Molecular and cytogenetic analyses of cryptic species within the Synbranchus marmoratus Bloch, 1795 (Synbranchiformes: Synbranchidae) grouping: species delimitations, karyotypic evolution and intraspecific diversification

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phylogeographic Structure and Karyotypic Diversity of the Brazilian Shrew Mouse (Blarinomys breviceps, Sigmodontinae) in the Atlantic Forest

Blarinomys breviceps possesses cryptic and burrowing habits with poorly documented genetics and life history traits. Due to its rarity, only a few specimens and DNA sequences have been deposited in collections worldwide. Here, we present the most comprehensive cytogenetic and molecular characterization of this rare genus. Phylogenetic analyses based on partial cytochrome b sequences were performed, attempting to establish the relationships among individuals with distinct karyotypes along the geographic distribution of the genus in the Atlantic Forest. Classical and molecular cytogenetics, using banding patterns and FISH of telomeric and whole chromosome X-specific painting probes (obtained from the Akodontini Akodon cursor) were used to characterize and compare the chromosomal complements. Molecular phylogenetic analyses recovered 2 main geographically structured clades, northeastern and southeastern with pair-wise sequence divergences among specimens varying between 4.9 and 8.4%. Eight distinct karyomorphs are described: (A) 2n = 52 (50A, XX), (B) 2n = 52 (48A, XY+2Bs), (C) 2n = 45 (42A, XY+1B), (D) 2n = 43 (37A, XX+4Bs), (E) 2n = 37 (34A, XY+1B), (F) 2n = 34 (32A, XX), (G) 2n = 31 (27A, XX+2Bs), (H) 2n = 28 (26A, XY), all with the same number of autosomal arms (FNA = 50). Variation of 0-4 supernumerary chromosomes (Bs) presenting heterogeneity in morphology and distribution of interstitial telomeric sequences (ITSs) is reported. ITSs are also found in some metacentric autosomes. The phylogeographic separation between 2 major lineages with high levels of genetic divergence, and the wide karyotypic diversity indicate that B. breviceps is a diverse group that warrants taxonomic re-evaluation. Copyright (C) 2012 S. Karger AG, Basel

Sedimentary processes on the NW Iberian Continental Shelf since the Little Ice Age

The OMEX core CD110 W90, retrieved from the Douro Mud Patch (DMP) off the River Douro in the north of Portugal, records the period since the beginning of Little Ice Age (LIA). The core chronology is based upon the data attributes for Pb-210, Cs-137 and a C-14 dating from a level near the core base. Geochemical, granulometric, microfaunal (benthic foraminifera) and compositional data suggest the occurrence of precipitation changes which may have been, at least partially, influenced by the North Atlantic Oscillation (NAO), that contributes to the regulation of the ocean-atmosphere dynamics in the North Atlantic. Southwesterly Atlantic storm track is associated with the negative phases of the NAO, when the Azores High is anomalously weak, higher oceanographic hydrodynamism, downwelling events and increased rainfall generally occurs. Prevalence of these characteristics during the LIA left a record that corresponds to phases of major floods. During these phases the DMP received a higher contribution of relatively coarse-grained terrigenous sediments, enriched in quartz particles, which diluted the contribution of other minerals, as indicated by reduced concentrations of several lithogenic chemical elements such as: Al, As, Ba, Ce, Co, Cu, Fe, K, La, Li, Mg, Mn, Mo, Na, Ni, P, Rb, Sc, Sn, Th, V and Y. The presence of biogenic carbonate particles also underwent dilution, as revealed by the smaller abundance of foraminifera and correlative lower concentrations of Ca and Sr. During this period, the DMP also received an increased contribution of organic matter, indicated by higher values of lignin remains and a benthic foraminifera high productivity index, or BFHP, which gave rise to early diagenetic changes with pyrite formation. Since the beginning of the 20th century this contribution diminished, probably due to several drier periods and the impact of human activities in the river basins, e.g. construction of dams, or, on the littoral areas, construction of hard-engineering structures and sand extraction activities. During the first half of the 20th century mainly positive phases of the NAO prevailed, caused by the above normal strengthening of the subtropical high pressure centre of the Azores and the deepening of the low pressure centre in Iceland. These phases may have contributed to the reduction in the supply of both terrigenous sediments and organic matter from shallow water to the DMP. During the positive phases of the NAO, sedimentation became finer. The development of mining and industrial activities during the 20th century is marked, in this core, by higher concentrations of Pb. Furthermore, the erosion of heaps resulting from wolfram exploitation leaves its signature as a peak of W concentrations recorded in the sediments of the DMP deposited between the 1960s and the 1990s. Wolfram exploitation was an important activity in the middle part of the 20th century, particularly during the period of the Second World War. (C) 2012 Elsevier Ltd. All rights reserved.

