My dissertation research utilizes next-generation sequencing (NGS) technology and associated bioinformatics processing to answer systematic and evolutionary questions in Opiliones (harvestmen) at different taxonomic scales, focusing on the Laniatores superfamily Travunioidea and particularly the travunioid genus Sclerobunus. Opiliones are a diverse group of arachnids with over 6500 described species distributed on every continent except Antarctica. Despite relatively high diversity (e.g., more described species than mammals), harvestmen are poorly studied.

This dissertation research has three main projects. The first chapter is a higher-level phylogenetics and taxonomic study of the Travunioidea, a clade of ~80 species of harvestmen distributed throughout the Holarctic. Here I utilized ultraconserved elements (UCE) for phylogenomic reconstruction using multiple types of phylogenetic reconstruction methods. Based on results, a new taxonomic classification is proposed for the Travunioidea, including the identification and diagnosis of a new family, and I reassess the phylogenetic utility of morphological characters used to differentiate and diagnose travunioid taxa. The second chapter focuses on species delimitation of the western North American travunioid genus Sclerobunus. I utilized modern integrative taxonomic methods, using both discovery-based and validation-based approaches by combining morphometrics, mitochondrial genetic data, genitalic morphology, and nuclear genetic data derived from newly developed genes based on a comparative transcriptomics approach. This research resulted in a revision of the genus including synonymy of the genus Cyptobunus, elevation of four subspecies, and the description of five new species. The third chapter is a phylogeographic analysis of Sclerobunus robustus, a species distributed throughout the southwestern United States. A hypothesis-based framework was adopted, where stable habitats (i.e., potential refugia) were identified through ecological niche modeling, and hypotheses regarding genetic patterns associated with these refugia were developed. Hypotheses were tested by using genetic data in the form of loci and SNPs derived from double-digest RAD sequencing methods. Two large refugial regions were identified and population genomic analyses supported the presence of both.

Convergent evolution of a trait can occur at interspecific or intraspecific levels. Traits that are convergent within species are predicted to have a similar genetic basis due to recently shared ancestry. However, it is unclear how likely it is to find similar genetics during cases of rapid evolution or evolution on historic timescales. My dissertation examines evidence for phenotypic and genetic convergence in guppies, which are found in recently-introduced and historically-existing sites that differ in predation regime. I use ornamental tail coloration as it varies highly among developmental stages within an individual, males and females, individuals within a site, and between sites.

In Chapter one, I demonstrate that color develops in a consistent order among males from different predation regimes and I propose that early maturing males are the ideal stage for future experiments. In Chapter two, I determine that black melanin-related genes are likely candidates for genetic differences among fish varying in the presence of ornamental coloration. In Chapter three, I find evidence that phenotypic color pattern differences during rapid evolution follow a mixture of unique and similar trajectories based on current selective environment experienced. The color gene differences exhibit partial molecular convergence, indicating that guppies maintain variation within and between sites. In Chapter four, I discover phenotypic color pattern similarity among independently derived drainages and predator communities with substantial genetic divergence. I find a lack of molecular convergence in the identity of differentially expressed color genes among geographic locations. Taken together, these results suggest that the scale at which convergence is examined is very important. Sites may exhibit an overall signal for convergence, but may also follow slightly different trajectories upon closer examination. Convergent evolution of a trait should not be considered as being entirely present or not present, but rather a mixture of contingent and similar evolutionary processes acting at the level considered.

Silk production has independently evolved in several insect lineages, such as Hymenoptera (bees, ants and wasps), Siphonaptera (fleas), and Archeognatha (bristletails and silverfish). Primarily composed of proteins, silks are used for functions, such as protection, prey capture, dispersal, and reproduction. Because of the ecological significance of silks, natural selection can act on silk fibers and thus the underlying silk proteins (fibroins). Lepidoptera (moths and butterflies) and Embioptera (webspinners) are two insect orders that utilize silk mostly for protection. Characterizing fibroins from distantly related insect lineages may reveal convergent evolutionary patterns and provide insight into the functional elements of their fibroins. Within a lineage, conserved characteristics may yield a better understanding of silk evolution and fiber formation.

I obtained fibroin sequence data through silk gland cDNA libraries of Lepidoptera and Embioptera. This was accomplished through construction of species-specific silk gland libraries for the lepidopteran Hepialus californicus (ghost moth), and five embiopteran species: Antipaluria urichi, Archembia n. sp., parthenogenic Haploembia solieri, Oligotoma nigra, and Saussurembia davisi. After screening the libraries, transcripts for fibroins were identified for each species. Hepialus californicus heavy chain fibroin and light chain fibroin were compared to other lepidopteran and trichopteran heavy and light chain fibroins. Analyses revealed that both heavy and light chain fibroins have historically experienced stabilizing selection and light chain fibroin has potential as a phylogenetic marker. For the embiopteran fibroins, many similar features were found to be shared across species, such as glycine/serine rich, repetitive motifs, and a short, non-repetitive carboxyl-terminal region. Conservation of these elements suggests that these regions are important for fiber formation and mechanical properties. Furthermore, the conserved carboxyl-terminal regions imply that embiopteran fibroins have undergone stabilizing selection. Additional characterizations were performed on embiopteran silks to determine amino acid composition, secondary structural conformations, and mechanical properties. These features were found to have a high degree of similarity across taxa, suggesting that embiopteran silk has remained largely unchanged since it arose over 100 million years ago.

