Cytochrome c oxidase subunit 1 (Cox 1) is the barcode for many animal groups, protists, and macroalgae. Previously in fungi, the efficiency of Cox 1 as a genetic marker was only analysed in Penicillium subgenus Penicillium and Leohumicola spp. In this thesis, two species isolated from the intestinal tracts of caterpillars from Costa Rica, and two potential species complexes, P. sclerotiorum and P. oxalicum belonging to Penicillium subgenus Aspergilloides and Furcatum, were studied using the Genealogical Concordance Concept (Gee) recognition criterion and barcoding methods. Analyses with beta-tubulin (BenA), the nuclear internal transcriber spacer (ITS) region, Cox 1, translation elongation factor 1-alpha (TEF1-alpha), and calmodulin (CaM) revealed that the Penicillium species isolated from Costa Rica are undescribed, and that P. sclerotiorum is a complex of seven phylogenetic species (including the Costa Rican species) that fit the prevailing morphological concept of P. sclerotiorum. The phylogenetic species were compared and newly discovered diagnostic morphological characters were used to create a taxonomic key to the species of the complex. The new species are formally described as P. guanacastense, P. mallochii, P. krugii, P. cainii, P. jacksonii and P. ciebiessum. Analyses of multiple strains of P. oxalicum revealed that it is a single phylogenetic species, despite having a world wide distribution, an unusually high degree of morphological variation, and a diversity of ecological roles. Cox1 proved to be a good barcode for identifying the selected Penicillium groups, and provided a species level resolution of 83.3%. ITS provided the same resolving ability. BenA (91.7%), TEF1-alpha (100%) and CaM (100%) provided a higher species level resolution than Cox1, but BenA, TEF1-alpha, and CaM were difficult to amplify or sequence for some Penicillium groups. A secondary barcode marker is suggested in addition to Cox1 for Penicillium.

Astragalus robbinsii var. fernaldii (Fernald's Milkvetch) is a federally listed taxon of "special concern" due for re-assessment by COSEWIC. Restricted to the Strait of Belle Isle region of Atlantic Canada, its taxonomy in respect to two co-occurring congeners, Astragalus eucosmus and Astragalus robbinsii var. minor, has been questioned due to the variable states of the varieties' diagnostic characters. To clarify phylogenetic relationships within this species complex we studied variation in chloroplast DNA, AFLPs and morphology. Chloroplast DNA sequence data distinguished A. r. var. minor from A. eucosmus and most A. r. var. fernaldii. Three A. r. var. fernaldii populations possessed the A. r. var. minor haplotype, and AFLPs revealed no population structure, indicative of gene flow between taxa and populations. Morphometric analysis indicates that A. r. var. fernaldii is closer to A. eucosmus than A. r. var. minor. Based on these results taxonomic recognition of A. r. var. fernaldii is unwarranted, and we recommend that the taxon be merged into A. eucosmus.

<p> The brachyuran superfamily Palicoidea &Scaron;tev&ccaron;i&cacute;, 2005, commonly referred to as stilt-crabs, currently includes the families Crossotonotidae Moosa and Ser&eacute;ne, 1981 and Palicidae Bouvier, 1898, consisting of two and nine genera, respectively. Both genus and species level relationships remain largely enigmatic and are based primarily upon highly variable morphological characters. Molecular phylogenetic analyses, based on COI and 16S mitochondrial gene sequences, are used to clarify relationships among primarily western Atlantic species and to independently validate diagnostic morphological characters. The resultant molecular phylogenetic tree resolves three major clades: one grouping <i>Palicus affinis, Palicus alternatus,</i> and <i> Palicus bahamensis;</i> a second grouping <i>Palicus faxoni</i> and <i>Palicus obesus;</i> and the third grouping <i>Crossotonotus </i> sp., <i>Pseudopalicus</i> sp., <i>Palicus cristatipes, </i> and <i>Palicus sica.</i> Putative specimens of <i> P. floridanus</i> were positioned as sister species to other groups. Molecular phylogenetic evidence infers intrageneric evolutionary history of <i> Palicus</i> Philippi, 1838, concordant with relationships suggested by gonopod morphology of congeners. Gonopod morphology was found to be highly conserved within species, moderately conserved among species sharing a common clade, and divergent among species in different well-separated clades. Conversely, many morphological characters that have historically been applied to describe and identify palicids were found to be highly variable within species, inconsistently variable among species, and in some cases relatively conserved across divergent clades. On the basis of present molecular phylogenetic analyses, separation of the families Crossotonotidae and Palicidae may be supported only if further revisions to membership of the family Palicidae were to be undertaken. These revisions are deferred pending more robust genetic analyses.</p>

