Biology Theses and Dissertations
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Item GENETIC AND MOLECULAR ANALYSIS OF GERMLINE SEX DETERMINATION IN CAENORHABDITIS BRIGGSAE, A MODEL FOR THE CONVERGENT EVOLUTION OF HERMAPHRODITISM(2010) Doty, Alana V.; Haag, Eric S.; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Though sex determination and differentiation are critical biological processes, genetic mechanisms that specify sex have undergone profound and rapid evolutionary change across taxa. We may be able to infer processes that generate sex determination diversity by examining closely related species. Within the nematode genus Caenorhabditis, two species, C. elegans and C. briggsae, are androdioecious, producing self-fertile hermaphrodites and males; other Caenorhabditis species generate males/females. Interestingly, phylogenies reveal that C. elegans and C. briggsae independently acquired hermaphroditism, a relatively rare adaptation among animals. In this work, I describe differences in germline sex determination between C. elegans and C. briggsae that may help reveal the molecular basis of their convergent evolution of hermaphroditism. I first describe mutations in the pleiotropic, STAR family RNA-binding protein Cbr-GLD-1 that affect germline sex in C. briggsae. I find that C. briggsae gld-1 mutant hermaphrodites have a sex determination phenotype opposite to that of C. elegans: masculinized versus feminized germlines. I demonstrate that Cbr-GLD-1 coding-plus-regulatory sequences can rescue Ce-gld-1 null animals, arguing that this change in sex determination is not due to changes in GLD-1 function or expression. I further show that gld-1's role in regulating oogenesis is conserved across the Elegans group of Caenorhabditis, demonstrating that the oogenesis function of gld-1 is likely ancient, whereas its sperm-repressing role in C. briggsae has evolved recently. To identify mRNA targets of Cbr-GLD-1 that might be responsible for its sex determination function in C. briggsae, I use an in vivo genome-wide approach to isolate mRNAs associated with Cbr-GLD-1, including potential sex determination targets. I identify 800 putative mRNA targets and confirm specificity of this gene set via qRT-PCR and RNAi. Next, to reveal the roles of GLD-1 in evolutionary context, I create a phylogeny of STAR proteins across metazoans. Finally, I characterize a single feminizing allele recovered through forward screens in C. briggsae for germline sex determination mutants. This work thus begins to dissect the molecular and genetic basis of hermaphroditism in C. briggsae and contributes to a growing body of research on the evolution of germline sex determination differences between C. elegans and C. briggsae.Item Elucidating the Macro- and Micro-evolutionary Relationships of the Federally Listed Endangered Species Agalinis acuta (Orobanchaceae)(2010) Pettengill, James Beaton; Neel, Maile C; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Agalinis acuta (Orobanchaceae) is a federally listed endangered plant species native to the mid-Atlantic and northeastern coastal plains of the United States. Due to morphological ambiguity and molecular similarity between A. acuta and Agalinis tenella and Agalinis decemloba a conservation priority is to determine whether A. acuta represents an evolutionarily distinct entity worthy of protection under the Endangered Species Act. To resolve this question, a phylogenetic study was first conducted based on seven chloroplast DNA loci and the nuclear DNA locus ITS from 79 individuals representing 29 Agalinis species. A study evaluating the utility of those cpDNA loci and three analytical techniques for the purpose of DNA barcoding was also conducted. The phylogenetic study indicated that A. acuta was perhaps evolutionarily indistinct from A. decemloba and A. tenella. Based on the results of subsequent analyses of 21 microsatellite loci and morphological data evaluated under myriad species concepts, A. acuta, A. decemloba, and A. tenella best represent a single species with two subspecies; the former two putative species would constitute a subspecies called A. decemloba ssp. decemloba and A. tenella would be A. decemloba ssp. tenella. With evolutionary distinct entities described, a phylogeographic study was conducted to determine the extent to which historical processes rather than contemporaneous events can explain extant patterns of genetic and phenotypic diversity within A. decemloba. The dispersal of a few individuals out of southern refugial populations likely represents the process through which northern populations were established; however, recent anthropogenic effects that disproportionately affected northern populations may have also contributed to extant patterns of diversity. Neutral or adaptive explanations for phenotypic variation among populations are also investigated. The conservation implications of population genetic analyses were assessed for members of A. decemloba ssp. decemloba. Despite the evidence that this taxon is self-compatible, the high levels of inbreeding and low levels of heterozygosity are of such a magnitude in certain populations that genetic