Population structure of spotted sunfish (Lepomis punctatus) in the Florida Everglades as revealed by DNA microsatellite analysis

Genetic diversity can be used to describe patterns of gene flow within and between local and regional populations. The Florida Everglades experiences seasonal fluctuations in water level that can influence local population extinction and recolonization dynamics. In addition, this expansive wetland has been divided into water management regions by canals and levees. These combined factors can affect genetic diversity and population structure of aquatic organisms in the Everglades. We analyzed allelic variation at six DNA microsatellite loci to examine the population structure of spotted sunfish (Lepomis punctatus) from the Everglades. We tested the hypothesis that recurrent local extinction and recent regional divisions have had an effect on patterns of genetic diversity. No marked differences were observed in comparisons of the heterozygosity values of sites within and among water management units. No evidence of isolation by distance was detected in a gene flow and distance correlation between subpopulations. Confidence intervals for the estimated F-statistic values crossed zero, indicating that there was no significant genetic difference between subpopulations within a region or between regions. Notably, the genetic variation among subpopulations in a water conservation area was greater than variation among regions (Fsp>FPT). These data indicate that the spatial scale of recolonization following local extinction appears to be most important within water management units.

Connectivity of fragmented amphibian populations in a neotropical landscape

A high proportion of amphibian species are threatened with extinction globally, and habitat loss and degradation are the most frequently implicated causes. Rapid deforestation for the establishment of agricultural production is a primary driver of habitat loss in tropical zones where amphibian diversity is highest. Land-cover change affects native assemblages, in part, through the reduction of habitat area and the reduction of movement among remnant populations. Decreased gene flow contributes to loss of genetic diversity, which limits the ability of local populations to respond to further environmental changes. The focus of this dissertation is on the degree to which common land uses in Sarapiquí, Costa Rica impede the movement of two common amphibian species. First, I used field experiments, including displacement trials, and a behavioral landscape ecology framework to investigate the resistance of pastures to movement of Oophaga pumilio. Results from experiments demonstrate that pastures do impede movement of O. pumilio relative to forest. Microclimatic effects on movement performance as well as limited perceptual ranges likely contribute to reduced return rates through pastures. Next, I linked local processes to landscape scale estimates of resistance. I conducted experiments to measure habitat-specific costs to movement for O. pumilio and Craugastor bransfodrii, and then used experimental results to parameterize connectivity models. Model validation indicated highest support for resistance estimates generated from responses to land-use specific microclimates for both species and to predator encounters for O. pumilio. Finally, I used abundance and experiment-derived resistance estimates to analyze the effects of prevalent land uses on population genetic structure of the two focal species. While O. pumilio did not exhibit a strong response to landscape heterogeneity and was primarily structured by distances among sites, C. bransfordii genetic variation was explained by resistance estimates from abundance and experiment data. Collectivity, this work demonstrates that common land uses can offer different levels of resistance to amphibian movements in Sarapiquí and illustrates the value of investigating local scales processes to inform interpretation of landscape-scale patterns.^

An Integrated Flow and Transport Model to Study the Impact of Mercury Remediation Strategies for East Fork Poplar Creek Watershed, Oak Ridge, Tennessee

An integrated flow and transport model using MIKE SHE/MIKE 11 software was developed to predict the flow and transport of mercury, Hg(II), under varying environmental conditions. The model analyzed the impact of remediation scenarios within the East Fork Poplar Creek watershed of the Oak Ridge Reservation with respect to downstream concentration of mercury. The numerical simulations included the entire hydrological cycle: flow in rivers, overland flow, groundwater flow in the saturated and unsaturated zones, and evapotranspiration and precipitation time series. Stochastic parameters and hydrologic conditions over a five year period of historical hydrological data were used to analyze the hydrological cycle and to determine the prevailing mercury transport mechanism within the watershed. Simulations of remediation scenarios revealed that reduction of the highly contaminated point sources, rather than general remediation of the contaminant plume, has a more direct impact on downstream mercury concentrations.

Variabilidade genética de Leishmania spp. circulantes entre seres humanos e cães e infecção de flebotomíneos em áreas endêmicas para as leishmanioses no Rio Grande do Norte

Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector’s species and the parasite’s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.

Variabilidade genética de Leishmania spp. circulantes entre seres humanos e cães e infecção de flebotomíneos em áreas endêmicas para as leishmanioses no Rio Grande do Norte

Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector’s species and the parasite’s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.