Molecular Characterization and Physical Mapping of Two Classes of 5S rDNA in the Genomes of Gymnotus sylvius and G. inaequilabiatus (Gymnotiformes, Gymnotidae)

The nucleotide sequences of the 5S rRNA multigene family and their distribution across the karyotypes in 2 species of Gymnotiformes, genus Gymnotus (G. sylvius and G. inaequilabiatus) were investigated by means of fluorescence in situ hybridization (FISH). The results showed the existence of 2 distinct classes of 5S rDNA sequences in both species: class I and class II. A high conservative pattern of the codifying region of the 5S rRNA gene was identified, contrasting with significant alterations detected in the nontranscribed spacer (NTS). The presence of TATA-like sequences along the NTS of both species was an expected occurrence, since such sequences have been associated with the regulation of the gene expression. FISH using 5S rDNA class I and class II probes revealed that both gene classes were collocated in the same chromosome pair in the genome of G. sylvius, while in that of G. inaequilabiatus, class II appeared more disperse than class I. Copyright (C) 2012 S. Karger AG, Basel

Treatment of rice straw hemicellulosic hydrolysates with advanced oxidative processes: a new and promising detoxification method to improve the bioconversion process

Abstract Background The use of lignocellulosic constituents in biotechnological processes requires a selective separation of the main fractions (cellulose, hemicellulose and lignin). During diluted acid hydrolysis for hemicellulose extraction, several toxic compounds are formed by the degradation of sugars and lignin, which have ability to inhibit microbial metabolism. Thus, the use of a detoxification step represents an important aspect to be considered for the improvement of fermentation processes from hydrolysates. In this paper, we evaluated the application of Advanced Oxidative Processes (AOPs) for the detoxification of rice straw hemicellulosic hydrolysate with the goal of improving ethanol bioproduction by Pichia stipitis yeast. Aiming to reduce the toxicity of the hemicellulosic hydrolysate, different treatment conditions were analyzed. The treatments were carried out according to a Taguchi L16 orthogonal array to evaluate the influence of Fe+2, H2O2, UV, O3 and pH on the concentration of aromatic compounds and the fermentative process. Results The results showed that the AOPs were able to remove aromatic compounds (furan and phenolic compounds derived from lignin) without affecting the sugar concentration in the hydrolysate. Ozonation in alkaline medium (pH 8) in the presence of H2O2 (treatment A3) or UV radiation (treatment A5) were the most effective for hydrolysate detoxification and had a positive effect on increasing the yeast fermentability of rice straw hemicellulose hydrolysate. Under these conditions, the higher removal of total phenols (above 40%), low molecular weight phenolic compounds (above 95%) and furans (above 52%) were observed. In addition, the ethanol volumetric productivity by P. stipitis was increased in approximately twice in relation the untreated hydrolysate. Conclusion These results demonstrate that AOPs are a promising methods to reduce toxicity and improve the fermentability of lignocellulosic hydrolysates.

Halogenated aliphatic and aromatic aerobic biodegradation via direct metabolism and cometabolism: batch and continuous tests

In this thesis the application of biotechnological processes based on microbial metabolic degradation of halogenated compound has been investigated. Several studies showed that most of these pollutants can be biodegraded by single bacterial strains or mixed microbial population via aerobic direct metabolism or cometabolism using as a growth substrates aromatic or aliphatic hydrocarbons. The enhancement of two specific processes has been here object of study in relation with its own respective scenario described as follow: 1st) the bioremediation via aerobic cometabolism of soil contaminated by a high chlorinated compound using a mixed microbial population and the selection and isolation of consortium specific for the compound. 2nd) the implementation of a treatment technology based on direct metabolism of two pure strains at the exact point source of emission, preventing dilution and contamination of large volumes of waste fluids polluted by several halogenated compound minimizing the environmental impact. In order to verify the effect of these two new biotechnological application to remove halogenated compound and purpose them as a more efficient alternative continuous and batch tests have been set up in the experimental part of this thesis. Results obtained from the continuous tests in the second scenario have been supported by microbial analysis via Fluorescence in situ Hybridisation (FISH) and by a mathematical model of the system. The results showed that both process in its own respective scenario offer an effective solutions for the biological treatment of chlorinate compound pollution.

