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- Nutrient limitation is the main regulatory factor for carotenoid accumulation and for Psy and Pds steady state transcript levels in Dunaliella salina (Chlorophyta) exposed to high light and salt stressPublication . Coesel, Sacha; Baumgartner, Alexandra; Teles, Lícia; Ramos, Ana; Henriques, Nuno; Cancela, Leonor; Varela, J.Dunaliella salina (Dunal) Teodoresco (1905) is a green unicellular alga able to withstand severe salt, light, and nutrient stress, adaptations necessary to grow in harsh environments such as salt ponds. In response to such growth conditions, this microalga accumulates high amounts of β-carotene in its single chloroplast. In this study, we show that carotenoid accumulation is consistently inhibited in cells grown in nutrient-supplemented media and exposed either to high-light or medium-low-light conditions. Likewise, carotenogenesis in cells shifted to higher salinity (up to 27% NaCl) under medium-low-light conditions is inhibited by the presence of nutrients. The steady-state levels of transcripts encoding phytoene syn- thase and phytoene desaturase increased substantially in D. salina cells shifted to high light or high salt under nutrient- limiting conditions, whereas the presence of nutrients inhibited this response. The regulatory effect of nutrient availability on the accumulation of carotenoids and mes- senger RNA levels of the first two enzymes committed to carotenoid biosynthesis is discussed.
- Performing fish counts with a wide-angle camera, a promising approach reducing divers' limitationsPublication . Assis, J.; Claro, B.; Ramos, Ana; Boavida, Joana; Serrão, EsterVisual standardised methods for census of reef fishes have long been used in fisheries management and biological surveys. However, these tools have inherent sources of bias and the SCUBA divers who perform them are highly constrained in terms of survey time,maximumdepth and frequency of sampling. Alternatives like underwater video are thus being recommended in a wide range of configurations. Yet, all these techniques are still limited in field of view, particularly when compared to the ability of a SCUBA diver performing censuses. In this scope, we evaluated the potential of an underwater wide-angle camera (UWC) to survey fish assemblages by testing it against instantaneous underwater visual census (UVC). Our results showed minimal yet significant differences between methods, mainly because of the camera's loss of resolution when under extreme visibility conditions. Both approaches had the same consistency and ability to detect changes in fish assemblages but, to estimate total species richness, the UWC needed less field effort. Moreover, a SCUBA diver performing census had an effect on fish assemblages which introduced differences of greater magnitude than those found between methods. The removal of the diver effect, the proven ability to detect changes in fish assemblages and the verified gain in field effort, pointed the wide-angle camera as a promising tool to perform census of reef fishes.
- Genetic implications of phylogeographical patterns in the conservation of the boreal wetland butterfly Colias palaeno (Pieridae)Publication . Kramp, Katja; Cizek, Oldrich; Madeira, Pedro M.; Ramos, Ana; Konvicka, Martin; Castilho, Rita; Schmitt, ThomasThe boreo-montane wetland butterfly species Colias palaeno has a European distribution from the Alps to northern Fennoscandia. Within its European range, the species' populations have shrunk dramatically in recent historical times. Therefore, detailed baseline knowledge of the genetic makeup of the species is pivotal in planning potential conservation strategies. We collected 523 individuals from 21 populations across the entire European range and analyzed nuclear (20 allozyme loci) and mitochondrial (600bp of the cytochrome c oxidase subunit I gene) genetic markers. The markers revealed contrasting levels of genetic diversity and divergence: higher in allozymes and lower in mitochondrial sequences. Five main groups were identified by allozymes: Alps, two Czech groups, Baltic countries, Fennoscandia, and Poland. The haplotype mitochondrial network indicates a recent range expansion. The most parsimonious interpretation for our results is the existence of a continuous Wurm glacial distribution in Central Europe, with secondary disjunction during the Last Glacial Maximum into a south-western and a north-eastern fragment and subsequent moderate differentiation. Both groups present signs of postglacial intermixing in the Czech Republic. However, even a complete extinction in this region would not considerably affect the species' genetic basis, as long as the source populations in the Alps and in northern Europe, comprising the most relevant evolutionary units for conservation, are surviving.
