Caracterização biológica e molecular de quatro cepas de Trypanosoma cruzi Chagas,1909 (Kinetoplastida, Trypanosomatidae) isoladas de pacientes chagásicos crônicos

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Susceptibility to deltamethrin in the predatory mites Neoseiulus californicus and Phytoseiulus macropilis (Acari: Phytoseiidae) populations in protected ornamental crops in Brazil

Knowledge of inter and intra-specific variation in the susceptibility of natural enemies to pesticides could help to better design integrated pest management strategies. The objective of this research was to evaluate the susceptibility to deltamethrin in populations of the predatory mites Neoseiulus californicus (McGregor) and Phytoseiulus macropilis (Banks) populations collected from protected ornamental crops in Brazil. The susceptibility to deltamethrin was characterized against immature and adult stages of both species. The impact of this insecticide was also measured by estimating the intrinsic rate of increase (r (i)). The immature and adult stages of N. californicus were approximately 3,600 and 3,000-fold more tolerant to deltamethrin than those of P. macropilis. However, high variability in the susceptibility to this insecticide was detected among P. macropilis populations, with resistance ratios of up to 3,500-fold. The selection of deltamethrin-resistant strains of P. macropilis could be exploited in applied biological control programs.

Demersal communities in the Mediterranean Sea: a case study of Triglidae (Osteichthyes, Scorpaeniformes) on the conservation and sustainable use of marine resources

An appropriate management of fisheries resources can only be achieved with the continuous supply of information on the structure and biology of populations, in order to predict the temporal fluctuations. This study supports the importance of investigating the bio-ecology of increasingly exploited and poorly known species, such as gurnards (Osteichthyes, Triglidae) from Adriatic Sea (Mediterranean), to quantify their ecological role into marine community. It also focuses on investigate inter and intra-specific structuring factor of Adriatic population. These objectives were achieved by: 1) investigating aspects of the population dynamics; 2) studying the feeding biology through the examination of stomach contents; 3) using sagittal otoliths as potential marker of species life cycle; 4) getting preliminary data on mDNA phylogeny. Gurnards showed a specie-specific “critical size” coinciding with the start of sexual maturity, the tendency to migrate to greater depths, a change of diet from crustaceans to fish and an increase of variety of food items eaten. Distribution of prey items, predator size range and depth distribution were the main dimensions that influence the breadth of trophic niche and the relative difference amongst Adriatic gurnards. Several feeding preferences were individuated and a possible impact among bigger-size gurnards and other commercial fishes (anchovy, gadoids) and Crustacea (such as mantis prawn and shrimps) were to be necessary considered. Otolith studies showed that gurnard species have a very fast growth despite other results in other areas; intra-specific differences and the increase in the variability of otolith shape, sulcus acusticus shape, S:O ratios, sulcus acusticus external crystals arrangement were shown between juveniles and adults and were linked to growth (individual genetic factors) and to environmental conditions (e.g. depth and trophic niche distribution). In order to facilitate correct biological interpretation of data, molecular data were obtained for comparing morphological distance to genetic ones.

Multi-locus microsatellite analysis supports the hypothesis of an autochthonous focus of Echinococcus multilocularis in northern Italy

Echinococcus multilocularis is characterised by a wide geographical distribution, encompassing three continents (North America, Asia and Europe) yet very low genetic variability is documented. Recently, this parasite has been detected in red foxes (Vulpes vulpes) circulating in an Alpine region of Italy, close to Austria. This finding raised the question as to whether an autochthonous cycle exists in Italy or whether the infected foxes originated from the neighbouring regions of Austria. Studies have shown that multi-locus microsatellite analysis can identify genomic regions carrying mutations that result in a local adaptation. We used a tandem repeated multi-locus microsatellite (EmsB) to evaluate the genetic differences amongst adult worms of E. multilocularis collected in Italy, worms from neighbouring Austria and from other European and extra-European countries. Fluorescent PCR was performed on a panel of E. multilocularis samples to assess intra-specific polymorphism. The analysis revealed four closed genotypes for Italian samples of E. multilocularis which were unique compared with the other 25 genotypes from Europe and the five genotypes from Alaska. An analysis in the Alpine watershed, comparing Italian adult worms with those from neighbouring areas in Austria, showed a unique cluster for Italian samples. This result supports the hypothesis of the presence of an autochthonous cycle of E. multilocularis in Italy. EmsB can be useful for 'tracking' the source of infection of this zoonotic parasite and developing appropriate measures for preventing or reducing the risk of human alveolar echinococcosis.

A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.

Chapter Six – Individual Trait Variation and Diversity in Food Webs

In recent years, there has been a renewed interest in the ecological consequences of individual trait variation within populations. Given that individual variability arises from evolutionary dynamics, to fully understand eco-evolutionary feedback loops, we need to pay special attention to how standing trait variability affects ecological dynamics. There is mounting empirical evidence that intra-specific phenotypic variation can exceed species-level means, but theoretical models of multi-trophic species coexistence typically neglect individual-level trait variability. What is needed are multispecies datasets that are resolved at the individual level that can be used to discriminate among alternative models of resource selection and species coexistence in food webs. Here, using one the largest individual-based datasets of a food web compiled to date, along with an individual trait-based stochastic model that incorporates Approximate Bayesian computation methods, we document intra-population variation in the strength of prey selection by different classes or predator phenotypes which could potentially alter the diversity and coexistence patterns of food webs. In particular, we found that strongly connected individual predators preferentially consumed common prey, whereas weakly connected predators preferentially selected rare prey. Such patterns suggest that food web diversity may be governed by the distribution of predator connectivity and individual trait variation in prey selection. We discuss the consequences of intra-specific variation in prey selection to assess fitness differences among predator classes (or phenotypes) and track longer term food web patterns of coexistence accounting for several phenotypes within each prey and predator species.

