitats in the Cameroonian sunbird Cyanomitra olivacea. I use SNP data and methylation data to contrast patterns of genetic and epigenetic variation, finding that methylation frequency levels shift with changes in environment, and are higher overall in human-altered habitats. Then, I develop a decision-making framework to enable more effective research-to-policy translation, and apply it to a particular case study: preserving adaptive genetic variation in Cameroon. This study is one of the first to explore genetic and epigenetic variation in concert, and reveals the potential ways organisms adapt to anthropogenic change. It is also an attempt to bridge the much-maligned science-policy gap and develop policy tools to bolster conservation.

Species across the globe are faced with unprecedented pressures due to human impacts. In what is now recognized as the Sixth Mass Extinction, habitat loss and climate change have driven many populations to and over the brink of extinction. Drawing from recent advances in genomic sequencing and analyses, I aimed to facilitate the conservation and recovery of two declining species, the Tricolored Blackbird (Agelaius tricolor) and the Western Burrowing Owl (Athene cunicularius hypugaea). I analyzed population structure and genetic diversity in both species, the former with both reduced representation and whole genome sequencing and the latter with low coverage, whole genome data. For the Tricolored Blackbird, I found no indications of population structure in the species, an important finding for conservation as this indicates there is neither a need for individual management units for the species nor a concern about barriers to gene flow. Burrowing Owls are more complicated in having both resident and migratory breeding groups. I found genetic structure was linked to the migratory phenotype, with residents being highly structured and migrants having no structure. Furthermore, I found indications of high inbreeding in resident breeding populations. Using numerous metrics of environmental variation, I found significant genetic differences between residents and migrants. Among those regions of the genome most differentiated and correlating with environmental differences, genes associated with the processing of fats, or lipophagy, were found to be significantly more represented. Revisiting Tricolored Blackbirds with whole genome data, I found indications that perhaps the population is not in complete panmixia as previously indicated by reduced representation data analyses, but that any restrictions to gene flow are indeed low. Further, the higher resolution dataset detected that the recent known decline of the species is apparent in changes in effective population size over time. My work provided essential information for conservation efforts for both species and should be a foundation for continuing genomics research on these and other species of conservation concern as well.

How new species evolve is one of the most fundamental questions in biology. In particular, there has been considerable debate about the relative roles of genetic drift and natural selection in speciation. A salient example is the controversy surrounding the origins of Amazonian biodiversity. The Pleistocene has long considered one of the most important periods of diversification in the Amazon. According to the Pleistocene refugia hypothesis rainforest contracted into small refugia and genetic drift played the dominate role in driving speciation. However, the centers of diversity that would have provided evidence for these supposed refugia were never found. In addition, phylogenies and fossil records show that most lineages are older than the Pleistocene, refuting this simple allopatric model of speciation. Although multiple mechanisms such as geographical barriers, marine incursions, and ecological factors have been hypothesized to explain the Amazon's hyperdiversity there have been relatively few tests of these hypotheses.

Here I examined the importance of ecological factors in driving population divergence in the lesser tree frog (D. minutus) between the Amazon and Cerrado and along the gradient between these two biomes. Substantial temperature and precipitation differences between these biomes raise the possibility that environmental factors drive population differentiation, potentially resulting in speciation.

In chapter one, I examined the importance of environmental variables in driving reproductive divergence in the lesser tree frog. Water availability and temperature emerged as important drivers of body size variation, with body size found to be negatively correlated with vocal traits. For frogs, vocalization is one of the most important traits for mating success. Thus, these results suggest that the contrasting selective pressures on body size between the Amazon and Cerrado influence vocalization, which may result in reproductive divergence and suggests how reproductive isolation may evolve as a result of divergent natural selection pressures in these distinct habitats.

In chapter two, I evaluated the relative roles of genetic drift and natural selection in driving divergence in the lesser tree frog along the Amazon-Cerrado gradient. I showed that environmental variables are more important than geographical distance for driving divergence in reproductive and morphological traits. This suggests that ecological differences along the gradient result in adaptive divergence. Such divergence in phenotypic traits indicates the dominant role of natural selection over neutral processes for speciation events in this region.

Finally, in the third chapter, I investigated the sources of phenotypic variation in response to environmental variation between the Amazon and Cerrado biomes. The goal was to understand the contributions of genetic and environment to D. minutus phenotypic variation in larval development between these two biomes. I conducted a common garden experiment to test whether phenotypic variation in D. minutus has a genetic component, whether tadpoles from the Cerrado develop faster due to a more seasonal habitat, and whether D. minutus shows plastic responses to different thermal conditions. This experimental work showed that both environment and genetics play a role in phenotypic variation. Moreover, tadpoles from Cerrado populations developed faster than those from the Amazon populations. This is indicative of local adaptation to a more seasonal habitat by the Cerrado populations.

Examining patterns of biodiversity in the Tropical Andes provides insight into ecological and evolutionary processes leading to accumulation of lineages in montane regions. In the Tropical Andes, the dramatic changes in topographic and environmental conditions over short distances constitute an ideal system for studying morphological adaptation and geographic discontinuities. These patterns of biodiversity along elevational gradients in the Tropical Andes remains intriguing because there is no comprehensive theory that explains, species richness, high levels of endemism, and the extraordinary variation in traits that are ecologically relevant, in a region that occupies less than 1% of global surface. In particular, I examine phylogenies, eco morphology and population genetics of birds in the Tropical Andes.