<p> The integration of advances in computing technology with major innovations in sequence data collection and phylogenetic inference has revolutionized evolutionary biology in the 21^st century. In particular, the continual development of both theory and software that allow for more flexibility in utilizing molecular clock methods has radically transformed our understanding of the mode and tempo of diversification across the Tree of Life. Over the course of five chapters, this dissertation explores methodological challenges to phylogenetic inference with the aim of better understanding the evolutionary history of the Holocentridae (squirrelfishes and soldierfishes). </p><p> Chapter 1 begins by focusing on the problem of accommodating clade specific rate heterogeneity in molecular clock analyses. While various nucleotide substitution models have been developed to accommodate among lineage rate heterogeneity, recently developed "uncorrelated relaxed clock" and "random local clock" models are predicted to perform better in the presence of lineage specific rate heterogeneity as these models relax assumptions of inheritance of nucleotide substitution rates between descendant lineages. Using simulations and two cetacean (whale and dolphin) datasets as a case study, we demonstrate abrupt changes in rate isolated to one or a few lineages in the phylogeny can mislead rate and age estimation, even when the node of interest is calibrated; and provide suggestions for diagnosing extreme clade specific rate heterogeneity.</p><p> Homoplasy is another important, yet often overlooked, source of error in phylogenetic studies. Chapters 2 and 3 utilize phylogenetic informative approaches to screen nucleotide sequence data for homoplasious site patterns. Using phylogenetic informativeness profiles, chapter 2 reconciles two competing hypotheses of ray-finned fish divergence times by highlighting that mitogenomic based Jurassic and Triassic divergence time estimates for most major lineages of spiny-rayed (acanthomorph) fishes were an artifact of tree extension. Evolutionary relationships of early diverging acanthomorph fishes are also contentious, with molecular data supporting either holocentrids or a clade comprised of holocentrids and primarily deep-sea fishes as the sister lineage to the species-rich percomorpha. Chapter 3 reveals this conflict to also be largely driven by homoplasy and reconciles results based on previously published data with a 132 gene next-generation sequence dataset to identify the sister lineage of percomorph and the phylogenetic placement of holocentrid fishes.</p><p> Chapter 4 continues to explore holocentrid evolutionary relationships. Using a multi-locus dataset that includes all but one holocentrid genus, this chapter provides the first molecular phylogeny of the group. The systematics of holocentrid fishes has unstable for over 100 years. We demonstrate several of the key synapomorphies for holocentrid genera are in fact homoplasious. Likewise, several genera of holocentrine (squirrelfish) are rendered consistently paraphyletic by a series of maximum-likelihood and Bayesian analyses and we propose taxonomic revisions to reflect shared ancestry.</p><p> Chapter 5 further investigates the temporal history of holocentrid evolution. Contemporary holocentrid species richness is concentrated in the Indo-Australian Archipelago (IAA), yet these fishes also represent some of the most numerous fossil taxa in deposits of the Eocene West Tethyan biodiversity hotspot. Using likelihood-based methods integrated with a molecular timetree that incorporates fossils as tip taxa, we reconstruct the history of range evolution for these fishes. Following the collapse of the West Tethys, holocentrids exhibit a signature of increased range fragmentation, becoming isolated between the Atlantic and Indo-Pacific Ocean basins. However, rather than originating within the emerging IAA hotspot, the IAA appears to have acted as a reservoir for holocentrid diversity that originated in adjacent regions over deep evolutionary timescales. By integrating extinct lineages, these results provide a necessary historic perspective on the formation and maintenance of global marine biodiversity. </p>

