Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

Benthic nematode assemblage composition and diversity during a natural recovery process of Zostera noltii seagrass beds,

In 2008, the stable seagrass beds of the Mira estuary (SW Portugal) disappeared completely; however, during 2009, they have begun to present early symptoms of natural recovery, characterised by a strongly heterogeneous distribution. This study was designed to investigate the spatial and temporal variability patterns of species composition, densities and trophic composition of the benthic nematode assemblages in this early recovery process, at two sampling sites with three stations each and at five sampling occasions. Because of the erratic and highly patchy seagrass recovery and the high environmental similarity of the two sampling sites, we expected within-site variability in nematode assemblages to exceed between-site variability. However, contrary to that expectation, whilst nematode genus composition was broadly similar between sites, nematode densities differed significantly between sites, and this between-site variability exceeded within-site variability. This may be linked to differences in the Zostera recovery patterns between both sites. In addition, no clear temporal patterns of nematode density, trophic composition and diversity were evident.

Genetic diversity and population structure of the sea lamprey (Petromyzon marinus L.) across its distributional range

Lampreys are a group of ancient vertebrates with 360 million years of existence. Throughout their evolution, they have acquired local adaptations to the colonized habitats, showing high plasticity and adaptive capacities. The sea lamprey (Petromyzon marinus L.) is a parasitic and anadromous species that occurs in both sides of the North Atlantic. The aims of this study were to analyse, using microsatellite markers, the genetic diversity and population structure of sea lamprey throughout its distributional range. Analyses demonstrated consistent signs of high population differentiation between European and North American samples (two-groups structure), most probably due to isolation by distance, but low differentiation among populations from the same coast. The apparent lack of homing in this species is in line with its high evolutive success, as homing may bring adults back to natal habitats that have changed, or that are intermittently unfavourable. Analyses also demonstrated higher levels of genetic diversity in North American samples; DIVERSIDADE GENÉTICA E ESTRUTURA POPULACIONAL DA LAMPREIA-MARINHA (PETROMYZON MARINUS L.) AO LONGO DA SUA ÁREA DE DISTRIBUIÇÃO Resumo: As lampreias são organismos ancestrais com cerca de 360 milhões de anos de existência. No decorrer da longa escala evolutiva têm vindo a adquirir adaptações aos locais que colonizaram, tendo uma forte capacidade evolutiva e adaptativa. A lampreia-marinha (Petromyzon marinus L.) é uma espécie parasita e anádroma que ocorre em ambas as costas do Atlântico Norte. Este estudo teve como principal objetivo estudar a diversidade genética e a estrutura populacional desta espécie ao longo da sua área de distribuição, através do uso de microssatélites. Os resultados demonstraram forte divergência entre populações das costas Este e Oeste do Atlântico Norte, provavelmente devido à elevada distância entre populações, mas pouca diferenciação entre populações da mesma costa. A ausência de homing nesta espécie terá contribuído para o seu sucesso evolutivo, uma vez que o homing pode levar indivíduos a reproduzirem-se em habitats que se tornaram desfavoráveis ou intermitentemente inapropriados. Os resultados demonstraram também uma maior variabilidade genética nas populações americanas.

Can birds play a role as High Nature Value indicators of montado system?

Montados form a heterogeneous landscape of wooded matrix dominated by cork and/or holm oak with open areas characterized by fuzzy boundaries. Montado supports a high biological diversity associated to low intensity management and a landscape diversity provided by a continuous gradient of land cover. Among other features this permits the classification of montados as a High Nature Value (HNV) system. We assessed the role of birds as HNV indicators for montado, and tested several bird groups—farmland, edge, forest generalists and forest specialists species; and some universal indicators such as species conservation status, Shannon’s diversity index and species richness. Our study areas covered the North–South distribution of cork oak in Portugal, and we surveyed the breeding bird communities across 117 sampling sites. In addition to variables related to management and sanitary status, we considered variables that characterize the landscape heterogeneity inside the montado—trees and shrub density and richness of woody vegetation. Our results suggest that specific bird guilds can be used as HNV indicators of particular typologies of montado, and highlight the need to develop an indicator that could be transversally applied to all types of montado.

Genetic diversity of Bursaphelenchus cocophilus in South America

Molecular characterisation of Bursaphelenchus cocophilus, the causal agent of ‘red ring disease’, is imperative for efficient identification procedures in Brazil and Colombia, because quarantine species such as B. xylophilus and B. mucronatus are already listed in both countries. ITS-1/2 region and D2-D3 segment of LSU rDNA were used to characterise isolates of B. cocophilus obtained from coconut plantations in Brazil and Colombia. Results from ITS-1/2 and LSU rDNA regions showed that all isolates of B. cocophilus from Brazil and Colombia formed a monophyletic group. The LSU rDNA region indicated that all isolates formed a single monophyletic group with high Bayesian posterior probability (100%). This is the first study on ITS-1/2 for the characterisation of B. cocophilus populations. A species-specific primer was designed for identification of B. cocophilus.