In chapter one, I use phylogenetic-based methods to assess slowdown in evolution of body size, changes among lineages in elevational-range overlap and rate-shifts in cladogenesis. Interspecific phylogenetic comparative analysis of the Core Tanager clade supports an overall density-dependent cladogenesis consistent with tenets of adaptive radiation. In chapter two, I use explicit spatial hypotheses of the distribution of the Andean Purplish-Mantled Tanager for present-day and to the past ice-age 18,000-22,000 years ago to test for concordant patterns between population genetic structure using microsatellites loci and mtDNA sequence variation, and potential effects of climate change. Topographical and environmental determinants explain a large proportion of spatial genetic variation in this tanager, but demographic history does not appear to be responding to the expansion of suitable area since last ice-age. In the third chapter, I use coalescent methods for a multilocus data set to examine demographic history between of The Olive-striped and Streak-necked Flycatcher (the montane Mionectes) and between populations for each of these species. The amount of differentiation between species that barely overlap ranges along elevation is similar to the differentiation observed between populations within the same species across the Andean ranges. Because divergence times broadly overlapped between and within the same species, discordant patterns of differentiation in plumage and vocal traits between populations within the same species as compared with distinctive plumage and vocal phenotypes between species cannot be simply explained by rapid differentiation due to genetic drift alone.

Gene regulation is tightly regulated to produce individual phenotypes appropriate to a animal’s placement in time and space. How specific genetic and environmental factors shape gene regulation has been extensively studied in laboratory model species. However, far less is known about sources of variance in gene regulation in the wild, in populations where natural selection operates. My dissertation research aimed to identify factors that influence individual variation in gene regulation by evaluating transcriptome-wide gene expression levels within and across populations. In my first chapter, I evaluated gene expression changes in captive Swainson’s thrushes (Catharus ustulatus) originating from two populations as they transitioned from the non-migratory to migratory condition. Birds exhibited significant seasonal differences in the expression of genes involved in cellular development in the brain, providing support for seasonal neural plasticity in migratory birds. In my second chapter, I focused on a single population, the North American gray wolves (Canis lupus) of Yellowstone National Park, to evaluate the relative impacts of intrinsic (age and sex) and environmental variables (social status and presence of sarcoptic mange) on gene expression variation in blood. I found that, unexpectedly, animal age, but not sex, social status, or sarcoptic mange infestation, impacted gene expression, suggesting that age may have pervasive, evolutionarily conserved effects on gene expression variation in natural populations of mammals. Finally, in my third chapter, I adopted an in vitro experimental approach to study how gene regulation is impacted by genotype at the CBD103 gene, which encodes a protein with antimicrobial properties, exists in two forms (alleles) in North American gray wolves, and is associated with lifetime reproductive success. I established keratinocyte lines from 24 gray wolves and utilized CRISPR/Cas9 to generate one heterozygous and homozygous line. To test whether the fitness effects of CBD103 genotype are explained by their roles in immune defense, I challenged the cells with synthetic antigens and live canine distemper virus. No effect of CBD103 genotype was detected, but immune challenge altered expression of thousands of genes. This work demonstrated the ability to establish a population panel of cell lines from a wild mammal, a potentially powerful method for studying gene regulation in natural populations.

We live on a predominantly microbial planet. I it is estimated that more than a billion microorganisms can live in a gram of soil. Microorganisms comprise the largest pool of genetic diversity on the planet and drive global biogeochemical cycles. Since microbial ecology is intimately associated with environment, changes in environmental conditions can have profound effects on the microbial diversity and function of microbial communities. In this dissertation I study; 1) the relationship between environmental heterogeneity and microbial diversity, 2) the relationship between the environment, microbial diversity, and microbial functional traits, and 3) microbial secession related to changing environmental conditions during anaerobic decomposition.

Chapter 1

Annual grassland invasions can increase environmental heterogeneity and reduce the biological diversity of plants and animals. There is a generally positive relationship between environmental heterogeneity and biodiversity, and more specifically, soil heterogeneity is known to influence plant diversity. Here I test if the diversity of soil microorganisms, like that of plants, displays a positive relationship with soil environmental heterogeneity. Specifically, I test to see if invasive annual grasses lead to reductions in soil heterogeneity and microbial alpha- and beta- diversity. I sampled the soil profile across invasive annual grassland, oak woodland, and coastal sage scrub habitat and characterized environmental heterogeneity (soil percent carbon, nitrogen, water content, total dissolved solids, and pH in addition to litter percent carbon, nitrogen and C:N), alpha and beta diversity. I found that invasive annual grassland habitat has greater soil environmental homogeneity than native woody habitats throughout the soil profile. Annual grassland communities have lower alpha-, but not beta-, diversity than native woody species. Patterns of alpha diversity with depth differ between grassland and woody habitat, and although not significant, woody habitats have higher community heterogeneity. Alpha diversity and beta diversity show positive relationships with several measures of environmental heterogeneity, suggesting that like plants, soil microbial diversity increases with environmental heterogeneity. Annual grassland invasions into native woody habitats reduce soil microbial diversity. This is particularly true in deep soil communities.