factors may be negatively impacting fitness. Because of the small effective population sizes and degree of isolation, all populations should be managed to reduce the risk of extinction associated with demographic and environmental stochasticity.Item Genetic Differentiation of Selected Eastern Box Turtle (Terrapene carolina) Populations in Fragmented Habitats, and a Comparison of Road-based Mortality Rates to Population Size(2009) Hagood, Susan; Adams, Lowell; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The decline of eastern box turtle populations is associated with habitat loss and fragmentation, lack of recruitment into breeding populations, removal of individuals from the wild for pets, and road mortality. Box turtle populations in many areas of the eastern United States may effectively be isolated as high traffic volumes on roads adjacent to turtle habitats prevent successful dispersal. If so, populations surrounded by heavily used roads may be less genetically diverse than those in relatively intact habitats. I investigated whether populations in three Montgomery County, Maryland parks that were surrounded by roads were genetically differentiated relative to populations in two larger habitats in Maryland's Prince George's and Anne Arundel counties. Sampling was conducted between 2005 and 2008. I used 10 microsatellite markers to compare these populations, and sampled in an additional five sites (two in Maryland, and one each in Pennsylvania, the District of Columbia, and Florida), to better assess population structure. I found little evidence of genetic differentiation among central Maryland populations regardless of the extent of isolation. I attribute these findings to the slow rate of change in turtle evolution; the observed similarities in genetic diversity may reflect past rather than present gene flow. I found moderate to great differentiation in populations separated by substantial distances. To determine whether road mortality exceeds additive mortality levels believed to be a threat to population persistence, I estimated population size in the three Montgomery County, Maryland, parks using mark-recapture techniques, and compared these estimates to the number of dead, injured, and live turtles in or very near roads observed during walking and driving surveys conducted in 2006. Road-based morality rates fell within the range estimated to be inconsistent with population growth in one of the parks. Road mortality appeared to affect females out of proportion to their abundance in the population. Suggestions for reducing box turtle mortality in areas associated with high mortality rates are included.Item Myosins IIIa and IIIb regulate stereocilia length by transporting espin 1 to the polymerizing end of actin filaments(2009) Merritt, Raymond Clyde; Kachar, Bechara; Popper, Arthur N; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mutations in the myosin IIIa gene are linked to DFNB30 late onset deafness, in which those affected individuals can hear for the first twenty years of life. Mutations in the espin gene are linked to DFNB36 congenital deafness. Both myosin IIIa and espin 1 are expressed in the inner ear hair cells and colocalize at stereocilia tips, the site of actin polymerization. Overexpression of these proteins in hair cells produce an increase in stereocilia length, and when both are co-expressed together they produce a length increase greater than when each one are overexpressed individually. These results suggest that these proteins interact and influence stereocilia length regulation. We confirmed the interaction of these two proteins in heterologous COS-7 cells. We observed that when co-expressed, these proteins promote elongation of filopodial actin protrusions in a synergistic manner. Mutational analyses show that myosin IIIa3THDI binds to the ankyrin repeats of espin 1. Live and fixed cell imaging shows that myosin IIIa transports espin 1 to the filopodia tips where espin 1 promotes actin polymerizations through its WH2 domain. Because of the late onset of deafness associated with myosin IIIa, it has been speculated that the lack of myosin IIIa function is partially compensated by the paralogous protein, myosin IIIb. Myosin IIIb, encoded by a distinct gene, lacks a C-terminal actin-binding domain shown to be essential for myosin IIIa filopodia tip localization. We observed that myosin IIIb localizes at stereocilia tips and is expressed at an earlier stage than myosin IIIa. We confirmed that myosin IIIb transports espin 1 to stereocilia tips and promotes actin polymerization, consistent with the hypothesis that it partially compensates for myosin IIIa. We found that binding to espin 1 is required for myosin IIIb movement and localization. We observed that myosin IIIb can downregulate the myosin IIIa localization in vestibular, but not in cochlear, hair cells. The interplay of myosins IIIa, IIIb, and espin 1 and their influence on stereocilia length unravels a novel molecular complex at the polymerizing end of F-actin and a framework to understand the cause of DFNB30 and DFNB36 deafnesses.Item MATING COSTS, MALE CHOICE DISPLACEMENT, AND THE EFFECTS ON HYBRIDIZATION