Why some organisms reproduce just once in their lifetime (semelparity), while others reproduce more than once (iteroparity), has been a central question of life history theory since it was posed by Cole (1954). I used comparative studies at large (phylogenetic) and small (demographic) scales to address this question in a group of evening primroses (the Sections Anogra and Kleinia, genus Oenothera, Onagraceae) found in the arid and semiarid region west of the 100th meridian in North America. In the phylogenetic study, I found that changes in habit were not associated with the changes in aridity that I expected, based on the classic demographic model of Charnov and Schaffer (1973). Instead, this study suggested that changes to the annual habit were associated with increased temperature. I propose that temperature is an important factor influencing the favorability of the annual habit via the effect that temperature has on growth rate. The remaining two studies, comparing the performance of a closely-related desert annual and desert perennial in natural and experimental settings, also indicated that temperature correlated with habit. Using demographic data from natural populations, I evaluated seed banking and iteroparity as alternative means of bet hedging. I found evidence that bet hedging occurs via seed banking in both populations, and may occur via post-reproductive survival in the perennial populations. The demographic data did not clearly show the patterns expected to favor one form of bet hedging over the other. Based on an analysis of climate data, I suggest that cold temperatures are unfavorable to the annual habit. I compared the performance of the same species pair directly in two common environments. In this reciprocal common garden experiment, the annual outperformed the perennial when conditions were good, and when conditions became stressful relatively early. The annual, with lower leaf mass per area, more rapid above ground growth, and accelerated phenology, exhibits the classic stress-avoiding strategy of desert annuals, explaining the conditions under which it excelled. Relative to the annual, I describe the perennial as a stress-tolerator, and discuss water and temperature stress as two forms of stress it may excel at tolerating.

Reproduction constitutes the principle determinant of organismal fitness and, therefore, a central theme in biology is devoted to understanding variation in the mode of reproduction and its effects within and among species. Different breeding systems lead to varying levels of inbreeding, outcrossing, and sex ratios---with concomitant effects on patterns of genetic variation, effective population size, and adaptation by natural selection. Theoretical studies have proposed many mechanisms to explain the diversity of breeding systems observed in nature, several models of which invoke deleterious effects of mutation as a dominant force in patterning breeding system evolution. These notions motivated the present investigations into (1) the factors contributing to outcrossing rates and sex ratio in Caenorhabditis elegans and their population genetic effects, and (2) the general importance of deleterious mutation in the evolution of breeding system, emphasizing the Caenorhabditis clade. Nematodes of the genus Caenorhabditis provide a convenient system for studying these issues because species vary in breeding system, self-fertile C. elegans hermaphrodites can be genetically transformed into females to create dioecious populations, and complete genomic sequences of two species allow genomic analysis. With this context, I constructed a quantitative model describing sex ratio and outcrossing as a function of male copulatory ability and sex-chromosome non-disjunction (as a consequence of the XO sex-determination mechanism). The sex ratio and amount of outcrossing in C. elegans were then predicted from laboratory experiments and nucleotide polymorphism data, by applying population genetic theory of selection at linked sites when partial selfing is taken into account. These analyses suggest that outcrossing occurs on the order of one percent. Subsequent computational and multigeneration experimental evolution studies of the potential influence of deleterious mutations on breeding system evolution demonstrated that (1) selfing species do not exhibit lower rates of deleterious mutation than outcrossing species, (2) a higher deleterious mutation rate does not prevent the invasion of selfing alleles into a population, and (3) adaptive modulation of sex-chromosome non-disjunction is unlikely to underlie the persistence of males in androdioecious species. These results indicate that mutational theories are unlikely to fully explain the evolution of breeding system and sex.