Molecular analysis of evolutionary relationships of Phaseolus dumosus with the gene pool of common bean

Valuable genetic variation for bean breeding programs is held within the common bean secondary gene pool which consists of Phaseolus albescens, P. coccineus, P. costaricensis, and P. dumosus. However, the use of close relatives for bean improvement is limited due to the lack of knowledge about genetic variation and genetic plasticity of many of these species. Characterisation and analysis of the genetic diversity is necessary among beans' wild relatives; in addition, conflicting phylogenies and relationships need to be understood and a hypothesis of a hybrid origin of P. dumosus needs to be tested. This thesis research was orientated to generate information about the patterns of relationships among the common bean secondary gene pool, with particular focus on the species Phaseolus dumosus. This species displays a set of characteristics of agronomic interest, not only for the direct improvement of common bean but also as a source of valuable genes for adaptation to climate change. Here I undertake the first comprehensive study of the genetic diversity of P. dumosus as ascertained from both nuclear and chloroplast genome markers. A germplasm collection of the ancestral forms of P. dumosus together with wild, landrace and cultivar representatives of all other species of the common bean secondary gene pool, were used to analyse genetic diversity, phylogenetic relationships and structure of P. dumosus. Data on molecular variation was generated from sequences of cpDNA loci accD-psaI spacer, trnT-trnL spacer, trnL intron and rps14-psaB spacer and from the nrDNA the ITS region. A whole genome DArT array was developed and used for the genotyping of P. dumosus and its closes relatives. 4208 polymorphic markers were generated in the DArT array and from those, 742 markers presented a call rate >95% and zero discordance. DArT markers revealed a moderate genetic polymorphism among P. dumosus samples (13% of polymorphic loci), while P. coccineus presented the highest level of polymorphism (88% of polymorphic loci). At the cpDNA one ancestral haplotype was detected among all samples of all species in the secondary genepool. The ITS region of P. dumosus revealed high homogeneity and polymorphism bias to P. coccineus genome. Phylogenetic reconstructions made with Maximum likelihood and Bayesian methods confirmed previously reported discrepancies among the nuclear and chloroplast genomes of P. dumosus. The outline of relationships by hybridization networks displayed a considerable number of interactions within and between species. This research provides compelling evidence that P. dumosus arose from hybridisation between P. vulgaris and P. coccineus and confirms that P. costaricensis has likely been involved in the genesis or backcrossing events (or both) in the history of P. dumosus. The classification of the specie P. persistentus was analysed based on cpDNA and ITS sequences, the results found this species to be highly related to P. vulgaris but not too similar to P. leptostachyus as previously proposed. This research demonstrates that wild types of the secondary genepool carry a significant genetic variation which makes this a valuable genetic resource for common bean improvement. The DArT array generated in this research is a valuable resource for breeding programs since it has the potential to be used in several approaches including genotyping, discovery of novel traits, mapping and marker-trait associations. Efforts should be made to search for potential populations of P. persistentus and to increase the collection of new populations of P. dumosus, P. albescens and P. costaricensis that may provide valuable traits for introgression into common bean and other Phaseolus crops.

Connectivity of fragmented amphibian populations in a Neotropical landscape

A high proportion of amphibian species are threatened with extinction globally, and habitat loss and degradation are the most frequently implicated causes. Rapid deforestation for the establishment of agricultural production is a primary driver of habitat loss in tropical zones where amphibian diversity is highest. Land-cover change affects native assemblages, in part, through the reduction of habitat area and the reduction of movement among remnant populations. Decreased gene flow contributes to loss of genetic diversity, which limits the ability of local populations to respond to further environmental changes. The focus of this dissertation is on the degree to which common land uses in Sarapiquí, Costa Rica impede the movement of two common amphibian species. First, I used field experiments, including displacement trials, and a behavioral landscape ecology framework to investigate the resistance of pastures to movement of Oophaga pumilio. Results from experiments demonstrate that pastures do impede movement of O. pumilio relative to forest. Microclimatic effects on movement performance as well as limited perceptual ranges likely contribute to reduced return rates through pastures. Next, I linked local processes to landscape scale estimates of resistance. I conducted experiments to measure habitat-specific costs to movement for O. pumilio and Craugastor bransfodrii, and then used experimental results to parameterize connectivity models. Model validation indicated highest support for resistance estimates generated from responses to land-use specific microclimates for both species and to predator encounters for O. pumilio. Finally, I used abundance and experiment-derived resistance estimates to analyze the effects of prevalent land uses on population genetic structure of the two focal species. While O. pumilio did not exhibit a strong response to landscape heterogeneity and was primarily structured by distances among sites, C. bransfordii genetic variation was explained by resistance estimates from abundance and experiment data. Collectivity, this work demonstrates that common land uses can offer different levels of resistance to amphibian movements in Sarapiquí and illustrates the value of investigating local scales processes to inform interpretation of landscape-scale patterns.