AND SPECIATION IN THE HAWAIIAN CRICKET LAUPALA (SUBFAMILY:TRIGONIDIINAE)(2009) Jadin, Jenna; Shaw, Kerry L; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Contact zones between two closely-related species provide unique laboratories for studying the processes of speciation. This is because, within these zones, species barriers will be reinforced and speciation will reach completion, or the barriers will break down, causing the two species to become one. Which of these two alternatives will occur depends on the degree of genetic differentiation and behavioral isolation between the species. If there is significant and non-combinable genetic variation between species, but behavioral isolation between the two incipient taxa is incomplete and allows hybrid offspring to be produced, these hybrid offspring will have lower fitness relative to parental types and selection should act directly to eliminate those offspring and indirectly against parents with broad mating preferences or traits. If however the genetic architecture is similar and behavioral isolation is incomplete, the populations would be expected to turn into a hybrid swarm and eventually become one species. Patterns of behavioral isolation and genetic variation in several Laupala species pairs suggest that contact zones between closely related species are marked by conflicting patterns of behavioral isolation and genetic differentiation. Evidence also suggests that the complex courtship system of Laupala may allow male choice to play an important role in sexual selection and speciation. Therefore I tested several hypotheses about the genetic differentiation, sexual selection, and behavioral isolation in a contact zone between the closely-related and morphologically indistinguishable L. tantalus and L. pacifica species pair. First, by using the mitochondrial COI gene and AFLPs as genetic markers, I demonstrated that there appears to be mitochondrial DNA introgression between sympatric, but not allopatric congeners, which suggests contemporary hybridization in the contact zone. Next, I found that males experience post-mating resource-limitation and show a significant tendency to invest less into a second mating, however, their investment is dependent upon female size. Finally, I found that there is apparent displacement of male choice, decreased variation in spermatophore production, and asymmetrical mating isolation within the contact zone. This evidence all suggests that there is increased behavioral isolation in this contact zone, which may be consistent with a hypothesis of speciation by reinforcement. However, this evidence also suggests that male costs may result in male choice conflicting with other isolating mechanisms. If so, this study may be another putative case of reinforcement, or it may be an entirely novel report of conflicting selection pressures within a hybrid zone. I suggest that further studies are needed to measure hybrid fitness as well as to evaluate relative male and female mating costs within the complex mating system of this rapidly-diversifying genus.Item THE MITOCHONDRIAL GENOME OF THE BLUE CRAB (CALLINECTES SAPIDUS), AN INFORMATIVE GENETIC MARKER FOR THE EVOLUTIONARY BIOLOGY AND POPULATION GENETICS OF THE SPECIES(2009) Feng, Xiaojun; Place, Allen R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The blue crab (Callinectes sapidus) is a widely distributed decapod which ranges from Nova Scotia to the northern Argentina coasts. It is one of the most abundant estuarine invertebrates, supporting both commercial and recreational fisheries along the Atlantic and Gulf coasts. This thesis presents data clearly establishing the unprecedented hyper-variability in the mitochondrial genome of C. sapidus. This variation extended to multiple regions, including the cox1, nad2, and nad4 protein coding loci as well as ribosomal 12s RNA molecule. The haplotype diversity of the nad2 gene approached 1, with a nucleotide diversity approaching 1%. This hyper-variability in the mtDNA allows using a single mtDNA gene (nad2) to distinguish hatchery-produced crabs from wild crabs after release to the wild. I found no dominant mtDNA haplotypes in wild populations but instead a distribution of a few low-frequency recurrent haplotypes with a large number of singletons. Because of this high diversity and extensive population mixing, the geographic structure in wild populations exhibits panmixia from the Atlantic to Gulf of Mexico. Some of the high genetic diversity found seems to stem from the heteroplasmic nature of the blue crab mtDNA. By cloning high fidelity PCR products, I confirmed single individual crab and megalopa harbored dozens of copies of mitochondrial haplotypes. A copy number analysis indicates discovery of unique haplotypes was probably not saturated with the possibility of inadequate sampling. The heteroplasmy in the blue crab appears to be under maternal inheritance without paternal contribution. While minor haplotypes are represented in wild populations, other minor haplotypes contained stop codons and/or non-synonymous substitutions which may influence the viability of the mitochondria. Given the blue crab inhabits