Coevolución virus-planta: impacto de las infecciones virales en poblaciones silvestres de Chiltepín (capsicum annuum var. Aviculare (dierbach) d’arcy & eshbaugh) en México.

El impacto negativo que tienen los virus en las plantas hace que estos puedan ejercer un papel ecológico como moduladores de la dinámica espacio-temporal de las poblaciones de sus huéspedes. Entender cuáles son los mecanismos genéticos y los factores ambientales que determinan tanto la epidemiología como la estructura genética de las poblaciones de virus puede resultar de gran ayuda para la comprensión del papel ecológico de las infecciones virales. Sin embargo, existen pocos trabajos experimentales que hayan abordado esta cuestión. En esta tesis, se analiza el efecto de la heterogeneidad del paisaje sobre la incidencia de los virus y la estructura genética de sus poblaciones. Asimismo, se explora como dichos factores ambientales influyen en la importancia relativa que los principales mecanismos de generación de variabilidad genética (mutación, recombinación y migración) tienen en la evolución de los virus. Para ello se ha usado como sistema los begomovirus que infectan poblaciones de chiltepín (Capsicum annuum var. aviculare (Dierbach) D´Arcy & Eshbaugh) en México. Se analizó la incidencia de diferentes virus en poblaciones de chiltepín distribuidas a lo largo de seis provincias biogeográficas, representando el área de distribución de la especie en México, y localizadas en hábitats con diferente grado de intervención humana: poblaciones sin intervención humana (silvestres); poblaciones toleradas (lindes y pastizales), y poblaciones manejadas por el hombre (monocultivos y huertos familiares). Entre los virus analizados, los begomovirus mostraron la mayor incidencia, detectándose en todas las poblaciones y años de muestreo. Las únicas dos especies de begomovirus que se encontraron infectando al chiltepín fueron: el virus del mosaico dorado del chile (Pepper golden mosaic virus, PepGMV) y el virus huasteco del amarilleo de venas del chile (Pepper huasteco yellow vein virus, PHYVV). Por ello, todos los análisis realizados en esta tesis se centran en estas dos especies de virus. La incidencia de PepGMV y PHYVV, tanto en infecciones simples como mixtas, aumento cuanto mayor fue el nivel de intervención humana en las poblaciones de chiltepín, lo que a su vez se asoció con una menor biodiversidad y una mayor densidad de plantas. Además, la incidencia de infecciones mixtas, altamente relacionada con la presencia de síntomas, fue también mayor en las poblaciones cultivadas. La incidencia de estos dos virus también varió en función de la población de chiltepín y de la provincia biogeográfica. Por tanto, estos resultados apoyan una de las hipótesis XVI clásicas de la Patología Vegetal según la cual la simplificación de los ecosistemas naturales debida a la intervención humana conduce a un mayor riesgo de enfermedad de las plantas, e ilustran sobre la importancia de la heterogeneidad del paisaje a diferentes escalas en la determinación de patrones epidemiológicos. La heterogeneidad del paisaje no solo afectó a la epidemiología de PepGMV y PHYVV, sino también a la estructura genética de sus poblaciones. En ambos virus, el nivel de diferenciación genética mayor fue la población, probablemente asociado a la capacidad de migración de su vector Bemisia tabaci; y en segundo lugar la provincia biogeográfica, lo que podría estar relacionado con el papel del ser humano como agente dispersor de PepGMV y PHYVV. La estima de las tasas de sustitución nucleotídica de las poblaciones de PepGMV y PHYVV mostró una rápida dinámica evolutiva. Los árboles filogenéticos de ambos virus presentaron una topología en estrella, lo que sugiere una expansión reciente en las poblaciones de chiltepín. La reconstrucción de los patrones de migración de ambos virus indicó que ésta expansión parece haberse producido desde la zona central de México siguiendo un patrón radial, y en los últimos 30 años. Es importante tener en cuenta que el patrón espacial de la diversidad genética de las poblaciones de PepGMV y PHYVV es similar al descrito previamente para el chiltepín lo que podría dar lugar a la congruencia de las genealogías del huésped y la de los virus. Dicha congruencia se encontró cuando se tuvieron en cuenta únicamente las poblaciones de hábitats silvestres y tolerados, lo que probablemente se debe a una codivergencia en el espacio pero no en el tiempo, dado que la evolución de virus y huésped han ocurrido a escalas temporales muy diferentes. Finalmente, el análisis de la frecuencia de recombinación en PepGMV y PHYVV indicó que esta juega un papel importante en la evolución de ambos virus, dependiendo su importancia del nivel de intervención humana de la población de chiltepín. Este factor afectó también a la intensidad de la selección a la que se ven sometidos los genomas de PepGMV y PHYVV. Los resultados de esta tesis ponen de manifiesto la importancia que la reducción de la biodiversidad asociada al nivel de intervención humana de las poblaciones de plantas y la heterogeneidad del paisaje tiene en la emergencia de nuevas enfermedades virales. Por tanto, es necesario considerar estos factores ambientales a la hora de comprender la epidemiologia y la evolución de los virus de plantas.XVII SUMMARY Plant viruses play a key role as modulators of the spatio-temporal dynamics of their host populations, due to their negative impact in plant fitness. Knowledge on the genetic and environmental factors that determine the epidemiology and the genetic structure of virus populations may help to understand the ecological role of viral infections. However, few experimental works have addressed this issue. This thesis analyses the effect of landscape heterogeneity in the prevalence of viruses and the genetic structure of their populations. Also, how these environmental factors influence the relative importance of the main mechanisms for generating genetic variability (mutation, recombination and migration) during virus evolution is explored. To do so, the begomoviruses infecting chiltepin (Capsicum annuum var. aviculare (Dierbach) D'Arcy & Eshbaugh) populations in Mexico were used. Incidence of different viruses in chiltepin populations of six biogeographical provinces representing the species distribution in Mexico was determined. Populations belonged to different habitats according to the level of human management: populations with no human intervention (Wild); populations naturally dispersed and tolerated in managed habitats (let-standing), and human managed populations (cultivated). Among the analyzed viruses, the begomoviruses showed the highest prevalence, being detected in all populations and sampling years. Only two begomovirus species infected chiltepin: Pepper golden mosaic virus, PepGMV and Pepper huasteco yellow vein virus, PHYVV. Therefore, all the analyses presented in this thesis are focused in these two viruses. The prevalence of PepGMV and PHYVV, in single and mixed infections, increased with higher levels of human management of the host population, which was associated with decreased biodiversity and increased plant density. Furthermore, cultivated populations showed higher prevalence of mixed infections and symptomatic plants. The prevalence of the two viruses also varied depending on the chiltepin population and on the biogeographical province. Therefore, these results support a classical hypothesis of Plant Pathology stating that simplification of natural ecosystems due to human management leads to an increased disease risk, and illustrate on the importance of landscape heterogeneity in determining epidemiological patterns. Landscape heterogeneity not only affected the epidemiology of PepGMV and PHYVV, but also the genetic structure of their populations. Both viruses had the highest level of genetic differentiation at the population scale, probably associated with the XVIII migration patterns of its vector Bemisia tabaci, and a second level at the biogeographical province scale, which could be related to the role of humans as dispersal agents of PepGMV and PHYVV. The estimates of nucleotide substitution rates of the virus populations indicated rapid evolutionary dynamics. Accordingly, phylogenetic trees of both viruses showed a star topology, suggesting a recent diversification in the chiltepin populations. Reconstruction of PepGMV and PHYVV migration patterns indicated that they expanded from central Mexico following a radial pattern during the last 30 years. Importantly, the spatial genetic structures of the virus populations were similar to that described previously for the chiltepin, which may result in the congruence of the host and virus genealogies. Such congruence was found only in wild and let-standing populations. This is probably due to a co-divergence in space but not in time, given the different evolutionary time scales of the host and virus populations. Finally, the frequency of recombination detected in the PepGMV and PHYVV populations indicated that this mechanism plays an important role in the evolution of both viruses at the intra-specific scale. The level of human management had a minor effect on the frequency of recombination, but influenced the strength of negative selective pressures in the viral genomes. The results of this thesis highlight the importance of decreased biodiversity in plant populations associated with the level of human management and of landscape heterogeneity on the emergence of new viral diseases. Therefore it is necessary to consider these environmental factors in order to fully understand the epidemiology and evolution of plant viruses.