Speciation reversal and biodiversity dynamics with hybridization in changing environments

A considerable fraction of the world's biodiversity is of recent evolutionary origin and has evolved as a by-product of, and is maintained by, divergent adaptation in heterogeneous environments. Conservationists have paid attention to genetic homogenization caused by human-induced translocations (e.g. biological invasions and stocking), and to the importance of environmental heterogeneity for the ecological coexistence of species. However, far less attention has been paid to the consequences of loss of environmental heterogeneity to the genetic coexistence of sympatric species. Our review of empirical observations and our theoretical considerations on the causes and consequences of interspecific hybridization suggest that a loss of environmental heterogeneity causes a loss of biodiversity through increased genetic admixture, effectively reversing speciation. Loss of heterogeneity relaxes divergent selection and removes ecological barriers to gene flow between divergently adapted species, promoting interspecific introgressive hybridization. Since heterogeneity of natural environments is rapidly deteriorating in most biomes, the evolutionary ecology of speciation reversal ought to be fully integrated into conservation biology.

Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains

We describe a rational approach to simultaneously test Escherichia coli strains for the presence of known virulence genes in a reverse dot blot procedure. Specific segments of virulence genes of E. coli designed to have similar hybridization parameters were subcloned on plasmids and subsequently amplified by PCR as unlabeled probes in amounts sufficient to be bound to nylon membranes. Various pathogenic isolates and laboratory strains of E. coli were probed for the presence of virulence genes by labeling the genomic DNA of these strains with digoxigenin and then hybridizing them to the prepared nylon membranes. These hybridization results demonstrated that besides the E. coli K-12 safety strain derivatives, E. coli B and C strains are also devoid of genes encoding any of the investigated virulence factors. In contrast, pathogenic E. coli control strains, used to evaluate the method, showed typical hybridization patterns. The described probes and their easy application on a single filter were shown to provide a useful tool for the safety assessment of E. coli strains to be used as hosts in biotechnological processes. This approach might also be used for the identification and characterization of clinically significant E. coli isolates from human and animal species.

Chloroplast DNA evidence of colonization, adaptive radiation, and hybridization in the evolution of the Macaronesian flora.

Most evolutionary studies of oceanic islands have focused on the Pacific Ocean. There are very few examples from the Atlantic archipelagos, especially Macaronesia, despite their unusual combination of features, including a close proximity to the continent, a broad range of geological ages, and a biota linked to a source area that existed in the Mediterranean basin before the late Tertiary. A chloroplast DNA (cpDNA) restriction site analysis of Argyranthemum (Asteraceae: Anthemideae), the largest endemic genus of plants of any volcanic archipelago in the Atlantic Ocean, was performed to examine patterns of plant evolution in Macaronesia. cpDNA data indicated that Argyranthemum is a monophyletic group that has speciated recently. The cpDNA tree showed a weak correlation with the current sectional classification and insular distribution. Two major cpDNA lineages were identified. One was restricted to northern archipelagos--e.g., Madeira, Desertas, and Selvagens--and the second comprised taxa endemic to the southern archipelago--e.g., the Canary Islands. The two major radiations identified in the Canaries are correlated with distinct ecological habitats; one is restricted to ecological zones under the influence of the northeastern trade winds and the other to regions that are not affected by these winds. The patterns of phylogenetic relationships in Argyranthemum indicate that interisland colonization between similar ecological zones is the main mechanism for establishing founder populations. This phenomenon, combined with rapid radiation into distinct ecological zones and interspecific hybridization, is the primary explanation for species diversification.

Evaluation of reproductive barriers contributes to the development of novel interspecific hybrids in the Kalanchoe genus