<p> Viral infection and the subsequent immune responses such as the expression of interferon beta (<i>ifnb1</i>) show extreme levels of cell to cell variability. A fraction of cells get infected and a fraction of infected cells induce an <i>ifnb1</i> response. These responding cells then signal to coordinate appropriate immune responses required to clear infection. The mechanism of propagation of this response at the single cell level is critical to generate an appropriate defense against the virus, yet is incompletely understood. </p><p> Interesting work on cell to cell variability has been done using transfected <i>ifnb1</i> reporter constructs. However, this approach has several limitations. The reporter systems introduce multiple copies of the reporter construct in each cell, which does not reflect the conditions in the intact cells where only two of the interferon promoter and gene are present. This alters the <i>ifnb1</i> enhanceosome stoichiometry from the one present in a normal physiological environment, and potentially distorts the patterns of single cell responses observed. In addition, reporter constructs integrate the response that occur over many hours, which makes it difficult to measure the expression dynamics that occur early after exposure to infection. Such measurements in the intact cells would be helpful for understanding the mechanisms underlying the propagation of this immune response. </p><p> In order to obtain sensitive and accurate measurements of changes in gene expression in infected single cells, we used single-cell single-molecule mRNA imaging to directly and simultaneously count the transcripts of <i> ifnb1</i> and that of a virus (Newcastle disease virus) gene Hemagglutinin-neuraminidase hn . This experimental approach enabled us to measure the single-cell responses from the very early stages of infection, in primary immune cells. Simultaneous measurement of <i>ifnb1</i> and the viral gene hn high lighted the variation in responses across cells, the temporal evolution of the expression of the two genes and their single cell correlation following infection. </p><p> We find that the single cell <i>ifnb1</i> response to virus infection shows a temporally dispersed (asynchronous) pattern. A small fraction of infected cells respond very early and more <i>ifnb1</i> expressing cells are recruited at later time after infection. In contrast, the single <i> ifnb1</i> response to the toll like receptor stimulant LPS, follows a highly synchronous pattern, where in a large number of cells showed an <i> ifnb1</i> response around the same time after treatment. These results suggest that the temporal evolution of single cell <i>ifnb1</i> responses was likely dependent on the type of the inducing stimuli. Furthermore we observed that extracellular signaling plays an important role in introducing cell-to-cell variability in <i>ifnb1</i>gene induction in response to virus infection. Inhibition of extracellular signaling converted the response to virus infection into an early synchronous LPS-like response. Thus extracellular signaling shapes the temporally dispersed pattern of single cell <i> ifnb1</i> response to viral infection. </p><p> The pattern of <i>ifnb1</i> responses to virus infection involves an increase in the amplitude of the response per cell as well as an increase in the number of responding cells over time of infection. These properties may enable cells to fine tune the <i>ifnb1</i> responses gradually. This strategy of mounting an antiviral cytokine response may be useful in calibrating the immune response such that an appropriate antiviral response is generated and cellular toxicity resulting from excessive cytokine expression is usually avoided.</p>

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 71-02, Section: B, page: . Adviser: Chao Tang. Includes supplementary digital materials.

