Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

This thesis includes two studies: The first examined patterns of neutral genetic diversity within Bromus tectorum L. across the IMW region, and uses patterns of microsatellite (SSR) genotype distribution to make inferences about the respective roles of adaptively significant genetic variation, adaptive phenotypic plasticity, and facultative outcrossing in the ongoing invasion and recent range expansion of B. tectorum. It has been previously demonstrated that, due to extremely low outcrossing rates, it is possible to characterize individual genotypes of this species using four SSR loci. We sampled 20 individuals from each of 96 B. tectorum populations (classified by region and habitat) from throughout the IMW and used these SSR markers to characterize each individual. We found 131 four-locus SSR genotypes; however, the 14 most common genotypes collectively accounted for 79.2% of the individuals sampled. Individuals with certain SSR genotypes sorted strongly into warm or salt desert habitats (stringent habitats) and flowered earlier than individuals with genotypes from more mesic habitats, providing evidence of adaptively significant genetic variation associated with these genotypes. Other SSR genotypes were found across a wide range of habitats though they tended to be less prevalent in stringent habitats, providing evidence that adaptive phenotypic plasticity may be important for the distribution of some common genotypes. We observed very few heterozygous individuals, consistent with the highly inbreeding reproductive strategy of B. tectorum. Because specialist genotypes dominating recently invaded areas within the IMW region contained unique alleles, they are not likely to have resulted from recombination, leading us to doubt the role of facultative outcrossing as a significant mechanism facilitating the current range expansion of B. tectorum in the IMW.Previous research investigating the population and ecological genetics of Bromus tectorum L. in the North American invaded range has relied on either allozyme or microsatellite (SSR) genetic analyses, both of which have proven to have shortcomings. In order to overcome the issues associated with these other marker types, in the second study of this thesis we developed single nucleotide polymorphism (SNP) markers for B. tectorum by 1) obtaining normalized cDNA, 2) sequencing normalized cDNA using 454 sequencing, 3) aligning resultant contigs and looking for SNPs, 4) designing assays for SNP validation and genotyping using KASPar, 5) converting working KASPar assays for use with the Fluidigm EP1 platform using the 96.96 Dynamic ArrayTM IFC. Sequencing resulted in 1258041 reads, which assembled into 65486 contigs (20782 large contigs exceeding 500 base pairs). Using selection criteria of at least 10x coverage and 30% of the minor allele, 3333 putative SNPs were identified. We developed KASP assays for 255 putative SNPs, which resulted in 101 working polymorphic assays. Ninety-six assays were then successfully converted for use with KASP on the Fluidigm EP1 genotyping platform using 96.96 dynamic arrays.

The research field of genetics provides useful tools to investigate the biology of species that are difficult to observe and study and are especially valuable in guiding the conservation of endangered species. The Chatham Island Tāiko (Tchāik, Pterodroma magentae) is the world’s most endangered seabird with an estimated population size of just 120-150 birds, including only 8-15 breeding pairs. This thesis used genetic techniques to investigate aspects of Tāiko biology and relationships in order to aid Tāiko conservation. The mitochondrial cytochrome b gene and duplicated regions of domain I of the mitochondrial control region were DNA sequenced in almost the entire known Tāiko population. The level of genetic variation revealed in Tāiko was unexpectedly high considering endangered species typically exhibit low genetic diversity. Sequencing of ancient DNA from subfossil Tāiko bones allowed an investigation of the past level of genetic variation and the species’ previous geographic distribution. A large proportion of the genetic diversity of the extinct Tāiko populations was retained in the remnant population. However, genetic variation in Tāiko chicks was low, thus genetic diversity in the population could be lost in just a few generations. There are many nonbreeding Tāiko so DNA sexing was used to examine sex ratios in the population. Almost all unpaired birds were male, which signified a potential Allee effect (i.e. that a reduced density of potential mates is decreasing population productivity). Further understanding of the Tāiko mating system and behaviour was obtained by parentage, sibship and pairwise relatedness analyses of genotypes at eight microsatellite DNA loci. It is important that Tāiko are found so they can be protected from introduced predators. The results of mitochondrial DNA sequencing and microsatellite DNA genotyping indicated that there are likely to be more Tāiko breeding in undiscovered areas. Analysis of philopatry using both mitochondrial and nuclear markers can assist conservation by the identification of areas to search for these undiscovered individuals. Tāiko may have once and could still be found on islands near South America since DNA sequencing showed the Magenta Petrel type specimen (collected in 1867 in the South Pacific Ocean) is a Tāiko.