Forward-in-Time, Spatially Explicit Modeling Software to Simulate Genetic Lineages Under Selection

SELECTOR is a software package for studying the evolution of multiallelic genes under balancing or positive selection while simulating complex evolutionary scenarios that integrate demographic growth and migration in a spatially explicit population framework. Parameters can be varied both in space and time to account for geographical, environmental, and cultural heterogeneity. SELECTOR can be used within an approximate Bayesian computation estimation framework. We first describe the principles of SELECTOR and validate the algorithms by comparing its outputs for simple models with theoretical expectations. Then, we show how it can be used to investigate genetic differentiation of loci under balancing selection in interconnected demes with spatially heterogeneous gene flow. We identify situations in which balancing selection reduces genetic differentiation between population groups compared with neutrality and explain conflicting outcomes observed for human leukocyte antigen loci. These results and three previously published applications demonstrate that SELECTOR is efficient and robust for building insight into human settlement history and evolution.

Anthropogenic impact on diazotrophic diversity in the mangrove rhizosphere revealed by nifH pyrosequencing

Diazotrophs in the mangrove rhizosphere play a major role in providing new nitrogen to the mangrove ecosystem and their composition and activity are strongly influenced by anthropogenic activity and ecological conditions. In this study, the diversity of the diazotroph communities in the rhizosphere sediment of five tropical mangrove sites with different levels of pollution along the north and south coastline of Singapore were studied by pyrosequencing of the nifH gene. Bioinformatics analysis revealed that in all the studied locations, the diazotroph communities comprised mainly of members of the diazotrophic cluster I and cluster III. The detected cluster III diazotrophs, which were composed entirely of sulfate-reducing bacteria, were more abundant in the less polluted locations. The metabolic capacities of these diazotrophs indicate the potential for bioremediation and resiliency of the ecosystem to anthropogenic impact. In heavily polluted locations, the diazotrophic community structures were markedly different and the diversity of species was significantly reduced when compared with those in a pristine location. This, together with the increased abundance of Marinobacterium, which is a bioindicator of pollution, suggests that anthropogenic activity has a negative impact on the genetic diversity of diazotrophs in the mangrove rhizosphere.

Genetic effect of domestication selective pressures on Pacific oyster at larval stage

Among bivalve species, the Pacific oyster, Crassostrea gigas, is the most economically important bivalve production over the world. Today, C. gigas is subject to an important production effort that leads to an intensive artificial selection. Larval stage is relatively unknown, specifically in a domestication context. Genetic consequence of artificial selection is still at a preliminary study. We aimed to tackle the consequence of inconscient domestication on the variance reproductive success focusing on larval stage, keystone of the life cycle. We studied two kinds of specific selective processes that common hatchery rearing practices exert : the effect of discarding the smallest larvae on genetic diversity and the artificial environment rearing effect via the temperature providing a contrast resembling wild versus hatchery conditions (20 and 26°C). In order to monitor the effect of the selection of fast growing larvae by sieving, growth variability and genetic diversity in a larval population descended from a factorial breeding was studied. We used a mixed-family approach to reduce potentially confounding environmental biais. The retrospective assignment of individuals to family groups has been performed using a three microsatellite markers set. Two different rearing were carried out in parallel. For three (replicates) 50-l tanks, the smallest larvae were progressively discarded by selective sieving, whereas for the three others no selective sieving was performed. The intensity of selective sieving was adjusted so as to discard 50% of the larvae over the whole rearing period in a progressive manner. As soon as the larvae reached the pediveliger stage, ready to settle larvae were sampled for genetic analysis. Regarding the artificial environment rearing effect via the temperature, we used a similar mixed-family approach. The progeny from a factorial breeding design was divided as follows: three (replicates) 50-l tanks were dedicaced to a rearing at 26°C versus 20°C for three others 50-l tanks. The whole size variability was preserved for this experiment. Individual growth measurements for larvae genetically identified have been performed at days 22 and 30 after fertilization for both conditions. In a same way, we collected individual measurements for genotyped juvenile oysters (80 days after fertilization). At a phenotypic scale, relative survival and settlement success for larvae with sieving were higher. Sieving appears as a time-saving process associated with a better relative survival ratio. But in the same time, our results confirm that a significant genetic variability exist for early developmental traits in the Pacific oyster. This is congruent with the results already obtained that investigated genetic variability and genetic correlations in early life-history traits of Crassostrea gigas. Discarding around 50% of the smallest larvae can lead to significant selection at the larval stage.