Chapter 2

Plant invasions frequently alter ecosystem processes in part because they modify soil microbial communities. These communities decompose the bulk of terrestrial organic matter by producing and releasing extracellular enzymes. California's native Mediterranean habitats (e.g. Oak woodland and coastal sage scrub) are invaded by annual grasses and are converted to invasive annual grasslands. I investigated the relationship between extracellular enzyme activities and microbial community composition in these habitats by examining 1) how extracellular enzyme activities differed between native and invasive habitats, 2) whether changes in microbial community correlate with changes in extracellular activity, and 3) if the composition of bacterial phyla that contain genes for extracellular enzymes differ between habitats. I found that annual grassland enzyme activities are much different from those of woody habitat, and the differences in enzyme activities between habitats generally declined with depth as did enzyme activities. There was also a strong correlation between community composition and extracellular enzyme activity. This correlation was not influenced by soil environmental variables. The relative abundance of phyla with genes for extracellular enzymes were similar between habitats and those genes are contained in distinct assemblages of phyla. Habitat change through annual grassland invasion modifies soil communities and their functions thought out the soil profile. Future studies on the effects of annual grassland invasion on ecosystem processes in deep soil are needed to fully understand the consequences of these invasions.

Chapter 3.

Natural tar seeps are the source of millions of fossils from animals that became entrapped, died and were decomposed over the millennia. The microbial communities responsible for the anaerobic decomposition of these entrapped animals are not known. However, microbial communities likely play a role in the rapid time to skeletonization of animal components submerged in tar. I hypothesized that high energy animal tissue would support fast growing taxa and support lower microbial diversity, and that microbial succession across different locations in the tar environment and animal tissue decay would resemble known patterns of microbial decomposition in similar habits. I sampled different locations in a tar seep and also bobcat limbs that were experimentally submerged in the seep and left to decay until skeletonization. Microbial communities were characterized using 16S rDNA sequencing of the V4 region. I found that decay communities had lower diversity than tar environment communities and that microbial succession proceeded similarly to that in analogous habitats. The addition of animal tissue into this tar seep appeared to lead to rapid microbial community succession. This microbial succession likely affected the rate of decomposition of this tissue. Future experiments are required to understand the role of microbial succession in determining the rate of decomposition and time to skeletonization in tar environments.

This doctoral dissertation focuses on genomic approaches to study evolution and adaptation in birds. Recent advances in next-generation sequencing (NGS) technology and analytical tools are making the production of genomic data more accessible than ever before. As such, the accumulation of genomic data is occurring at an ever-increasing pace. This genomic data harbors a trove of information waiting to be unlocked. For birds, an initiative called the Bird 10,000 Genomes Project strives to produce genomes for all living species of birds. This growing availability of bird genomes is fueling our ability to understand the genetic architecture underlying evolutionary and adaptative processes. In my dissertation, I use genomic data and analytical tools to understand color evolution and genomic signals of climate adaptation in birds. My first two chapters explore color and visual system evolution in birds. In the first chapter, I introduce the R package, charisma, for categorizing color from digital images for high-throughput analyses of color evolution. Current color categorization of digital images often entails manual classification of color for each image or a single estimate of colors for the entire group. I present charisma a novel efficient and standardized method to identify biologically relevant colors in thousands of images. In the second chapter, I explore the evolution of avian visual system sensitivity. I align and trim SWS1 opsin sequences to predict the visual sensitivity of 418 bird species across the bird tree of life. I estimate rates and reconstruct ancestral states of visual system sensitivity. I found that a violet-sensitive visual system is ancestral in birds and that an ultraviolet-sensitive visual system has evolved at least 18 times across the bird tree of life. In the last two chapters of my dissertation, I present a reference genome and investigate climate adaptation in Yellow Warblers, Setophaga petechia. In my third chapter, I present a highly contiguous reference genome assembly for Yellow Warbler using HiFi long-read and Hi-C proximity sequencing technologies. I generated a 1.22 Gb assembly including 687 scaffolds with a contig N50 of 6.80 Mb, scaffold N50 of 21.18 Mb, and a BUSCO completeness score of 96.0%. This high-quality reference provides a key resource for understanding gene flow, divergence, and local adaptation and informing conservation management. In my fourth chapter, I investigate genomic signals of climate adaptation in Yellow Warblers in California. Climate change is an ongoing threat to biodiversity and species are being forced to respond or face extinction. This response is dependent on their ability to adapt to rapidly changing environments. I use a whole genome sequencing approach to examine genomic signals of local adaptation in California breeding Yellow Warblers to identify populations most vulnerable to climate change. Despite low genetic structure in Yellow Warblers breeding in California, I identified unique genotype-environment associations and 2,972 putatively adaptive single-nucleotide polymorphisms across 137 individuals. This study highlights the importance of understanding neutral and adaptive genetic variation in bird populations.