Ariidae (Siluriformes), widely distributed in world tropical shelves, includes 150-200 species, one third endemic to the New World. Males practice mouthbrooding of eggs and embryos. To study the phylogeny of neotropical ariids, a morphological matrix [26 species (three outgroup, 23 ingroup), 56 characters) was used. The strict consensus tree of 12 parsimonious trees is 85 steps long. Ariidae includes two subfamilies. Galeichthyinae includes one species endemic to the eastern Pacific, Galeichthys peruvianus. Ariinae has three New World lineages. Notarius includes seven species in the western Atlantic, Colombia to southern Brasil, and seven in the eastern Pacific, Baja California to Peru. Cathorops has two lineages, a monotypic for C. dasycephalus, from eastern Pacific, and other for the rest. There are six species of Cathorops in western Atlantic and seven in eastern Pacific. The tribe Ariopsini, 24 species, is defined by two states from the otolith. The tribe is divided in two unities. The first includes Bagre, four species, and Occidentarius platypogon, endemic to the eastern Pacific. Western Atlantic Bagre are known from Massachussetts to Brasil; the eastern Pacific species go from California to Peru. The other ariopsin unity includes Ariopsis, five species, and Sciades, six species. Ariopsis ranges in western Atlantic from Massachussetts to Venezuela and in eastern Pacific from Mexico to Peru. Sciades includes five western Atlantic species, Colombia to Brasil, and one eastern Pacific species, Mexico to Peru. The freshwater genus Potamarius , four species, three from western Atlantic rivers and one from Ecuadorian rivers, is the sister taxa to Ariopsis. Genidens , four western Atlantic species, Brasil to Tierra del Fuego, is related to ariopsins. The hypothesis for Bagre produced with the morphological matrix coincides with the topology from morphometric techniques; three phylogenetic hypotheses found with molecular techniques were different. Bagre pinnimaculatus is the sister species to B. bagre, both are the most derived species; B. marinus is the most generalized species. Other transisthmian speciation events were found within the genera Notarius, Cathorops, and Ariopsis. In the New World three lineages have independently returned to freshwaters. Some New World ariid lineages show tendency to occur mainly in marine waters.

It is axiomatic that genomic stability is dependent upon regulatory pathways, termed checkpoints, which sense perturbations of cell cycle execution including damage to chromosomal DNA. In Saccharomyces cerevisiae, the principal DNA damage checkpoint is at G2/M. Heretofore, this and other checkpoints, such as the spindle assembly checkpoint, which is also operative at the metaphase/anaphase transition, have been viewed as essentially linear pathways, responding to a specific type of damage, signaling via sui generis proteins, and targeting a limited number of effectors for arrest. In a 1999 report, our laboratory reported bifurcation of the pathway downstream from Mec1 activation; this established the genetic basis of a previously unexplained phenotype: partial arrest defects of rad53 and pds1 strains. Moreover, the bifurcated pathway model established the framework for subsequent studies which determined the molecular targets of each. Here, I present evidence that the DNA damage and spindle checkpoint pathways are part of a network which is capable of bilaterally responding to damage. After DNA damage the Mec1-centric pathway is initially preeminent; the spindle pathway is redundant. After prolonged damage, however, the spindle checkpoint components become required for arrest. In studies of overexpression of the Mec1 homologue Tel1, I delineated the pathway responsible for the resultant constitutive delay; strikingly, the spindle components Mad1 and Mad2 are activated, not from the kinetochore, but from the nuclear periphery. This off-kinetochore pool of Mad proteins, anchored by the myosin-like proteins, Mlp1 and Mlp2, is likewise activated by the DNA damage response. Tel1 physically interacts with Xrs2 of the Mre11·Rad50·Xrs2 complex; evidence that Xrs2 participates in these same responses is also presented. Finally, the sensitivity of xrs2 to a microtubule poison, benomyl, suggests that M R·X may also participate in sensing spindle disruption. From a screen for novel checkpoint genes, I isolated Gtr1 (and later, Gtr2), which are negative regulators of the Ran cycle. Here, I provide evidence that deletion of either produces an identical partial arrest defect, which is independent of the Mec1-centric pathway. Because Gtr2 physically interacts with Esp1, I surmise that Gtr1/Gtr2 may enforce cytosolic localization of Pds1/Esp1 after DNA damage.

The actin cytoskeleton in yeast plays a role in many morphological events such as cell growth, secretion, polarity, bud emergence, and endocytosis. Some proteins have been identified that regulate the elongation, nucleation, and stability of the actin cytoskeleton. One of the many proteins controlling the state of the actin cytoskeleton is an actin bundling protein encoded by the gene SAC6 (suppressor of actin). In this study, I have examined some of the phenotypic consequences of overexpression of Sac6p and analyzed interactions between Sac6p and actin in vitro. I have investigated the molecular basis of allele-specific suppression observed between combination of act1 mutations and sac6 mutations by analyzing in vitro the interaction between wild-type and mutant actin and wild-type and mutant Sac6p. The biochemical assays show in vitro suppression of binding and crosslinking activity between Sac6 suppressor protein and mutant actin protein. In addition, the Sac6 suppressor proteins tested have an increased affinity to wild-type actin. This analysis, as well as previous genetic data, is consistent with the idea that suppression results from an overall increase in affinity to actin rather than a strict "lock and key" mechanism previously hypothesized. Overexpression of Sac6p under the control of a galactose inducible promoter results in growth inhibition, accumulation of multinucleated cells, and altered actin cytoskeletal organization. To better understand why overexpression of Sac6p has detrimental effects, I devised a screen to isolate genomic mutations that can suppress the growth defects resulting from elevated levels of Sac6p. I have found that an act1-3 strain is able to suppress Sac6p overexpression and one of the mutants isolated in the screen is also an act1 mutant allele. In addition, the temperature sensitive and osmotic sensitive phenotypes are not complemented by an act1-3 strain. These results suggest that the lethality is mediated through the interaction of Sac6p with actin. In addition, this analysis shows that the proper stoichiometry of Sac6p is critical for proper function of actin in vivo.