- Evolution at a different pace: distinctive phylogenetic patterns of cone snails from two ancient oceanic archipelagosPublication . Cunha, Regina L.; Lima, Fernando P.; Tenorio, Manuel J.; Ramos, Ana A.; Castilho, Rita; Williams, Suzanne T.Ancient oceanic archipelagos of similar geological age are expected to accrue comparable numbers of endemic lineages with identical life history strategies, especially if the islands exhibit analogous habitats. We tested this hypothesis using marine snails of the genus Conus from the Atlantic archipelagos of Cape Verde and Canary Islands. Together with Azores and Madeira, these archipelagos comprise the Macaronesia biogeographic region and differ remarkably in the diversity of this group. More than 50 endemic Conus species have been described from Cape Verde, whereas prior to this study, only two nonendemic species, including a putative species complex, were thought to occur in the Canary Islands. We combined molecular phylogenetic data and geometric morphometrics with bathymetric and paleoclimatic reconstructions to understand the contrasting diversification patterns found in these regions. Our results suggest that species diversity is even lower than previously thought in the Canary Islands, with the putative species complex corresponding to a single species, Conus guanche. One explanation for the enormous disparity in Conus diversity is that the amount of available habitat may differ, or may have differed in the past due to eustatic (global) sea level changes. Historical bathymetric data, however, indicated that sea level fluctuations since the Miocene have had a similar impact on the available habitat area in both Cape Verde and Canary archipelagos and therefore do not explain this disparity. We suggest that recurrent gene flow between the Canary Islands and West Africa, habitat losses due to intense volcanic activity in combination with unsuccessful colonization of new Conus species from more diverse regions, were all determinant in shaping diversity patterns within the Canarian archipelago. Worldwide Conus species diversity follows the well-established pattern of latitudinal increase of species richness from the poles towards the tropics. However, the eastern Atlantic revealed a striking pattern with two main peaks of Conus species richness in the subtropical area and decreasing diversities toward the tropical western African coast. A Random Forests model using 12 oceanographic variables suggested that sea surface temperature is the main determinant of Conus diversity either at continental scales (eastern Atlantic coast) or in a broader context (worldwide). Other factors such as availability of suitable habitat and reduced salinity due to the influx of large rivers in the tropical area also play an important role in shaping Conus diversity patterns in the western coast of Africa.
- Three in one-multiple faunal elements within an endangered european butterfly speciesPublication . Junker, Marius; Zimmermann, Marie; Ramos, Ana A.; Gros, Patrick; Konvicka, Martin; Neve, Gabriel; Rakosy, Laszio; Tammaru, Toomas; Castilho, Rita; Schmitt, ThomasIce ages within Europe forced many species to retreat to refugia, of which three major biogeographic basic types can be distinguished: "Mediterranean", "Continental" and "Alpine / Arctic" species. However, this classification often fails to explain the complex phylogeography of European species with a wide range of latitudinal and altitudinal distribution. Hence, we tested for the possibility that all three mentioned faunal elements are represented within one species. Our data was obtained by scoring 1,307 Euphydryas aurinia individuals (46 European locations) for 17 allozyme loci, and sequencing a subset of 492 individuals (21 sites) for a 626 base pairs COI fragment. Genetic diversity indices, F statistics, hierarchical analyses of molecular variance, individual-based clustering, and networks were used to explore the phylogeographic patterns. The COI fragment represented 18 haplotypes showing a strong geographic structure. All but one allozyme loci analysed were polymorphic with a mean F-ST of 0.20, supporting a pronounced among population structure. Interpretation of both genetic marker systems, using several analytical tools, calls for the recognition of twelve genetic groups. These analyses consistently distinguished different groups in Iberia (2), Italy, Provence, Alps (3), Slovenia, Carpathian Basin, the lowlands of West and Central Europe as well as Estonia, often with considerable additional substructures. The genetic data strongly support the hypothesis that E. aurinia survived the last glaciation in Mediterranean, extra-Mediterranean and perialpine refugia. It is thus a rare example of a model organism that combines attributes of faunal elements from all three of these sources. The observed differences between allozymes and mtDNA most likely result from recent introgression of mtDNA into nuclear allozyme groups. Our results indicate discrepancies with the morphologically-based subspecies models, underlining the need to revise the current taxonomy.