Identificación de áreas prioritarias para la conservación de razas de maíz en la sierra de Ecuador

En Ecuador el maíz es el cultivo más importante en superficie y es base de la alimentación para la población rural que vive en los Andes. A diferencia de lo que sucede en la Costa, en la región Sierra todavía se cultivan numerosas variedades tradicionales que se agrupan en veinticuatro razas. Mantener esta diversidad es, pues, de gran importancia no solo para la seguridad alimentaria, sino también como fuente de genes para tolerancia a factores abióticos que podrían ser incorporados a las variedades modernas. Si bien parte de esta diversidad fue recolectada a mediados del siglo pasado y está siendo conservada en distintos bancos de germoplasma, es deseable que su conservación in situ también esté asegurada, entre otras razones, porque de esta manera el cultivo puede seguir evolucionando. Para poder implementar un plan de conservación en finca que contribuya a preservar este patrimonio, resulta imprescindible identificar áreas idóneas donde concentrar los recursos y conocer las características y tipologías de los agricultores que manejan la diversidad actual. Generar esta información es el objetivo principal de esta investigación y para lograrlo se han llevado a cabo cuatro estudios: (1) Análisis de la diversidad a nivel de razas e identificación de áreas de alta riqueza de razas, alta diversidad morfológica y/o alta diversidad ecogeográfica en la Sierra de Ecuador, (2) Identificación del perfil y las características de los agricultores que conservan y manejan las variedades tradicionales de maíz en la Sierra de Ecuador, (3) Análisis del conocimiento local, manejo y usos de variedades tradicionales de maíz en la Sierra de Ecuador, y (4) Identificación de áreas de alta diversidad y bajo riesgo de pérdida para la conservación en finca de maíz en la Sierra de Ecuador. Para el primer estudio se visitaron 303 fincas distribuidas a lo largo de la Sierra y se recolectaron 636 muestras que fueron caracterizadas morfológicamente mediante 14 variables: 8 relacionadas con la mazorca (forma, longitud y diámetro de la mazorca, color y diámetro de olote y número y disposición de hileras) y 7 referidas el grano (número total de granos, color, forma, longitud, anchura y grosor de grano y tipo de endospermo). Adicionalmente, las fincas donde se tomaron las muestras fueron caracterizadas ecogeográficamente mediante 5 variables climáticas (temperatura media estacional, rango de temperatura media anual, temperatura mínima de diciembre, precipitación estacional y precipitación de octubre), 2 geofísicas (altitud y pendiente) y 5 edáficas (textura principal del suelo, profundidad a roca, pH, contenido en materia orgánica y fertilidad). A partir de esta información y mediante técnicas de sistemas de información geográfica (SIG), se generaron mapas de distribución por raza en formato vectorial y un mapa de riqueza de razas, un mapa de diversidad morfológica y un mapa de diversidad ecogeográfica en formato ráster con celdas de 10 km x 10 km. Los resultados permitieron constatar que, en los últimos 60 años, no se ha perdido ninguna raza. Sin embargo, Canguil, Chaucho y Clavito han dejado de cultivarse en algunas provincias con la consiguiente erosión genética del cultivo. La caracterización morfológica detectó diferencias en el grado de variabilidad intra-raza, siendo Patillo Ecuatoriano, Racimo de Uva y Uchima las razas más heterogéneas tanto para los caracteres cualitativos como cuantitativos. A nivel climático y geofísico, también se detectaron diferencias en el grado de variación intra-raza; Cuzco Ecuatoriano, Kcello Ecuatoriano y Montaña Ecuatoriana fueron las razas que en promedio presentaron mayores rangos y coeficientes de variación para estas variables ecogeográficas. En cuanto a las condiciones edáficas todas las razas, excepto Cónico Dentado, presentaron una gran heterogeneidad, pudiendo crecer tanto en suelos ricos como pobres, con valores de pH entre ácido y moderadamente alcalino. La comparación entre razas reveló diferencias significativas en los rangos ambientales de algunas razas como Cónico Dentado, que tiende a cultivarse a menor altitud y, por tanto, en ambientes menos fríos y de mayor precipitación que Blanco Blandito, Patillo Ecuatoriano, Sabanero Ecuatoriano, Uchima y Zhima. Para la mayoría de las razas se encontraron materiales potencialmente adaptados a condiciones de estrés (precipitación estacional inferior a 500 mm y suelos con pH entre 4.5 y 5.5). Finalmente, los mapas de riqueza, de diversidad morfológica y de diversidad ecogeográfica mostraron 36 celdas de alta diversidad repartidas en las 10 provincias de la Sierra: 11 celdas en las provincias del norte, 11 en las provincias del centro y 14 en las provincias del sur. Para la caracterización e identificación de las tipologías de los agricultores que cultivan maíz en la Sierra de Ecuador y el análisis de los posibles factores de riesgo de pérdida de diversidad, se realizaron entrevistas individuales y semiestructuradas a los agricultores dueños de las fincas donde se recolectaron las muestras para el estudio de diversidad (254 en total). Las preguntas que se formularon (11 abiertas y 5 cerradas) estuvieron organizadas en seis bloques: datos del agricultor, características de la finca, diversidad y conocimiento del cultivo, manejo del cultivo, usos y flujo de semillas. Los resultados indicaron que la diversidad de maíz que hay en la Sierra de Ecuador es manejada mayoritariamente por agricultores mestizos, de entre 30 y 55 años, que cultivan una o dos variedades tradicionales para autoconsumo, en parcelas de menos de 0.5 ha y en asocio con fréjol. El análisis de segmentación mediante el algoritmo Chi-square automatic interaction detection (CHAID) permitió identificar un pequeño grupo de agricultores