Leaf shape is remarkably variable among plants, and hence likely has major consequence for ecological function and fitness. My thesis addresses the ecological significance of clinal variation for a leaf shape polymorphism in Ipomoea hederacea (lobed leaves dominate the north, entire-shaped leaves restricted to the south), and investigates the role of adaptation and demography in shaping its evolutionary history in its eastern North American range. To evaluate the adaptive value of the cline, I surveyed leaf shape genotypes from 77 populations , and found a steep latitudinal leaf shape cline that was not reflected in 173 neutral genetic markers. Furthermore, the leaf shape locus was a genomic outlier, implicating divergent selection in generating or maintaining the cline. I investigated the thermoregulatory and freezing tolerance properties of the leaf shape genotypes, and discovered that lobed leaves remain marginally warmer at night, and a 1°C decrease separated mildly damaged and severely frost damaged tissue, potentially suggesting that a critical ambient temperature could drive differential leaf shape damage. I further explored three additional hypothesized selective agents (insect herbivores, flowering phenology and growth), and showed that these putative agents impose selection on I. hederacea, but do not differentiate between leaf shapes. These studies highlighted the challenge of identifying selective agents, even for a polymorphic trait with hypothesized selective mechanisms. To understand the contribution of adaptation and demography in shaping I. hederacea’s evolutionary history, I sequenced 7 nuclear loci from 192 individuals sampled from 24 populations and characterized patterns of nucleotide diversity. I demonstrated that I. hederacea is genetically structured in patches consistent with long-distance dispersal, genetically depauperate, and undergoing range expansion, suggesting a recent founder event or metapopulation dynamics. My thesis represents a comprehensive evaluation of the key processes affecting a polymorphism that influences plant morphology, geographical distribution, and population history.

Leaf shape is remarkably variable among plants, and hence likely has major consequence for ecological function and fitness. My thesis addresses the ecological significance of clinal variation for a leaf shape polymorphism in Ipomoea hederacea (lobed leaves dominate the north, entire-shaped leaves restricted to the south), and investigates the role of adaptation and demography in shaping its evolutionary history in its eastern North American range. To evaluate the adaptive value of the cline, I surveyed leaf shape genotypes from 77 populations , and found a steep latitudinal leaf shape cline that was not reflected in 173 neutral genetic markers. Furthermore, the leaf shape locus was a genomic outlier, implicating divergent selection in generating or maintaining the cline. I investigated the thermoregulatory and freezing tolerance properties of the leaf shape genotypes, and discovered that lobed leaves remain marginally warmer at night, and a 1°C decrease separated mildly damaged and severely frost damaged tissue, potentially suggesting that a critical ambient temperature could drive differential leaf shape damage. I further explored three additional hypothesized selective agents (insect herbivores, flowering phenology and growth), and showed that these putative agents impose selection on I. hederacea, but do not differentiate between leaf shapes. These studies highlighted the challenge of identifying selective agents, even for a polymorphic trait with hypothesized selective mechanisms. To understand the contribution of adaptation and demography in shaping I. hederacea’s evolutionary history, I sequenced 7 nuclear loci from 192 individuals sampled from 24 populations and characterized patterns of nucleotide diversity. I demonstrated that I. hederacea is genetically structured in patches consistent with long-distance dispersal, genetically depauperate, and undergoing range expansion, suggesting a recent founder event or metapopulation dynamics. My thesis represents a comprehensive evaluation of the key processes affecting a polymorphism that influences plant morphology, geographical distribution, and population history.