<p> The Lesser Sunda Archipelago, also known as Nusa Tenggara, lies in the southeastern portion of Indonesia and extends between Bali in the west, and New Guinea in the east. While the Lesser Sundas themselves are oceanic islands that have never been land bridged to a continent the islands on either side do. Bali and the other Greater Sunda Islands of Java, Sumatra, and Borneo become periodically land bridged with Asia during glacial maxima forming the Sunda Shelf. New Guinea and Aru become periodically land bridged to Australia during glacial maxima and form the Sahul Shelf. Given their current orientation, the Lesser Sundas may act as &lsquo;stepping stones&rsquo; for animals and plants dispersing between the Sunda and Sahul Shelves and may act as a two-way filter for organisms dispersing between two of the world&rsquo;s great biogeographical realms. Alfred Russel Wallace&rsquo;s discovery of a pattern of clinal mixture of species from different biogeographical realms was a key insight leading to his identification of the Wallace Line and to his creation of the field of biogeography. Even though the Lesser Sundas played a critical role in the development of the field, this region has received little subsequent attention from historical biogeographers and our current understanding of Lesser Sunda biogeography has only modestly improved relative to what was known at the time of Wallace. The reptiles and amphibians of the Lesser Sundas represent a particularly interesting group of vertebrates from a biogeographical standpoint because they appear to show distributional patterns that are most consistent with a stepping-stone model of island colonization caused by the two-way filter zone. In Chapter 1, I review the geological and biogeographical literature for the Lesser Sundas and use these sources to formulate hypotheses concerning the colonization of the archipelago by rafting terrestrial vertebrates. In Chapters 2 through 4, I investigate the possibility that flying lizards, forest skinks, and fanged frogs have colonized the archipelago in a stepping-stone manner using a phylogenomic approach (using sequence data from mtDNA and hundreds of nuclear loci) whereby the relationships among island-specific lineages can be used to infer the sequence of island colonization. Flying lizards of the genus <i>Draco</i> form a monophyletic group that colonized the western Inner Arc islands of Lombok or Sumbawa from the Sunda Shelf around 10 million years ago when Lombok and Sumbawa first became land-positive. <i> Draco</i> continued expanding eastward through the Inner Arc until they reached Lembata, while a series of dispersal events from Flores south to Sumba, east to Timor, north to Wetar, west to Alor, and finally west to Pantar (the island immediately west of Lembata). The islands of Sumbawa and Flores contain multiple non-sister lineages that are parapatrically distributed and are exchanging migrants within an island. Forest Skinks of the genus <i> Sphenomorphus</i> show relatively little morphological divergence across their range yet exhibit large levels of genetic divergence. The oldest lineages of <i>Sphenomorphus</i> within the Lesser Sundas occur on the islands of Lombok and Flores and they expanded eastward through the Inner Arc until they reached Pantar. But rather than reaching Alor from neighboring Pantar, <i> Sphenomorphus</i> dispersed from Flores south to Sumba, then east to Timor, Alor, and Wetar. There are multiple non-sister lineages of <i> Sphenomorphus</i> on Lombok, Flores, and Sumba, and estimates of migration between lineages within each island suggest that these lineages are not interbreeding. Fanged frogs of the genus <i>Limnonectes</i> have colonized the Inner Arc of the Lesser Sundas from the Sunda Shelf. It is possible that <i> Limnonectes kadarsani</i> and <i>L. dammermani</i> diverged <i> in situ</i> on Lombok after which <i>L. kadarsani</i> dispersed east all the way to Lembata. But rather although a tree topology consistent with a stepping-stone pattern of island colonization is suggested by the mtDNA data, the phylogenomic results suggest a leap-frog pattern where Lembata is derived from West Flores, and these two lineages are closer related to Sumbawa than they are to Eastern Flores. The parapatrically distributed lineages on Flores are experiencing asymmetrical gene flow with successful migrants moving from west to east. In summary, the oldest islands of the western Inner Arc tend to harbor the most divergent lineages for all three focal taxa, a pattern expected from lineages originating from the Sunda Shelf. In <i>Draco </i> and <i>Sphenomorphus,</i> the islands of the eastern Inner Banda Arc are colonized by way of the &lsquo;Sumba Route&rsquo; where they disperse into the Outer Banda Arc island of Sumba and then move east to Timor, and finally north into the eastern Inner Arc. All three focal taxa show multiple non-sister lineages on some of the larger islands, suggesting either that multiple colonization events of a single island occurred, or possibly that formerly separated paleo-islands have since merged allowing for secondary contact of lineages that diverged in allopatry. These studies have shown that the biogeography of reptiles and amphibians within the Lesser Sundas is extremely complex. By examining biogeographical patterns across many co-distributed taxa these studies have the potential to provide insights into the geological history of the archipelago. From an evolutionary perspective, these studies highlight the presence of multiple independently evolving lineages within a currently described species occurring on the same island, which suggests that species diversity within reptiles and amphibians of the Lesser Sundas is underestimated.</p>

<p> This study investigates the eIF4E family members in Dinoflagellates. Dinoflagellates are eukaryotic algae with large genomes and a minimal role for transcriptional regulation. All mRNA in dinoflagellates is <i>trans </i>-spliced with a 22-nucleotide 5'-spliced-leader sequence bearing a multi-methylated cap. Like other eukaryotes, dinoflagellates encode multiple eIF4E family members that are anticipated to fulfill a range of functions. Three distinct and novel clades of eIF4E have been recognized in dinoflagellates that are separate from the three metazoan classes of eIF4E. The dinoflagellate <i> Amphidinium carterae</i> encodes eight eIF4E family members while <i> Karlodinium veneficum</i> encodes fifteen eIF4E family members. I assayed six of these family members from <i>A. carterae</i> for expression levels, m⁷GTP binding, yeast knockout complementation and affinity for three mRNA cap analogs using surface plasmon resonance (SPR). Transcripts of each are expressed through a diel cycle, but only eIF4E-1 family members and eIF4E-2a are expressed at the level of protein. Recombinant eIF4E-1 family members and eIF4E-3a, but not eIF4E-2a, are able to bind to m⁷GTP-agarose beads. Of the clade 1 eIF4Es, only eIF4E-1a and -1d1 complement a S. cerevisiae strain conditionally deficient in functional eIF4E, consistent with their function as translation initiation factors. However, only eIF4E-1a can be recovered from <i>A. carterae</i> extracts by m⁷GTP affinity binding. Using SPR analysis, the affinity of <i>A. carterae</i> eIF4E-1a for m⁷GTP is lower than that of murine eIF4E-1A. By the same analysis, <i>A. carterae</i> eIF4E-1a has a higher affinity for m⁷GpppG than m⁷GTP. In addition, <i>K. veneficum</i> eIF4E-1a1 displays many of the same characteristics as <i>A. carterae</i> eIF4E-1a. Four eIF4E-1 and one eIF4E-2 family members from <i>K. veneficum</i> were characterized for m⁷GTP binding capacity, only the eIF4E-1 family members can be pulled down with m⁷GTP. Three eIF4E family members were tested for their ability to interact with a putative eIF4E-interacting protein, although none interacted. Overall, the eIF4E-1a sub-clade emerges with characteristics consistent with the role of a prototypical translation initiation factor. These initial analyses will allow for a better understanding of specific translational control of gene expression through mRNA recruitment in the unique dinoflagellate lineage.</p>