Background: Interspecific hybridization is a useful tool in ornamental breeding to increase genetic variability and introduce new valuable traits into existing cultivars. The successful formation of interspecific hybrids is frequently limited by the presence of pre- and post-fertilization barriers. In the present study, we investigated the nature of hybridization barriers occurring in crosses between Kalanchoe species and evaluated possibilities of obtaining interspecific hybrids. Results: The qualitative and quantitative analyses of pollen tube growth in situ were performed following intra-and interspecific pollinations. They revealed occurrence of pre-fertilization barriers associated with inhibition of pollen germination on the stigma and abnormal growth of pollen tubes. Unilateral incongruity related to differences in pistil length was also observed. The pollen quality was identified as a strong factor influencing the number of pollen tubes germinating in the stigma. In relation to post-fertilization barriers, endosperm degeneration was a probable barrier hampering production of interspecific hybrids. Moreover, our results demonstrate the relation of genetic distance estimated by AFLP marker analysis of hybridization partners with cross-compatibility of Kalanchoe species. At the same time, differences in ploidy did not influence the success of interspecific crosses. Conclusions: Our study presents the first comprehensive analysis of hybridization barriers occurring within Kalanchoe genus. Reproductive barriers were detected on both, pre- and post-fertilization levels. This new knowledge will contribute to further understanding of reproductive isolation of Kalanchoe species and facilitate breeding of new cultivars. For the first time, interspecific hybrids between K. nyikae as maternal plant and K. blossfeldiana as well as K. blossfeldiana and K. marnieriana were generated.

Multilocus genetic analyses provide insight into speciation and hybridization in aquatic grasses, genus Ruppia

Aquatic plants of the genus Ruppia inhabit some of the most threatened habitats in the world, such as coastal lagoons and inland saline to brackish waters where their meadows play several key roles. The evolutionary history of this genus has been affected by the processes of hybridization, polyploidization, and vicariance, which have resulted in uncertainty regarding the number of species. In the present study, we apply microsatellite markers for the identification, genetic characterization, and detection of hybridization events among populations of putative Ruppia species found in the southern Iberian Peninsula, with the exception of a clearly distinct species, the diploid Ruppia maritima. Microsatellite markers group the populations into genetically distinct entities that are not coincident with geographical location and contain unique diagnostic alleles. These results support the interpretation of these entities as distinct species: designated here as (1) Ruppia drepanensis, (2) Ruppia cf. maritima, and (3) Ruppia cirrhosa. A fourth distinct genetic entity was identified as a putative hybrid between R. cf. maritima and R. cirrhosa because it contained a mixture of microsatellite alleles that are otherwise unique to these putative species. Hence, our analyses were able to discriminate among different genetic entities of Ruppia and, by adding multilocus nuclear markers, we confirm hybridization as an important process of speciation within the genus. In addition, careful taxonomic curation of the samples enabled us to determine the genotypic and genetic diversity and differentiation among populations of each putative Ruppia species. This will be important for identifying diversity hotspots and evaluating patterns of population genetic connectivity. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 00, 000–000.

GENETIC VARIATION, LOCAL ADAPTATION AND POPULATION STRUCTURE IN NORTH AMERICAN RED OAK SPECIES, QUERCUS RUBRA L. AND Q. ELLIPSOIDALIS E. J. HILL

Forest trees, like oaks, rely on high levels of genetic variation to adapt to varying environmental conditions. Thus, genetic variation and its distribution are important for the long-term survival and adaptability of oak populations. Climate change is projected to lead to increased drought and fire events as well as a northward migration of tree species, including oaks. Additionally, decline in oak regeneration has become increasingly concerning since it may lead to decreased gene flow and increased inbreeding levels. This will in turn lead to lowered levels of genetic diversity, negatively affecting the growth and survival of populations. At the same time, populations at the species’ distribution edge, like those in this study, could possess important stores of genetic diversity and adaptive potential, while also being vulnerable to climatic or anthropogenic changes. A survey of the level and distribution of genetic variation and identification of potentially adaptive genes is needed since adaptive genetic variation is essential for their long-term survival. Oaks possess a remarkable characteristic in that they maintain their species identity and specific environmental adaptations despite their propensity to hybridize. Thus, in the face of interspecific gene flow, some areas of the genome remain differentiated due to selection. This characteristic allows the study of local environmental adaptation through genetic variation analyses. Furthermore, using genic markers with known putative functions makes it possible to link those differentiated markers to potential adaptive traits (e.g., flowering time, drought stress tolerance). Demographic processes like gene flow and genetic drift also play an important role in how genes (including adaptive genes) are maintained or spread. These processes are influenced by disturbances, both natural and anthropogenic. An examination of how genetic variation is geographically distributed can display how these genetic processes and geographical disturbances influence genetic variation patterns. For example, the spatial clustering of closely related trees could promote inbreeding with associated negative effects (inbreeding depression), if gene flow is limited. In turn this can have negative consequences for a species’ ability to adapt to changing environmental conditions. In contrast, interspecific hybridization may also allow the transfer of genes between species that increase their adaptive potential in a changing environment. I have studied the ecologically divergent, interfertile red oaks, Quercus rubra and Q. ellipsoidalis, to identify genes with potential roles in adaptation to abiotic stress through traits such as drought tolerance and flowering time, and to assess the level and distribution of genetic variation. I found evidence for moderate gene flow between the two species and low interspecific genetic differences at most genetic markers (Lind and Gailing 2013). However, the screening of genic markers with potential roles in phenology and drought tolerance led to the identification of a CONSTANS-like (COL) gene, a candidate gene for flowering time and growth. This marker, located in the coding region of the gene, was highly differentiated between the two species in multiple geographical areas, despite interspecific gene flow, and may play a role in reproductive isolation and adaptive divergence between the two species (Lind-Riehl et al. 2014). Since climate change could result in a northward migration of trees species like oaks, this gene could be important in maintaining species identity despite increased contact zones between species (e.g., increased gene flow). Finally I examined differences in spatial genetic structure (SGS) and genetic variation between species and populations subjected to different management strategies and natural disturbances. Diverse management activities combined with various natural disturbances as well as species specific life history traits influenced SGS patterns and inbreeding levels (Lind-Riehl and Gailing submitted).