indígenas con parcelas medianas (entre 0.5 ha y 1.5 ha) que conservan un mayor número de variedades tradicionales por finca que el agricultor promedio. Los análisis estadísticos no detectaron diferencias significativas entre etnias (mestizo vs. indígena), géneros (hombre vs. mujer) y grupos de edad (jóvenes menores de 30 años, adultos entre 30 y 55 años y adultos mayores de 55 años) en lo que respecta al conocimiento del cultivo (criterios de reconocimiento y razones de preferencia) y manejo (tipo de cultivo), pero sí detectaron diferencias entre regiones, principalmente en el modo de cultivar el maíz; mientras que en el norte y sur tienden a sembrarlo en asocio y con un mayor número de especies, en el centro acostumbran a cultivarlo preferentemente solo. En cuanto a los usos, se recopilaron hasta 39 modos diferentes de consumir maíz, siendo Kcello Ecuatoriano y Zhima las razas para las que se registró un mayor número de usos. La comparación del número medio de usos por variedad entre etnias evidenció que los agricultores mestizos utilizan sus variedades tradicionales de forma más variada que los indígenas. Entre los factores de riesgo que se analizaron, el bajo porcentaje de jóvenes agricultores que se ocupan de las fincas podría suponer una amenaza a medio plazo por falta de relevo generacional. Adicionalmente, las numerosas sinonimias y homonimias que se detectaron y el bajo intercambio de semillas también podrían ser causa de pérdida de diversidad, bien por reemplazo o por envejecimiento de la semilla. Finalmente, se concluyó que las razas Chaucho, Complejo Chillo-Huandango, Complejo Mishca-Huandango, Cónico Dentado, Montaña Ecuatoriana y Sabanero Ecuatoriano son particularmente vulnerables, no solo por su baja presencia, sino también por el color de grano que tienen (los mismos que la mayoría de las razas más comunes) y carecer de nombres y usos específicos. Finalmente, para la priorización de áreas de conservación en finca para maíz en la Sierra de Ecuador, se utilizaron 13 criterios de diferente naturaleza: 2 ecogeográficos (precipitación, diversidad ecogeográfica), 6 biológicos (grado de presencia del cultivo, riqueza de razas, diversidad morfológica, presencia de mezclas, presencia de razas locales y riesgo de erosión genética), 3 culturales (abundancia de variedades por finca, diversidad de usos y frecuencia de intercambio) y 2 demográficos (tamaño de la población y distancia a núcleos urbanos). Mediante técnicas SIG y de evaluación multicriterio, los valores originales de las capas-criterio fueron transformados a una escala de 0 a 100. Posteriormente, las capas-criterio normalizadas fueron sumadas utilizando tres métodos de ponderación: (1) mismo peso, (2) diferente peso según la puntuación otorgada por 72 expertos, y (3) diferente peso según el método de comparación entre pares de criterios. Los resultados permitieron identificar ocho celdas de 10 km x 10 km con alta puntuación (> 65): tres celdas en el norte (una en cada una de las provincias), una celda en el centro (en la provincia de Cotopaxi), y cuatro celdas en la región sur (dos en Azuay y otras dos en Loja). ABSTRACT In Ecuador, the maize is the most important cultivation in surface and it is a base of the feeding for the rural population who lives in the Andes. In contrast to what it happens on the Coast, in the Sierra region still there are cultivated numerous traditional varieties that are grouped into twenty-four races. Maintaining this diversity is, therefore, of great importance not only for food security, but also as a source of genes for tolerance to abiotic factors could be incorporated into modern varieties. Although part of this diversity was collected in the middle of the last century and is still preserved in various germplasm banks, it is desirable for the in situ conservation also is assured, among other reasons, because in this way the crop can continue to evolve. To be able to implement a conservation plan on farm that contribute to preserving this heritage, it is essential to identify suitable areas where to concentrate resources and know the characteristics and typology of farmer who managed the current diversity. To generate this information is the main target of this investigation and to achieve this, four studies have been carried out: (1) Analysis of the diversity at races and identification of areas of high richness of races, high morphological diversity and / or ecogeographical high diversity in the Sierra of Ecuador, (2) Identification of the profile and characteristics of farmers who conserve and manage traditional varieties of maize in the Sierra of Ecuador, (3) Analysis of local knowledge, management and use of traditional varieties of maize in the Sierra of Ecuador, and (4) Identification of areas of high diversity and low risk of loss for the conservation of maize in the Sierra of Ecuador. For the first study were visited 303 farms distributed along the Sierra and collected 636 samples that were characterized morphologically by 14 variables: 8 related to the ear (shape, length and diameter of the cob, colour, and diameter of cob and number and arrangement of rows) and 7 referred to the grain (total number of grain, colour, shape, length, width, and thickness and type of grain endosperm). In addition, the farms where the samples were taken were characterized ecogeographically through 5 climatic variables (seasonal average temperature, range of average annual temperature, minimum temperature for December, seasonal precipitation and precipitation of October), 2 geophysical (altitude and slope) and edaphic 5 (main texture of the soil, deep rock, pH, content of organic matter and fertility). From this information and techniques of geographic information systems (GIS), maps were generated for distribution by race in vector format and a map of richness