- Isolation and characterization of a stress-inducible Dunaliella salina Lcy-β gene encoding a functional lycopene β-cyclasePublication . Ramos, Ana; Coesel, Sacha; Marques, Ana; Rodrigues, Marta; Baumgartner, Alexandra; Noronha, João; Rauter, Amélia; Brenig, Bertram; Varela, J.The halotolerant green alga Dunaliella salina accumulates large amounts of β-carotene when exposed to various stress conditions. Although several studies concerning accumulation and biotechnological production of β-carotene have been published, the molecular basis and regulation of the genes involved in carotenoid biosynthesis in D. salina are still poorly known. In this paper, we report the isolation and regulation of the lycopene β-cyclase (Lcy-β) gene by abiotic stress. The function of this gene was determined by heterologous genetic complementation in E. coli. Gene expression and physiological analyses revealed that D. salina Lcy-β steady-state transcript and carotenoid levels were up-regulated in response to all stress conditions tested (salt, light and nutrient depletion). The results presented here suggest that nutrient availability is a key factor influencing carotenogenesis as well as carotenoid biosynthesis-related gene expression in D. salina.
- Molecular and functional characterization of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Dunaliella salinaPublication . Ramos, Ana; Marques, A. R.; Rodrigues, M.; Henriques, Nuno; Baumgartner, Alexandra; Castilho, Rita; Brenig, Bertram; Varela, J.In green algae,the final step of the plastidial methylerythritol phosphate (MEP) pathway is catalyzed by 4-hydroxy-3-methylbut-2-enyldiphosphate reductase(HDR; EC: 1.17.1.2),an enzyme proposed to play a keyrole in the regulation of isoprenoid biosynthesis. Here we report the isolation and functional characterization of a 1959- bp Dunaliella salina HDR (DsHDR) cDNA encoding a deduced polypeptide of 474 amino acid residues. Phylogenetic analysis implied a cyanobacterial origin for plant and algal HDR genes. Steady-state DsHDR transcript levels were higher in D. salina cells submitted to nutritional depletion, high salt and/or high light, suggesting that DsHDR may respond to the same environmental cues as genes involved in carotenoid biosynthesis.
- Molecular basis of isoprenoid biosynthesis in Dunaliella salinaPublication . Ramos, Ana; Varela, J.
- Population structure and connectivity of the European conger eel (Conger conger) across the north-eastern Atlantic and western Mediterranean: Integrating molecular and otolith elemental approachesPublication . Correia, A. T.; Ramos, Ana; Barros, F.; Silva, Goncalo; Hamer, P.; Morais, P.; Cunha, R. L.; Castilho, RitaGenetic variation (mtDNA) of the European conger eel, Conger conger, was compared across five locations in the north-eastern Atlantic (Madeira, Azores, South Portugal, North Portugal and Ireland) and one location in the western Mediterranean (Mallorca). Genetic diversity of conger eel was high, and differentiation among regions was not significant. Additionally, comparisons of element:Ca ratios (Sr:Ca, Ba:Ca, Mn:Ca and Mg:Ca) in otolith cores (larval phase) and edges (3 months prior to capture) among the Azores, North Portugal, Madeira and Mallorca regions for 2 years indicated that variation among regions were greater for edges than cores. Therefore, while benthic conger may display residency at regional scales, recruitment may not necessarily be derived from local spawning and larval retention. Furthermore, data from otoliths suggest a separated replenishment source for western Mediterranean and NE Atlantic stocks. The combination of genetics and otolith chemistry suggests a population model for conger eel involving a broad-scale dispersal of larvae, with limited connectivity for benthic juvenile life stages at large spatial scales, although the existence of one or multiple spawning grounds for the species remains uncertain.
- Engaging children in geosciences through storytelling and creative dancePublication . Matias, Ana Catarina; Carrasco, A. Rita; Ramos, Ana A.; Borges, R.Natural sciences have traditionally been disseminated in outreach activities as formal, one-way presentations. Nevertheless, innovative strategies are being increasingly developed using arts, gaming, and sketching, amongst others. This work aimed at testing an alternative and innovative way to engage non-expert audiences in ocean and coastal geology through a combination of scientific concept explanations and creative dancing. An informal education activity focusing on ocean dynamics was designed for 10-yearold students. It combines coastal science concepts (wind, waves, currents, and sand), storytelling techniques (narrative arc), and creative dance techniques (movement, imaginative play, and sensory engagement). A sequence of six exercises was proposed, starting with the generation of offshore ocean waves and ending with sediment transport on the beach during storm/fair-weather conditions. Scientific concepts were then translated into structured creative movements, within imaginary scenarios, and accompanied by sounds or music. The activity was performed six times with a total of 112 students. It was an inclusive activity given that all students in the class participated, including children with several mild types of cognitive and neurological impairment. The science and art activity aroused emotions of enjoyment and pleasure and allowed for effective communication between scientists and school community. Moreover, the results provide evidence of the activity’s effectiveness in engaging children and developing their willingness to further participate in similar activities.