a broad variety of environments and that the mtDNA genome appears to be under selective pressure, the potential for mtDNA functional correlates with this genetic diversity maybe at the basis for the robust physiological capability of the species.Item CRYPTIC DIVERSITY, ECOLOGICAL DIFFERENTIATION AND POPULATION GENETICS OF AN ESTUARINE COPEPOD, ACARTIA TONSA DANA 1849 (COPEPODA: CALANOIDA)(2009) Chen, Gang; Hare, Matthew P; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Surprising genetic diversity has been discovered in marine holoplankton, organisms that "drift" in water currents throughout their life cycle. This discovery challenges our assumptions and suggests that holoplankton species may have limited dispersal and/or have adapted to small-scale oceanographic features. In this study, I investigated population genetics of Acartia tonsa, a holoplanktonic estuarine copepod containing deeply-diverged mitochondrial lineages, on the United States Atlantic coast. The study goals include: 1) assessing its cryptic species/genetic diversity; 2) inferring evolutionary and geographic origins of its cryptic lineages; 3) testing environmental associations of cryptic lineages; 4) inferring evolutionary and ecological processes/mechanisms underlying population diversification of A. tonsa. Phylogenetic analyses of DNA sequences from two gene loci, mitochondrial cytochrome c oxidase subunit I (mtCOI) and nuclear ribosomal internal transcribed spacer (nITS), resolved five morphologically cryptic, genetically diverged lineages that were reproductively isolated species based on genealogical concordance principle. Three co-distributed, deeply-diverged mtCOI lineages (X, S, F) showed significant population differentiation within lineages and contrasting phylogeographic patterns among lineages. Population structures and isolation by distance patterns detected for all lineages suggested that dispersal of Acartia lineages was more or less limited to adjacent estuaries; geographic isolation was a key mechanism underlying population diversification of A. tonsa. The highly diversified, relatively recent lineage F demonstrated a southern center of origin in Florida with northward stepwise diversification. Its distinct localized population structure and strong association with low-salinity environments suggested that environmental stressors (such as salinity) could act as physiological barriers to gene flow, facilitating diversification of Acartia populations. Co-existing Acartia lineages were parapatrically distributed along the estuarine gradient across systems on the US Atlantic coast. Genetic, morphological and ecological evidence indicated niche partitioning and ecological differentiation of A. tonsa within estuaries. Multiple factors may have contributed to the observed parapatric distribution and niche partitioning, including selection by salinity, biological competition, and/or local adaptation. These findings in one of the best known estuarine copepods reinforce the general conclusion that marine biodiversity is substantially underestimated, not only in terms of species numbers, but also with respect to niche partitioning and the potential importance of ecological divergence in marine holoplankton.Item LEUNIG, LEUNIG HOMOLOG, AND SEUSS ARE TRANSCRIPTIONAL CO-REPRESSORS THAT REGULATE FLOWER DEVELOPMENT, MUCILAGE SECRETION, AND PATHOGEN RESISTANCE(2009) Bui, Minh; Higgins, William J; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Transcriptional repression is an important regulatory mechanism for development. My thesis focuses on dissecting the function of Groucho (Gro)/Transducin-Like Enhancer of split (TLE) family of transcriptional repressors in plant development. My work characterizes two Arabidopsis thaliana genes, LEUNIG (LUG), first discovered to repress transcription of the floral homeotic gene AGAMOUS (AG), and LEUNIG_HOMOLOG (LUH), a gene with the highest sequence similarity to LUG. To investigate the functional redundancy between LUG and LUH, I constructed and analyzed lug; luh double mutants, and concluded that both LUG and LUH repress AG expression in the flower, with LUG playing a more prominent role than LUH. The double mutant also revealed a previously unknown function of LUG and LUH in embryogenesis because lug-3; luh-1 double mutants are embryo lethal, while the single mutants develop normal embryos. During the course of this study, I developed a new genotyping method called Simple Allele-discriminating PCR (SAP), which is cost-effective, quick, and easy to perform. This method has greatly facilitated my research as well as others in the lab. A second part of my thesis addresses the role of LUG and LUH in other developmental processes besides flower development. My data indicate that these two genes, like their counter parts in fungi and animals, act as "global co-repressors" in various developmental and physiological processes. My thesis work revealed that both co-repressors, together with its interacting protein SEUSS (SEU), repress the Salicylic Acid (SA) pathogen defense pathway. Although lug-3, luh-1, and seu-1> mutants induced PR1 expression at higher levels than wild-type, only lug-3 and