of races, a map of morphological diversity and a map of ecogeographical diversity in raster format with cells of 10 km x 10 km. The results allowed observing that, over the past 60 years, it has not lost any race. Nevertheless, Canguil, Chaucho and Clavito have stopped being cultivated in some provinces with the consequent genetic erosion of the cultivation. The morphological characterization detected differences in the degree of variability intra-race, being Patillo Ecuatoriano, Racimo de Uva and Uchima races more heterogeneous both for the qualitative and quantitative characters. At climate and geophysical level, also detected differences in the degree of variation intra-race; Cuzco Ecuatoriano, Kcello Ecuatoriano and Montaña Ecuatoriana were races that, on average, showed higher ranges and coefficients of variation for these geographical characters. In terms of the edaphic conditions, all races, except Cónico Dentado, showed a great heterogeneity, and can grow both in rich and poor soils, with pH values between acid and moderately alkaline. The comparison between races revealed significant differences in the environmental ranges in some races as Cónico Dentado, which tends to be grown at lower elevations and, therefore, in environments less cold and greater precipitation than Blanco Blandito, Patillo Ecuatoriano, Sabanero Ecuatoriano, Uchima and Zhima. For most of the races were found materials potentially adapted to stress conditions (seasonal precipitation less than 500 mm and soil with a pH between 4.5 and 5.5). Finally, the maps of richness, morphologic diversity and ecogeographical diversity showed 36 cells high diversity distributed in 10 provinces of the Sierra: 11 cells in the northern provinces, 11 in the central provinces and 14 in the southern provinces. For the characterization and identification of the typology of the farmers who cultivate corn in the Sierra of Ecuador and the analysis of the possible factors of risk of loss of diversity, there were realized interviews individual and semistructured to the farmers’ owners of the farms where the samples were gathered for the study of diversity (254 in whole). The questions that were formulated (11 opened ones and 5 closed ones) were organized in six blocks: data of the farmer, characteristics of the farm, diversity and knowledge of the crop, crop management, uses and seed flow. The results indicated that the maize diversity that exist in the Sierra of Ecuador is managed mainly by mestizo farmers, aged between 30 and 55, who cultivate one or two traditional varieties for self-consumption, on plots of less than 0.5 has and in associated with beans. The segmentation analysis algorithm using the Chi-square automatic interaction detection (CHAID technique), allowed to identify a small group of indigenous farmers with medium-sized plots (between 0.5 there is and 1.5 it is) that a major number of traditional varieties preserves for farm that the average farmer. The statistical analysis did not detect significant differences between ethnic groups (mestizos vs. indigenous), genres (man vs. women) and age groups (young people under 30 years of age, adults between 30 and 55 years and adults over 55 years old) in regards to the knowledge of the cultivation (recognition criteria and reasons of preference) and management (type of crop), but if detected differences between regions, mainly on the mode of cultivating the maize; while in the north and south they tend to sow in associate and with a greater number of species, in the center accustomed to cultivate it preferably only. In regards to the uses, they were compiled up to 39 different ways of consuming maize, being Kcello Ecuatoriano and Zhima the races for which a major number of uses registered. The comparison of the average number of uses per variety between ethnic groups showed that the mestizo farmers used their traditional varieties of form more varied than the indigenous people. Between the factors of risk that were analyzed, the low percentage of young farmers who deal with the farms might suppose a medium-term threat for lack of generational relief. In addition, the numerous synonyms and homonyms that were detected and the low seed exchange could also be a cause of loss of diversity, either by replacement or by aging of the seed. Finally, it was concluded that the races Chaucho, Complex Chillo-Huandango, Complex Mishca-Huandango, Cónico Dentado, Montaña Ecuatoriana and Sabanero Ecuatoriano are particularly vulnerable, not only because of their low presence, but also by the grain color they have (the same as the majority of races more common) and lack of names and specific uses. Finally, for the prioritization of maize conservation areas on farm in the Sierra of Ecuador, used 13 criteria of different nature: 2 ecogeographic (precipitation, diversity ecogeographical), 6 biological (degree of presence of the crop, races richness, morphological diversity, the presence of mixtures, presence of local races and risk of genetic erosion), 3 cultural (abundance of varieties per farm, diversity of uses and frequency of exchange) and 2 demographic (population size and distance to urban centers). Using GIS techniques and multicriteria evaluation, the original values of the layers-criterion were transformed to a scale of 0 to 100. Later, the normalized layers - criteria were added using three weighting methods: (1) the same weight, (2) different weight according to the score given by 72 experts, and (3) different weight according to the method of comparison between pairs of criteria. The results allowed to identify eight 10 km cells x 10 km with high punctuation (> 65): three cells in the north (one in each of the provinces), a cell in the center (in the Cotopaxi province), and four cells in the south region (two in Azuay and other two in Loja).