Regulation of transcription is an important molecular mechanism through which organisms can respond to environmental change. Environmentally-related transcriptional variation can play a significant role in evolution, potentially acting as a mechanism for the formation of new adaptive phenotypes. Organisms are most sensitive to the influence of external environmental variation during development, yet very few studies have explored environmentally-related transcriptional variation in early life history stages. Marine invertebrate metamorphosis, where intimate larval-environment interactions trigger settlement onto the benthos and a drastic morphological shift from larval to adult form, exemplifies the influence of environment on development. Variation in both the timing of metamorphosis and the form of specific metamorphic inductive cues, even at an intraspecific level, suggests that larvae have molecular mechanisms for surviving settlement and metamorphosis in a range of environmental conditions. The extent of transcriptional variation at metamorphosis remains largely unknown due to limited information on both the natural inductive cues and the molecular mechanisms directing metamorphosis in marine invertebrates. Contributing to current understanding of the interplay between genes, environment and phenotype during development, I explored molecular and ecological aspects of metamorphosis in a marine invertebrate, the tropical abalone Haliotis asinina. First, I employed cDNA microarray methods to identify candidate genes and document widespread transcriptional changes occurring in Haliotis asinina larvae during larval development and metamorphosis. Microarray results reveal that as the abalone veliger larva matures, it requires coordinated regulation of temporally different gene batteries involved in a wide range of physiological and developmental processes associated with the transition to a new, benthic habitat. All candidate genes showed changes in expression following exposure of larvae to coralline algae, an external inductive cue, demonstrating the remarkable effect of environment on transcription during marine invertebrate metamorphosis. 144 genes, ~40% of which are novel, were identified as candidates for a role in H. asinina metamorphosis. This high proportion of novel genes indicates that the conserved signaling pathways operating in marine invertebrate metamorphosis likely regulate the expression of taxon-specific genes. The relationship between abalone larvae and their natural inductive cue, coralline algae, is species-specific. To characterize the metamorphic cue preferences of Haliotis asinina larvae from Heron Island Reef, Australia, I documented larval induction response to a number of different coralline algae species commonly found in adult H. asinina habitat. H. asinina larvae exhibit highly specific responses to induction of metamorphosis by different coralline algae species, with 0 – 100% metamorphosis by 48 hours post induction depending on algae species. Unlike any other abalone species studied, the most effective inducers of Heron Island Reef H. asinina are articulated corallines of the genus Amphiroa. Comparing the response of different larval families to select species of coralline algae indicated that coralline algae community composition is likely to significantly impact H. asinina population structure. Additionally, I compared larval response to dead and live coralline algae to show that induction specificity is driven by chemical, not physical, properties. Characterization of the surface cell biomarkers of three different coralline algae species indicates that algal biomolecular composition relates to variations in H. asinina induction response. To explore the influence of variation in larval induction environment, I compared transcription patterns of 17 metamorphosis-related genes in Haliotis asinina larvae induced by three different species of coralline algae. H. asinina post-induction gene expression profiles vary according to the species of coralline algae inducer. This transcriptional variation occurs in genes with diverse functions and spatial expression patterns, highlighting the global nature of the impact of benthic microhabitat on gene expression. The environment-specific modulation of gene expression in H. asinina post-induction may be a means for marine invertebrates to cope with changes in their settlement environment at metamorphosis. Genes expressed in the larval sensory structures acting to detect external metamorphic cues may be particularly good candidates for studying environmentally-related transcriptional variation. I identified three novel genes expressed in putative sensory structures of Haliotis asinina larvae just prior to metamorphosis. The spatial and temporal expression patterns of these genes correlate with changes in larval ciliation patterns throughout metamorphosis, strongly suggestive of a role in metamorphic initiation. The three genes exhibit significant overlap in spatial expression profiles, indicative of genetic crosstalk between different sensory systems at metamorphosis. Transcriptional variation in gastropod sensory system genes may have assisted the evolution of different metamorphic inductive cues for different species. The results presented here establish an important role for transcriptional variation during marine invertebrate metamorphosis. Transcriptional variation underlies the morphological change from larval to adult body plan and also appears to assist larval recruitment in variable benthic habitats. Modulation of gene expression at metamorphosis in response to the environment may ultimately influence marine invertebrate species biogeography and evolution.