The chloroplast genome of Euglena gracilis contains at least 155 introns, accounting for 39.2% of the genome. Among them are 88 group II introns of size 277-671 nucleotide (nt). Questions about the processing of these introns and their origin and spread in Euglena genera is the subject of this thesis. My working hypothesis is that the Euglena chloroplast genome evolved from an intron less ancestral genome by the invasion of mobile genetic elements relying in part on internally encoded enzyme activities for mobility. These internal enzymes may also be group II maturases. My research target was the largest intron in Euglena gracilis chloroplast, psbC intron 2 (4144 nt). I characterized psbC intron 2 as a cis-spliced group II intron encoding a 758 codon maturase-like protein (mat 2) that is interrupted by three additional group II introns. I also identified a putative RNA-binding domain (X), that relates to fungal mitochondrial group II intron maturases, and a reverse transcriptase (RT) domain, that has been found in other group II intron maturases and has been shown to have intron-translocation activity (Lazowska, 1994, Moran, 1995). In order to examine the distribution of mat 2, I characterized psbC intron 2 homologues from several species. I found that the mat 2 locus well conserved in most of the Euglena species tested, indicative of a deep root and important function of mat 2. Interestingly, the entire intron is absent in E. myxocylindracea. In order to investigate the effect of mat 2 loss, I analyzed group II intron content in E. myxocylindracea chloroplast genome. No group II introns have been identified in the genome. These data also provided evidence that mat 2 might be responsible for the acquisition and the processing of most of the group II introns in Euglena chloroplast. The activity of the mat 2 protein has been difficult to examine since the protein is hard to obtain. The development of protocols to overexpress mat 2 in E. coli and yeast are reported in the thesis.

A comparative sequence analysis of mosquito vitellogenin genes was done to gain a better understanding of the evolution of vitellogenin genes in mosquitoes. Genomic clones of vitellogenin genes were isolated from Aedes aegypti , Ae. atropalpus, Culex quinquefasciatus , Toxorhynchites amboinensis, and Anopheles albimanus. Vitellogenin genes were also cloned using degenerate PCR primers from 34 species of mosquitoes representing genera (Aedes, Anopheles, Armigeres, Coquillettidia, Culex, Culiseta, Deinocerites, Psorophora, Mansonia, Mimomyia, Toxorhynchites, and Wyeomyia). Analysis of mosquito vitellogenin gene sequences suggests that a majority of nonsynonymous amino acid substitutions were due to conserved and moderately conserved changes. The vitellogenin genes of the three anautogenous mosquito species had very high synonymous codon usage biases. On the other hand, the vitellogenin genes of autogenous mosquitoes exhibited low synonymous codon usage bias. An unusual pattern of synonymous codon usage was observed in the first 15 amino acid residues encoding the signal peptide in the mosquito vitellogenin genes, where a significantly high number of rarely used synonymous codons have accumulated. A phylogenetic footprinting analysis detected several evolutionarily conserved sequence elements in the 5' regulatory regions of some of the mosquito vitellogenin genes. Mosquito phylogenetic trees reconstructed from maximum parsimony, maximum likelihood, and distance methods based on vitellogenin gene sequences are highly supported and robust, and vitellogenin gene sequences can be utilized to infer the phylogenetic history of mosquitoes, and perhaps other animals.