Seawater carbonate chemistry and biological processes of Emiliania huxleyi (strains RCC1256 and NZEH) during experiments, 2011

With respect to their sensitivity to ocean acidification, calcifiers such as the coccolithophore Emiliania huxleyi have received special attention, as the process of calcification seems to be particularly sensitive to changes in the marine carbonate system. For E. huxleyi, apparently conflicting results regarding its sensitivity to ocean acidification have been published (Iglesias-Rodriguez et al., 2008a; Riebesell et al., 2000). As possible causes for discrepancies, intra-specific variability and different effects of CO2 manipulation methods, i.e. the manipulation of total alkalinity (TA) or total dissolved inorganic carbon (DIC), have been discussed. While Langer et al. (2009) demonstrate a high degree of intra-specific variability between strains of E. huxleyi, the question whether different CO2 manipulation methods influence the cellular responses has not been resolved yet. In this study, closed TA as well as open and closed DIC manipulation methods were compared with respect to E. huxleyi's CO2-dependence in growth rate, POC- and PIC-production. The differences in the carbonate chemistry between TA and DIC manipulations were shown not to cause any differences in response patterns, while the latter differed between open and closed DIC manipulation. The two strains investigated showed different sensitivities to acidification of seawater, RCC1256 being more negatively affected in growth rates and PIC production than NZEH.

Spatial behavior of two coral reef fishes within a Caribbean Marine Protected Area

A better understanding of the key ecological processes of marine organisms is fundamental to improving design and effective implementation of marine protected areas (MPAs) and marine biodiversity. The movement behavior of coral reef fish is a complex mechanism that is highly linked to species life-history traits, predation risk and food resources. We used passive acoustic telemetry to study monthly, daily and hourly movement patterns and space use in two species, Schoolmaster snapper (Lutjanus apodus) and Stoplight parrotfish (Sparisoma viride). We investigated the spatial overlap between the two species and compared intra-specific spatial overlap between day and night. Presence-absence models showed different diel presence and habitat use patterns between the two species. We constructed a spatial network of the movement patterns, which showed that for both species when fish were detected by the array of receivers most movements were made around the coral reef habitat while occasionally moving to silt habitats. Our results show that most individuals made predictable daily crepuscular migrations between different locations and habitat types, although individual behavioral changes were observed for some individuals across time. Our study also highlights the necessity to consider multiple species during MPA implementation and to take into account the specific biological and ecological traits of each species. The low number of fish detected within the receiver array, as well as the intraspecific variability observed in this study, highlight the need to compare results across species and individuals to be used for MPA management.