seu-1 mutants were pathogen resistant. Furthermore, LUH functions as a positive regulator in seed mucilage secretion, a process important for proper seed germination, hydration, and dispersal. I propose a possible connection between the defect in mucilage secretion and pathogen defense in luh-1 mutant plants and seeds, which places the foundation for further investigation and may uncover mucilage secretion as a major defense mechanism. My thesis has provided important insights into how transcriptional co-repressors regulate diverse developmental and physiological pathways in higher plants.Item Breaking the A-P axis: Evolution of diverse asexual reproduction strategies in Convolutriloba acoels(2009) Sikes, James M.; Bely, Alexandra E.; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The defining characteristic of the Bilateria is the presence of a distinct head end and tail end, which defines the anterior-posterior (A-P) axis, a feature that is established during embryogenesis and generally remains unaltered during the lifetime of an organism. While a few bilaterians have evolved asexual reproduction strategies that allow them to subdivide the A-P axis, acoels in the genus Convolutriloba have an unparalleled ability to alter the A-P axis during modes of transverse fission, longitudinal fission, and reversed polarity budding. Convolutriloba acoels thus offer an exceptional opportunity to investigate the mechanisms that allow for the radical modification of an already established A-P body axis and to explore the evolution and development of diverse asexual reproduction strategies among related species. In this study, I reconstruct the evolutionary history of asexual reproduction in the Convolutriloba and compare the diverse modes of asexual reproduction at the level of body-wall musculature, nervous system development, and cell proliferation while also exploring the regenerative potentials of tissues across species with different modes of asexual reproduction. In addition, I further explore the unusual process of A-P axis reversal that occurs during reversed polarity budding in C. retrogemma through studies of body patterning and regeneration. The results of these analyses suggest that a rich developmental toolkit of regenerative abilities, including the ability to utilize both epimorphosis and morphallaxis, to regenerate all parts of its body even from a small fragment, and to produce bifurcated A-P axes were present in the ancestor of the Convolutriloba allowing for the evolution of A-P axis modifications unlike any other bilaterian group. This toolkit along with the evolution of a seemingly unpatterned zone of tissue within the body of C. retrogemma capable of generating new anterior axes appear to have allowed this species to evolve the ability to form reversed A-P axes during budding.Item Population genetics of the eastern oyster in Chesapeake Bay(2008) Rose, Colin G.; Hare, Matthew P; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The eastern oyster, Crassostrea virginica, plays an important role in the ecology of Chesapeake Bay. Its large population size, long larval dispersal stage and potential for high variance in reproductive success is representative of many marine invertebrates. Nevertheless, many important aspects of the oyster's biology remain unclear. I investigated how migration, natural selection, and effective population size have shaped the evolution of Chesapeake oysters. First, I examined aspects of genetic connectivity among oysters from rivers throughout the Bay. A correlation between geographic and genetic distance indicated that oyster larval dispersal tends to be local and that migration between Bay tributaries is rare over an ecological time scale. This result contributes to a growing body of literature indicating that larval dispersal is not passive. Next, I showed that a pattern of non-neutral mitochondrial evolution previously observed in different oyster populations also existed in Chesapeake Bay C. virginica. Tests of selection indicated that the pattern, in which there is an excess of high frequency and low frequency haplotypes and a deficit of intermediate frequency haplotypes, was the result of positive selection on the genome. Demographic explanations appear unlikely to account for the mitochondrial haplotype pattern because nuclear loci exhibited neutral patterns of sequence evolution. Estimates of effective population size were several orders of magnitude smaller than census size, indicating that there was variance in reproductive success (sweepstakes reproduction). Nevertheless sweepstakes reproduction was not so severe that individual cohorts of juvenile oysters exhibited reduced levels of variation compared to the adult population. Finally I evaluated the risks associated with a supplementation program in which hatchery-raised oysters bred for disease tolerance were released into wild oyster populations. The results indicated that following supplementation, the wild effective population size remained large despite the danger of severe genetic bottlenecks. Increased hatchery effective population is suggested to prevent future harm to the wild population.