abstract: How fast is evolution? In this dissertation I document a profound change that occurred around the middle of the 20th century in the way that ecologists conceptualized the temporal and spatial scales of adaptive evolution, through the lens of British plant ecologist Anthony David Bradshaw (1926–2008). In the early 1960s, one prominent ecologist distinguished what he called “ecological time”—around ten generations—from “evolutionary time”— around half of a million years. For most ecologists working in the first half of the 20th century, evolution by natural selection was indeed a slow and plodding process, tangible in its products but not in its processes, and inconsequential for explaining most ecological phenomena. During the 1960s, however, many ecologists began to see evolution as potentially rapid and observable. Natural selection moved from the distant past—a remote explanans for both extant biological diversity and paleontological phenomena—to a measurable, quantifiable mechanism molding populations in real time. The idea that adaptive evolution could be rapid and highly localized was a significant enabling condition for the emergence of ecological genetics in the second half of the 20th century. Most of what historians know about that conceptual shift and the rise of ecological genetics centers on the work of Oxford zoologist E. B. Ford and his students on polymorphism in Lepidotera, especially industrial melanism in Biston betularia. I argue that ecological genetics in Britain was not the brainchild of an infamous patriarch (Ford), but rather the outgrowth of a long tradition of pastureland research at plant breeding stations in Scotland and Wales, part of a discipline known as “genecology” or “experimental taxonomy.” Bradshaw’s investigative activities between 1948 and 1968 were an outgrowth of the specific brand of plant genecology practiced at the Welsh and Scottish Plant Breeding stations. Bradshaw generated evidence that plant populations with negligible reproductive isolation—separated by just a few meters—could diverge and adapt to contrasting environmental conditions in just a few generations. In Bradshaw’s research one can observe the crystallization of a new concept of rapid adaptive evolution, and the methodological and conceptual transformation of genecology into ecological genetics. / Dissertation/Thesis / Doctoral Dissertation Biology 2015

Orientador: Vera Nisaka Solferini / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T20:52:47Z (GMT). No. of bitstreams: 1 Alcantara_Suzana_M.pdf: 3488910 bytes, checksum: ea2df0c2f9926f7db5e7ad46a5df0d26 (MD5) Previous issue date: 2006 / Resumo: Populações espacialmente estruturadas tendem a apresentar alta diferenciação genética e forte evidência de processos micro-evolutivos. Indivíduos de Brugmansia suaveolens (Solanaceae) são restritos a manchas populacionais. Neste trabalho, a diversidade genética de nove populações de B. suaveolens foi estimada através de isoenzimas (capítulo 1). Nossos resultados demonstram alta variabilidade e estruturação genética entre as populações. O comportamento do polinizador e a dispersão hidrocórica parecem determinar a estruturação intrapopulacional. Existem evidências de que efeitos estocásticos (i.e.: fundação e migração) afetam o padrão de diferenciação interpopulacional. A maioria dos resultados pode ser esperada sob dinâmica de estabelecimento de metapopulações. Tais populações são bastante susceptíveis à diferenciação de caracteres quantitativos, seja devido à evolução fenotípica neutra (causada por deriva) ou ação de seleção (adaptação local). Para avaliar o papel da deriva e da seleção nessa diferenciação, a variação genética de caracteres pode ser comparada à variação neutra estimada por marcadores moleculares. Quatro caracteres foram avaliados em quatro das populações de B. suaveolens estudadas (capítulo 2), três deles relacionados à defesa química contra herbívoros (concentração de alcalóides). Nossos resultados mostram uma diferenciação genética ca. de três a quatro vezes maior que a estimada por marcadores neutros para três dos caracteres, embora essa diferença não seja significativa. A exceção ocorre para a razão de indução de alcalóides, que mostra sinais de seleção estabilizadora (ausência de diferenciação interpopulacional), contrariando o padrão esperado pela teoria / Abstract: Populations patchily distributed tend to show high genetic structure and strong micro-evolutionary process evidences. Brugmansia suaveolens (Solanaceae) individuals are restrict to population patches. In this work, the genetic diversity of