Source populations in coastal crabs: parameters affecting egg production

Benthic marine invertebrates may form metapopulations connected via propagule dispersal. Conservation efforts often target potential source coastlines to indirectly benefit areas depending on allochthonous offspring production. Besides population density, adult size structure, sex ratio, brooding frequency and the proportion of breeding individuals may significantly influence the reproductive output of benthic populations, but these effects have seldom been tested. We used rocky shore crabs to assess the spatial variability of such parameters at relevant scales for conservation purposes and to test their consistency over 2 consecutive years; we then used the data to address whether bottom-up processes or biological interactions might explain the patterns observed. We decomposed egg production rates into their components for the 2 most abundant brachyuran species inhabiting the intertidal rocky habitat. Adult density and brooding frequency varied consistently among shores for both species and largely explained the overall spatial trends of egg production. Temporally consistent patterns also included among-shore differences in the size of ovigerous females of the grapsid Pachygrapsus transversus and between-bay differences in the fecundity of the spider crab Epialtus brasiliensis. Sex ratio was remarkably constant in both. We found no positive or negative correlations between adult density and brooding frequency to support either the existence of a component Allee effect (lack of mate encounters) or an effect of intra-specific competition. Likewise, shore-specific potential growth in P. transversus does not negatively correlate with frequency of ovigerous individuals, as would be expected under a critical balance between these 2 processes. The patterns observed suggest that bottom-up drivers may best explain spatial trends in the reproductive output of these species.

Influence of historical landscapes, drainage evolution and ecological traits on patterns of genetic diversity in Southeast Asian freshwater snakehead fishes

Snakehead fishes in the family Channidae are obligate freshwater fishes represented by two extant genera, the African Parachannna and the Asian Channa. These species prefer still or slow flowing water bodies, where they are top predators that exercise high levels of parental care, have the ability to breathe air, can tolerate poor water quality, and interestingly, can aestivate or traverse terrestrial habitat in response to seasonal changes in freshwater habitat availability. These attributes suggest that snakehead fishes may possess high dispersal potential, irrespective of the terrestrial barriers that would otherwise constrain the distribution of most freshwater fishes. A number of biogeographical hypotheses have been developed to account for the modern distributions of snakehead fishes across two continents, including ancient vicariance during Gondwanan break-up, or recent colonisation tracking the formation of suitable climatic conditions. Taxonomic uncertainty also surrounds some members of the Channa genus, as geographical distributions for some taxa across southern and Southeast (SE) Asia are very large, and in one case is highly disjunct. The current study adopted a molecular genetics approach to gain an understanding of the evolution of this group of fishes, and in particular how the phylogeography of two Asian species may have been influenced by contemporary versus historical levels of dispersal and vicariance. First, a molecular phylogeny was constructed based on multiple DNA loci and calibrated with fossil evidence to provide a dated chronology of divergence events among extant species, and also within species with widespread geographical distributions. The data provide strong evidence that trans-continental distribution of the Channidae arose as a result of dispersal out of Asia and into Africa in the mid–Eocene. Among Asian Channa, deep divergence among lineages indicates that the Oligocene-Miocene boundary was a time of significant species radiation, potentially associated with historical changes in climate and drainage geomorphology. Mid-Miocene divergence among lineages suggests that a taxonomic revision is warranted for two taxa. Deep intra-specific divergence (~8Mya) was also detected between C. striata lineages that occur sympatrically in the Mekong River Basin. The study then examined the phylogeography and population structure of two major taxa, Channa striata (the chevron snakehead) and the C. micropeltes (the giant snakehead), across SE Asia. Species specific microsatellite loci were developed and used in addition to a mitochondrial DNA marker (Cyt b) to screen neutral genetic variation within and among wild populations. C. striata individuals were sampled across SE Asia (n=988), with the major focus being the Mekong Basin, which is the largest drainage basin in the region. The distributions of two divergent lineages were identified and admixture analysis showed that where they co-occur they are interbreeding, indicating that after long periods of evolution in isolation, divergence has not resulted in reproductive isolation. One lineage is predominantly confined to upland areas of northern Lao PDR to the north of the Khorat Plateau, while the other, which is more closely related to individuals from southern India, has a widespread distribution across mainland SE Asian and Sumatra. The phylogeographical pattern recovered is associated with past river networks, and high diversity and divergence among all populations sampled reveal that contemporary dispersal is very low for this taxon, even where populations occur in contiguous freshwater habitats. C. micropeltes (n=280) were also sampled from across the Mekong River Basin, focusing on the lower basin where it constitutes an important wild fishery resource. In comparison with C. striata, allelic diversity and genetic divergence among populations were extremely low, suggesting very recent colonisation of the greater Mekong region. Populations were significantly structured into at least three discrete populations in the lower Mekong. Results of this study have implications for establishing effective conservation plans for managing both species, that represent economically important wild fishery resources for the region. For C. micropeltes, it is likely that a single fisheries stock in the Tonle Sap Great Lake is being exploited by multiple fisheries operations, and future management initiatives for this species in this region will need to account for this. For C. striata, conservation of natural levels of genetic variation will require management initiatives designed to promote population persistence at very localised spatial scales, as the high level of population structuring uncovered for this species indicates that significant unique diversity is present at this fine spatial scale.