The evolution of the P element family was studied in members of the Drosophila willistoni and Drosophila saltans species groups (subgenus Sophophora). The transmission of P elements among species, their spread within species and the strength of selective constraints, as well as the level at which they are imposed on these elements, were investigated using DNA sequence data. Particular emphasis was placed on the evolution of the canonical P element subfamily. This subfamily includes the functional P element first isolated from Drosophila melanogaster, which was termed canonical. It includes also other P elements belonging to the saltans and willistoni groups that are closely related to it. Based on the divergence among canonical elements, it was estimated that they last shared a common ancestor 3 million years ago, and that a minimum of eleven horizontal transfer events among species have taken place since then. This indicates that horizontal transfer is more important than anticipated in the transmission of P elements among species. The evolution of P elements within species was studied in detail in Drosophila sturtevanti and Drosophila willistoni. First, the population structure of these species was inferred from nuclear (alcohol dehydrogenase) and mitochondrial (part of subunits 4 and 5 of NADH dehydrogenase, and the transfer RNA gene for histidine) markers. The results suggest that only peripheral populations of D. willistoni show significant genetic differentiation. In D. sturtevanti significant population subdivision was detected among populations in the central part of the distribution, as well as between these and peripheral populations. These results were used as a reference to which P element divergence among populations could be compared. No selective constraints were detected in the evolution of canonical P elements within these two species. However, those constraints are present when elements were compared between species. It is concluded that selection is mostly effective at the time of horizontal transmission between species. Furthermore, P elements are shown to spread faster among populations than do neutral markers. This suggests that the spread of P elements within species can be achieved quickly, and surpass barriers such as moderate levels of population structuring within a species.

The Havasupai Indians are a small (∼600 members), Yuman-speaking population that resides on a reservation in the Grand Canyon region of northern Arizona. Due to their location and cultural practices, they are subjected to extreme geographic and reproductive isolation. Additionally, an influenza epidemic at the turn of the century decreased the reproducing population to 43 females and 42 males. These observations suggest that the Havasupai should contain less genetic diversity than other Native American populations. They are also disproportionately affected by disease, having the third largest incidence of Non Insulin-Dependent Diabetes Mellitus (NIDDM) in the world. An extensive analysis of maternal and paternal variation of the Havasupai was undertaken. Maternal variation was assayed by sequencing the non-coding control region (CR) of mitochondrial DNA (mtDNA), whereas paternal variation was examined using single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) located on the non-recombining portion of the Y-chromosome (NRY). Due to the availability of familial pedigrees dating back to the mid-1800s and spanning eight generations, precise mutation rates were determined for maternal and paternal lineages. The Havasupai thus offer a unique opportunity to explore genetic variation in a small, homogenous Native American population for which extensive genealogical information is readily available. Examination of mtDNA sequences from the complete 1127 by CR of 43 Havasupai individuals along with SNP and STR data from the Y-chromosome of 48 male Havasupai revealed that contrary to our initial prediction, the Havasupai, historically a small population (as evidenced by the limited number of founder haplotypes and low estimates of π), probably maintained a relatively high level of diversity (as evidenced by the number of rare haplotypes, high haplotype diversity, and high estimate of E(ν)), probably a remnant of their association with the larger Pai population from which they are derived. As the level of diversity displayed by the Havasupai seems to have been maintained since the recent population bottleneck, it must have been too small and/or too short to have any detectable effect on the overall diversity of the tribe. Lastly, there appears to be some association between mtDNA mutations and NIDDM in the Havasupai population.

The actin cytoskeleton has been implicated in the structural and mechanical properties of the cytoplasmic matrix. Actin and a number of actin associated proteins work in concert to carry out the various functions of the actin cytoskeleton. However, it is unclear how actin associated proteins function in conjunction with actin in vivo. I used Saccharomyces cerevisiae to investigate the actin cytoskeleton (1, 2, 3). Sac6 protein (Sac6p) is an actin bundling protein that consists of a head domain and two homologous actin binding domains (ABDs) (4). Despite their homology, evidence exists that there are functional differences between the ABDs. To explore these differences I asked if either ABD could function in place of the other by creating chimeric proteins with different combinations of the ABDs. When tested for function in vivo, these chimeric proteins are unable to complement the temperature and osmotic sensitivity of the sac6 null. This suggested that the ABDs of Sac6p are functionally distinct. To explore what functional differences exist between the ABDs of Sac6p, I made several truncations of Sac6p: a C-terminal deletion of Sac6p that retain the head and the first ABD (N410), and two different N-terminal deletions that contain only the second ABD (C386 & C397). Overexpression of N410 gave rise to a different organization of the actin cytoskeleton than did C386 or C397. This suggested that the ABDs/actin interactions are different. To determine whether the differences observed between the ABDs is the result of their interaction with actin, a method was developed to use allele specific suppression of the overexpression phenotype to define the region of interaction between the ABDs and actin. I tested full length Sac6p, N410, C386, and C397. The regions of actin implicated by suppression of the Sac6p overexpression and by allele specific suppression of sac6 mutants were similar. This similarity supports the validity of these two methods in mapping the regions of interaction between two proteins. Overexpression of N410 was suppressed by different actin mutations than overexpression of C386 or C397 suggesting that differences exist between the Sac6p actin binding domains in their interaction with actin.