The evolution of adaptively significant gene families is an important subject in the field of evolutionary genetics. For spiders, the gene families encoding silk proteins have received considerable attention due to the high performance capabilities of the fibers they produce. However, silk gene research has largely focused on spiders of the infraorder Araneomorphae, leaving much of the phylogenetic diversity of spiders unsampled for their silk genes. Here, I sample silk genes from spiders of the suborder Mesothelae and infraorder Mygalomorphae, which are distantly related to araneomorph spiders. Phylogenetic analyses of the spidroin genes indicate that numerous duplications occurred in the spidroin gene family after opisthotheles (mygalomorphs plus araneomorphs) split from mesotheles. However, while mesotheles appear to possess a single spidroin gene, they possess numerous copies of genes homologous to Egg Case Proteins, which are currently only known from one araneomorph species. Together these results indicate that the common ancestor of extant spiders possessed a diversity of silk genes. In addition to higher level species sampling of silk genes, I sequence repetitive and carboxy terminal regions of spidroins from all species of the trapdoor spider genus, Aliatypus. Gene tree analyses and tests of selection suggest that contrasting evolutionary forces influenced the different regions of the spidroin gene. I also investigate the expression of silk transcripts from the tarsi and silk glands of tarantulas. Tarantulas exude silk-like secretions from their tarsi, which is hypothesized to increase surface adhesion. I discovered that while known spidroin silk genes are not expressed in the tarsi, novel silk-like genes are expressed in tarantula silk glands and tarsi. Gene families of adaptive significance may also show phylogenetic signal. Here, I sample hemocyanin gene family sequences from a phylogenetically diverse sample of spider species and infer gene trees and species trees. Phylogenetic analyses reveal that despite instances of lineage specific duplication and loss of hemocyanin paralogs, hemocyanins have phylogenetic utility for most spider groups. This dissertation shows the importance of research on gene family evolution, the roles of gene families in adaptation, and the utility of gene families in phylogenetics.

This dissertation lays the foundation for a research program that highlights the important contribution that poorly-dispersing short-range endemic (SRE) organisms can have on advancing empirical and theoretical evolutionary biology. Here, SREs are represented by members of the harvestmen (Opiliones) superfamily Ischyropsalidoidea. In Chapter 1, phylogenetic analyses of an ancient rapid diversification at the base of Ischyropsalidoidea uncovers considerable incongruence across gene trees. Incongruent gene trees are not recovered in frequencies expected from a simple multispecies coalescent model, and incomplete lineage sorting is rejected as the sole contributor to gene tree conflict. In Chapter 2, an integrative taxonomy tests existing, and puts forward novel species-level taxonomic hypotheses within the Acuclavella species endemic to the inland temperate rainforest of the U.S. northern Rocky Mountains. Incongruence among taxonomic disciplines suggests rapid morphological evolution across multiple recent diversifications. Results suggest at least two undescribed species exist within inland populations of Acuclavella. Alternative combinations of incongruence across taxonomic disciplines possibly represent different stages of the speciation continuum, and suggest that different evolutionary mechanisms underlie speciation in thorn harvestmen, thus highlighting the importance of integrative species delimitation for evolutionary research. The disjunct inland temperate rainforest has become a model for recent biogeography, with a complex and recent evolutionary history suggesting that this inland forest was a refuge from Pleistocene glaciation that was compartmentalized into variously isolated pockets during the last glacial maximum (LGM). In Chapter 3, genomic-scale data are used to evaluate the evolutionary consequences of the LGM. This research finds that rivers were a major cause of refugia compartmentalization, with populations and species structured by rivers well before the LGM. Additionally, a negative correlation between elevation and heterozygosity suggests that populations were confined to lower valleys during the LGM with post-glacial colonization of higher elevations. The combined effects of rivers and glaciers paints a picture of a complex Pleistocene glacial refugia system during the LGM. This relatively high-resolution inference of past evolutionary events within an established biogeographic model system points towards the undervalued importance of using poorly-dispersing short-range endemic taxa for biogeography and phylogeography.

Spiders are very diverse and inhabit most terrestrial environments. Spiders are renowned for their silk usage, which they rely on for an array of essential, fitness-related tasks such as reproduction, dispersal and prey-capture. Spider silks are proteinaceous and are largely composed of structural proteins called spidroins. Spidroins are encoded by a gene family that has undergone dramatic proliferation. To date, whether specific molecular modifications of silks are associated with success in particular habitats is still unknown. To better understand the specializations of silk sequence and expression levels, I examined the molecular composition of spidroins from terrestrial and aquatic (marine and freshwater) spiders to determine how aquatic spiders are using their silk to thrive in wet environments. I also compared silk gene expression levels of males and females within and across species.

Sex-biased silk expression was observed in genes associated with male- and female-specific tasks (e.g., egg-case production by females, wandering for mates by males). While all females highly expressed egg-case silk genes, male silk gene expression differed across species. Comparison of silks from a semi-aquatic spider to a terrestrial counterpart did not provide obvious evidence for unique specializations of silks that function underwater. Rather, the silks of the semi-aquatic spider appear to have been preadapted for aquatic use. Characterization of silk molecules from a terrestrial cribellate spider (constructs webs with cribellar silk), led to the identification of new protein sequence motifs, including a putative gene for cribellar silk. Finally, by comparing the spidroins from multiple species that independently evolved water-association, I found that they share a sequence motif composed of hydrophobic amino acids [(GV)n]. My research also addressed some of the structural aspects of silk fibers used by different spiders in terrestrial and aquatic settings. Characterization of silks from terrestrial, aquatic, and marine semi-aquatic spiders shows that spiders have developed different silk-related strategies to match their habitat and lifestyle. In addition to contributing to an integrated understanding of spider silks, my research expands the potential of spider silk as a novel biomaterial.