Evaluation of the iPhone with an acrylic sleeve versus the Scoliometer for rib hump measurement in scoliosis

Background. Vertebral rotation found in structural scoliosis contributes to trunkal asymmetry which is commonly measured with a simple Scoliometer device on a patient's thorax in the forward flexed position. The new generation of mobile 'smartphones' have an integrated accelerometer, making accurate angle measurement possible, which provides a potentially useful clinical tool for assessing rib hump deformity. This study aimed to compare rib hump angle measurements performed using a Smartphone and traditional Scoliometer on a set of plaster torsos representing the range of torsional deformities seen in clinical practice. Methods. Nine observers measured the rib hump found on eight plaster torsos moulded from scoliosis patients with both a Scoliometer and an Apple iPhone on separate occasions. Each observer repeated the measurements at least a week after the original measurements, and were blinded to previous results. Intra-observer reliability and inter-observer reliability were analysed using the method of Bland and Altman and 95% confidence intervals were calculated. The Intra-Class Correlation Coefficients (ICC) were calculated for repeated measurements of each of the eight plaster torso moulds by the nine observers. Results. Mean absolute difference between pairs of iPhone/Scoliometer measurements was 2.1 degrees, with a small (1 degrees) bias toward higher rib hump angles with the iPhone. 95% confidence intervals for intra-observer variability were +/- 1.8 degrees (Scoliometer) and +/- 3.2 degrees (iPhone). 95% confidence intervals for inter-observer variability were +/- 4.9 degrees (iPhone) and +/- 3.8 degrees (Scoliometer). The measurement errors and confidence intervals found were similar to or better than the range of previously published thoracic rib hump measurement studies. Conclusions. The iPhone is a clinically equivalent rib hump measurement tool to the Scoliometer in spinal deformity patients. The novel use of plaster torsos as rib hump models avoids the variables of patient fatigue and discomfort, inconsistent positioning and deformity progression using human subjects in a single or multiple measurement sessions.

Population structure of Bactrocera dorsalis s.s., B. papayae and B. philippinensis (Diptera: Tephritidae) in southeast Asia: evidence for a single species hypothesis using mitochondrial DNA and wingshape data

Background Bactrocera dorsalis s.s. is a pestiferous tephritid fruit fly distributed from Pakistan to the Pacific, with the Thai/Malay peninsula its southern limit. Sister pest taxa, B. papayae and B. philippinensis, occur in the southeast Asian archipelago and the Philippines, respectively. The relationship among these species is unclear due to their high molecular and morphological similarity. This study analysed population structure of these three species within a southeast Asian biogeographical context to assess potential dispersal patterns and the validity of their current taxonomic status. Results Geometric morphometric results generated from 15 landmarks for wings of 169 flies revealed significant differences in wing shape between almost all sites following canonical variate analysis. For the combined data set there was a greater isolation-by-distance (IBD) effect under a ‘non-Euclidean’ scenario which used geographical distances within a biogeographical ‘Sundaland context’ (r2 = 0.772, P < 0.0001) as compared to a ‘Euclidean’ scenario for which direct geographic distances between sample sites was used (r2 = 0.217, P < 0.01). COI sequence data were obtained for 156 individuals and yielded 83 unique haplotypes with no correlation to current taxonomic designations via a minimum spanning network. BEAST analysis provided a root age and location of 540kya in northern Thailand, with migration of B. dorsalis s.l. into Malaysia 470kya and Sumatra 270kya. Two migration events into the Philippines are inferred. Sequence data revealed a weak but significant IBD effect under the ‘non-Euclidean’ scenario (r2 = 0.110, P < 0.05), with no historical migration evident between Taiwan and the Philippines. Results are consistent with those expected at the intra-specific level. Conclusions Bactrocera dorsalis s.s., B. papayae and B. philippinensis likely represent one species structured around the South China Sea, having migrated from northern Thailand into the southeast Asian archipelago and across into the Philippines. No migration is apparent between the Philippines and Taiwan. This information has implications for quarantine, trade and pest management.

Mitochondrial DNA supports the identification of two endangered river sharks (Glyphis glyphis and Glyphis garricki) across northern Australia

The river sharks (genus Glyphis) are a small group of poorly known sharks occurring in tropical rivers and estuarine waters across northern Australia, south-east Asia and the subcontinent. The taxonomy of the genus has long been unclear due to very few individuals having been caught and examined, resulting in a paucity of data regarding their distribution, biology and ecology. Only recently has attention focussed on the two Australian species, G. glyphis and G. garricki. This study is a result of a rare opportunity to collate the few samples that have been collected from these species and the bull shark Carcharhinus leucas, which shares an overlapping range. These samples were analysed using the DNA barcoding approach (cox1 mitochondrial gene), compared with six other species of carcharhinids and evaluated in light of the current taxonomic classification. Nine species-specific nucleotide differences were found between G. glyphis and G. garricki and no intra-specific variation provides strong support for the separation into distinct species. Significant differences were also observed at the inter-generic level, with Glyphis forming a distinct clade from Carcharhinus. This study provides the basis for future molecular studies required to better address conservation issues confronting G. glyphis and G. garricki in Australia.