nine B. suaveolens populations was estimate by means of allozyme electrophoresis (chapter 1). Our results indicate high variability and genetic structure among populations. The pollinator behaviour and the hydrochoric dispersal seem determine the intrapopulation structure. There are signals that stochastic effects (i.e.: migration and foundation) affect the differentiation among populations. The most of genetic patterns found can be created by a metapopulational establishment. These populations are very susceptible to the quantitative traits differentiation, due to neutral phenotypic evolution (generated by drift) or selection action (local adaptation). To evaluate the relative action of drift and selection on population differentiation, the genetic variation in quantitative traits can be compared to neutral variation estimated by molecular markers. Four traits were evaluated in four of the B. suaveolens populations studied (chapter 2), and three of them were related to chemical defense against herbivores (tropane alkaloids concentrations). Our results show a genetic differentiation ca. three to four times higher than the differentiation estimated by molecular markers for three of characters analyzed, although no significant. The exception is ¿alkaloids induction¿ trait, which show stabilizing selection signals (absence of interpopulation differentiation), instead of differentiation pattern expected by theory / Mestrado / Mestre em Ecologia

Following its introduction to North America in the late nineteenth century, Bromus tectorum L., an inbreeding invasive winter annual grass, has become dominant on millions of hectares of sagebrush steppe habitat throughout Intermountain Western North America. It appears that within the last 30-40 years, B. tectorum has expanded its range southward into the Mojave Desert and also into more climatically extreme salt desert environments. Previous research using microsatellite markers and experimental studies has suggested that lineages found in desert habitats are genetically distinct from those found in the sagebrush-steppe habitat and possess suites of traits that pre-adapt them to these environments. To provide additional support for our hypothesis that desert habitat-specific haplotypes dominate and are widely distributed across warm and salt desert habitats, we genotyped approximately 20 individuals from each of 39 B. tectorum populations from these habitats and adjacent sagebrush steppe habitats using 71 single nucleotide polymorphic (SNP) markers. Our data clearly demonstrate that populations throughout the Mojave Desert region, as well as in salt desert habitats further north, are dominated by a small number of closely related SNP haplotypes that belong to the desert clade. In contrast, populations from adjacent environments are largely dominated by haplotypes of the common clade, which is widely distributed throughout the North American sagebrush steppe. Populations across all habitats were usually dominated by 1-2 SNP haplotypes. This suggests that inbreeding B. tectorum lineages can often maintain their genetic integrity. It also explains the strong association between marker fingerprints and suites of adaptive traits in this species.

Algodão geneticamente modificado que expressa a toxina Cry1Ac de Bacillus thuringiensis Berliner tem sido plantado no Brasil desde 2006. Entre as pragas-alvo da tecnologia, Alabama argillacea (Hüeb.) é uma espécie monófoga e apresenta alto potencial de risco de evolução da resistência. Para a implantação de um programa de manejo da resistência de A. argillacea à toxina Cry1Ac no Brasil, os principais objetivos do trabalho foram: a) estabelecer a linhas-básicas de suscetibilidade à toxina Cry1Ac em populações de A. argillacea e definir concentrações diagnósticas para o monitoramento da resistência e b) isolar e caracterizar locos microssatélites para avaliar a variabilidade e estruturação genética de populações de A. argillacea no Brasil. As linhas-básicas de suscetibilidade foram estimadas por meio de bioensaio de imersão de discos de folhas em soluções contendo a toxina Cry1Ac para populações de A. argillacea coletadas nos estados da Bahia, Goiás, Mato Grosso e Mato Grosso do Sul, durante as safras agrícolas de 2008 e 2009. Foram isolados e caracterizados dez locos microssatélites. Para avaliar a variabilidade genética foram estimadas as heterozigosidades observadas e esperadas. Para o estudo da estruturação genética foram estimadas as estatísticas F e feita a análise de agrupamento (distância de Nei) e análise Bayesiana. Baseado na estimativa da CL50 foram