Breaking Oil-in-water Emulsions Stabilized By Yeast.

Several biotechnological processes can show an undesirable formation of emulsions making difficult phase separation and product recovery. The breakup of oil-in-water emulsions stabilized by yeast was studied using different physical and chemical methods. These emulsions were composed by deionized water, hexadecane and commercial yeast (Saccharomyces cerevisiae). The stability of the emulsions was evaluated varying the yeast concentration from 7.47 to 22.11% (w/w) and the phases obtained after gravity separation were evaluated on chemical composition, droplet size distribution, rheological behavior and optical microscopy. The cream phase showed kinetic stability attributed to mechanisms as electrostatic repulsion between the droplets, a possible Pickering-type stabilization and the viscoelastic properties of the concentrated emulsion. Oil recovery from cream phase was performed using gravity separation, centrifugation, heating and addition of demulsifier agents (alcohols and magnetic nanoparticles). Long centrifugation time and high centrifugal forces (2h/150,000×g) were necessary to obtain a complete oil recovery. The heat treatment (60°C) was not enough to promote a satisfactory oil separation. Addition of alcohols followed by centrifugation enhanced oil recovery: butanol addition allowed almost complete phase separation of the emulsion while ethanol addition resulted in 84% of oil recovery. Implementation of this method, however, would require additional steps for solvent separation. Addition of charged magnetic nanoparticles was effective by interacting electrostatically with the interface, resulting in emulsion destabilization under a magnetic field. This method reached almost 96% of oil recovery and it was potentially advantageous since no additional steps might be necessary for further purifying the recovered oil.

Carotenóides: propriedades, aplicações e biotransformação para formação de compostos de aroma

Carotenoids are widely distributed in nature, providing yellow, orange or red color in a great number of vegetables, microorganisms and in some animals. Carotenoids act as biological antioxidants and seem to play an important role in human health by protecting cells and tissues from the damaging effects of free radicals and singlet oxygen. Several authors describe the oxidative cleavage of carotenoids in flavor compounds as occuring through chemical or photochemical degradations or through biotechnological processes. Biotransformation of carotenoids seems to be a reasonable alternative to produce flavor compounds since these compounds are considered 'natural' ingredients. In this work we describe the properties of some carotenoids, as well as biotechnological approaches to obtain its oxyfunctionalized derivatives.

Atividade de extratos de Arrabidaea chica (Humb. & Bonpl.) Verlot obtidos por processos biotecnológicos sobre a proliferação de fibroblastos e células tumorais humanas

Arrabidaea chica (H&B) Verlot is a plant popularly known as Pariri and this species is a known source of anthocyanins, flavonoids and tannins. This report describes an approach involving enzymatic treatment prior to extraction procedures to enhance A chica crude extract anticancer activity. Anticancer activity in human cancer cell lines in vitro using a 48 h SRB cell viability assay was performed to determine growth inhibition and cytotoxic properties. The final extraction yield without enzyme treatment was higher (24.28%) compared to the enzyme-treated material (19.03%), with an enhanced aglycones anthocyanin ratio as determined by HPLC- DAD and LC-MS with direct infusion.