Population genetic structure of Neogobius caspius (Eichwald ,1831) in the South Caspian Sea using PCR-RFLP Marker

Neogobius caspius is a small benthic fish that is native to the Caspian Sea. The importance of this fish is because of it is role as a main food resource of the sturgeon fish. The genetic diversity of N. caspius population in the Caspian Sea was studied using PCR- RFLP technique. A total of 135 samples of N. caspius were collected from coastal line in the north Caspian sea, including specimens from coasts of Anzali , Torkman Port and Chalus. Genomic DNA was extracted by phenol-chloroform method and then was amplified using a pair primer of cytochrom b gene, 2 tRNA gene and the control region sequences by a thermal cycler. D2 (5'-CCGGAGTATGTAGGGCATTCTCAC-3'), CY1 (5'-YYTAACCRRGACYAATGACTTGA-3') 12 restriction enzyme were used to digest the target gene region including: Alul HincII —Tas1 —Rsa1 -MboI -DraI -BSeNI(BSRI) Alw261(BsmAI). Bsul 51 Hin11 Bsh12851- BsuRI(HaeIII) digested PCR products were observed by silver staining method followed by Polyacrylamide gel electrophoresis (PAGE). The results were shown the same pattern among the species. There was no polymorphism and no differentiation in population in the Neogobius caspius fish and all individuals have shown homogenous genotype.

Populations genetics study of the Ammi Genus in the Azores

Dissertação de Mestrado, Biodiversidade e Biotecnologia Vegetal, 6 de Julho de 2016, Universidade dos Açores.

Genome-scale analysis of the non-cultivable Treponema pallidum reveals extensive within-patient genetic variation

Insights into the genomic adaptive traits of Treponema pallidum, the causative bacterium of syphilis, have long been hampered due to the absence of in vitro culture models and the constraints associated with its propagation in rabbits. Here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the context of human infection. This strategy has unveiled a scenario of discreet T. pallidum interstrain single-nucleotide-polymorphism-based microevolution, contrasting with a rampant within-patient genetic heterogeneity mainly targeting multiple phase-variable loci and a major antigen-coding gene (tprK). TprK demonstrated remarkable variability and redundancy, intra- and interpatient, suggesting ongoing parallel adaptive diversification during human infection. Some bacterial functions (for example, flagella- and chemotaxis-associated) were systematically targeted by both inter- and intrastrain single nucleotide polymorphisms, as well as by ongoing within-patient phase variation events. Finally, patient-derived genomes possess mutations targeting a penicillin-binding protein coding gene (mrcA) that had never been reported, unveiling it as a candidate target to investigate the impact on the susceptibility to penicillin. Our findings decode the major genetic mechanisms by which T. pallidum promotes immune evasion and survival, and demonstrate the exceptional power of characterizing evolving pathogen subpopulations during human infection.

Recruitment patterns and processes of coastal fish species in a temperate rocky reef

Tese de Doutoramento, Ciências do Mar, da Terra e do Ambiente, Ramo: Ciências do Mar, Especialização em Ecologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Phylogeographical history of the white seabream Diplodus sargus (Sparidae): Implications for insularity

Partial sequences of the mitochondrial control region and its comparison with previously published cytochrome b (cyt-b) and microsatellite data were used to investigate the influence of island isolation and connectivity on white seabream genetic structure. To achieve this, a total of 188 individuals from four island localities (Castellamare and Mallorca, Mediterranean Sea; Azores and Canary Islands, Atlantic Ocean) and five coastal localities (Banyuls, Murcia and Tunisia, Mediterranean Sea; Galicia and Faro, Atlantic Ocean) were analysed. Results showed high haplotype diversity and low to moderate nucleotide diversity in all populations (except for the Canary Islands). This pattern of genetic diversity is attributed to a recent population expansion which is corroborated by other results such as cyt-b network and demographic analyses. Low differentiation among Mediterranean/Atlantic and coastal/island groups was shown by the AMOVA and FST values, although a weak phylogeographic break was detected using cyt-b data. However, we found a clear and significant island/ distance effect with regard to the Azores islands. Significant genetic differentiation has been detected between the Azores islands and all other populations. The large geographical distance between the European continental slope and the Azores islands is a barrier to gene flow within this region and historic events such as glaciation could also explain this genetic differentiation.