encontradas variações naturais de até seis vezes na suscetibilidade à toxina Cry1Ac entre as populações testadas. A partir da análise conjunta dos dados de concentração-mortalidade das populações testadas, foram definidas as concentrações diagnósticas de 10 e 32 µg de Cry1Ac/ml de água para futuros programas de monitoramento da resistência. O número médio de alelos por loco foi de 7,1 (variando de dois a 23 alelos). As heterozigosidades observada e esperada médias foram de 0,532 e 0,329. O índice de fixação intrapopulacional médio (f FIS) foi de 0,268, com variação entre os locos de -0,008 a 0,736. O índice de fixação da espécie (FIS) estimado através da análise de variância foi de 0,244 (IC 95% de 0,093 a 0,418). O valor de FST estimado foi de 0,036 (IC 95% de 0,007 a 0,080). Esse valor de FST não diferiu significativamente de zero, indicando a ausência de estruturação genética. Contudo foi detectado certo grau de endogamia intrapopulacional. A estruturação espacial da variabilidade genética não foi detectada, pois as populações avaliadas apresentaram uma coesão que é mantida pela alta taxa de migração (6,7 migrantes por geração). Entretanto, foi identificada indícios de estruturação genética determinada pelo tempo, uma vez que tanto o agrupamento baseado em distâncias genéticas quanto à análise Bayesiana identificaram grupos que são formados por populações coletadas em safras agrícolas diferentes. As causas ligadas a essa mudança na variabilidade genética não puderam ser identificadas, entretanto pode se inferir que possivelmente causas naturais ou práticas de manejo estejam determinando eventos de gargalo genético. Devido ao intenso fluxo gênico entre populações de A. argillacea no Brasil, estratégias de manejo da resistência devem ser implantadas no âmbito nacional. / Genetically modified cotton expressing Cry1Ac toxin of Bacillus thuringiensis Berliner has been planted in Brazil since 2006. Among target pests of this technology, Alabama argillacea (Hüeb.) is a monophagous species and offers a high potential risk of resistance evolution. In order to implement a resistance management program of A. argillacea to Cry1Ac toxin in Brazil, the objectives of this research were: a) to establish baseline susceptibility to Cry1Ac toxin in A. argillacea populations and define diagnostic concentrations for resistance monitoring and b) to isolate and characterize microsatellite loci to evaluate the variability and genetic structure of A. argillacea populations in Brazil. The baseline susceptibility data were estimated with leaf-disc bioassays by dipping into different concentration of Cry1Ac solution. Populations of A. argillacea were collected in Bahia, Goiás, Mato Grosso and Mato Grosso do Sul States, during 2008 and 2009 cotton-growing seasons. Ten microsatellite loci were isolated and characterized. The genetic variability was evaluated estimating observed and expected heterozygosities. For the studied of genetic structure, the F statistics was estimated, and Cluster analysis (Nei´s distance) and Bayesian analysis were performed. Based on estimation of LC50, natural variation up to 6-fold was detected in the susceptibility to Cry1Ac among tested populations. Based on analysis of concentration-mortality data by combining all populations, diagnostic concentrations of 10 and 32 µg of Cry1Ac/ml of water were defined for monitoring resistance. The mean number of alleles per loci was 7.1 (varying from 2 to 23 alleles). The observed and expected heterozigosities was 0,523 e 0, 395. The mean intrapopulation fixation index (f FIS) 0,268, varying from -0.008 to 0.736 between loci. The species fixation index (FIS) estimated by analysis of variance was 0.244(95% CI of 0.093 to 0.418). The estimated value of FST was 0.036 (95% CI of 0.007 to 0.080). The FST value was not significantly different from zero, indicating absence of genetic structure However, some degree of intrapopulational inbreeding was detected. Spatial structure of genetic variability was not detected because tested populations showed cohesion kept by high migration rate (6.7 migrants per generation). However, evidence of genetic structure across time was detected by Cluster analysis of genetic distance as well as by Bayesian analysis with group formation by population collection seasons. Factors affecting changes in genetic variability were not identified; however, natural factors or management practices may be determining some genetic bottleneck events. Due to intense gene flow among A. argillacea populations in Brazil, resistance management strategies must be implemented in a national basis.