<p> The genus <i>Ophiocordyceps</i> contains the most diverse assemblage of fungi attacking ants worldwide and are remarkably well adapted to the specific ecologies of their hosts. <i>Desmidiospora myrmecophila</i> is closely related to ant-pathogenic species within <i>Ophiocordyceps</i>, possibly specific to queens, but the sheer infrequency of encounters and previously unsuccessful attempts to culture this fungus has precluded any meaningful assessment until now. A new record of <i>Desmidiospora myrmecophila</i> from Louisiana was found infecting a foundress <i>Camponotus pennsylvanicus </i> queen, the same host species favored by the more common and ubiquitous ant-pathogenic <i>Ophiocordyceps</i> unilateralis found in the same geographic locality. To evaluate a long-held assumption that these fungi represent synanamorphs of a single species, we sampled our Desmidiospora specimen along with the local <i>O. unilateralis</i> population for molecular comparison. We are able to present for the first time the <i>in vitro </i> characteristics and morphology of <i>Desmidiospora myrmecophila</i> as well as a phylogenetic context for this fungus based on combined molecular analysis of representative members of the Ophiocordycipitaceae. Our results place the <i>Desmidiospora myrmecophila</i> lineage within the genus <i>Ophiocordyceps</i> but with a basal affiliation to the ant-pathogen clade. These results further implicate <i>Desmidiospora myrmecophila</i> as an important and quintessential example of cryptic diversity among an already taxonomically diverse and ecologically important group of fungi.</p><p>

<p> Plumage color evolution in birds has been the focus of theoretical and empirical research on sexual selection since Darwin. Many of the yellow, orange, and red hues seen in bird plumage are the result of carotenoid pigmentation. While a great number of recent studies have examined the functions of carotenoid-based plumage coloration in a single species, few have examined the evolutionary history of this trait in a comparative phylogenetic context. Using the New World blackbirds as a model clade, I focus on two questions that a comparative phylogenetic approach can uniquely address. First, what is the history of evolutionary change in carotenoid color that led to the colors seen in extant blackbird taxa? Second, by what proximate mechanisms have carotenoid pigments evolved? In Chapter 1, I present an ancestral state reconstruction of carotenoid-based plumage coloration across the Icterid phylogeny, based on reflectance measurements of museum skins. My results show robust evidence that red coloration was gained repeatedly from a yellow common ancestor. In Chapter 2, I used pigment biochemistry of meadowlark (<i>Sturnella</i>) and Cacique (<i>Cacicus</i>) feathers to test whether independent gains of red coloration are the result of parallel or convergent metabolic mechanisms. Meadowlarks have evolved red coloration using a different set of carotenoids than caciques, but the caciques have evolved the same set of carotenoids twice. This suggests that red coloration evolved by convergent evolution among different blackbird clades, but evolved by parallel evolution within the caciques. Lastly, in Chapter 3 I examine the relationship between color and carotenoid pigmentation in orioles, a blackbird clade in which orange has been gained at least twice independently from a yellow common ancestor. I found red-producing keto-carotenoids only in orange species and never in yellow species. This result is a striking contrast to our expectation for a continuous gradient of a carotenoid pigment concentration. These results suggest that repeated gains of C4-oxygenation ability best explain evolutionary changes in orange coloration in orioles. To summarize, I showed using phylogenetic comparative methods that blackbirds have repeatedly evolved towards redder carotenoid coloration. Using HPLC biochemistry, I showed that each of these gains of orange and red coloration is likely the result of a gain of C4-oxygenation ability. The prevalence of gains of orange and red coloration suggests that there may be a directional bias towards evolving longer-wavelength carotenoid plumage. The research presented in these chapters provides the phylogenetic framework necessary for future studies to examine the functional causes underlying the repeated evolution of carotenoid-based coloration.</p>