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.

Enzymatic activity on sediments and nematode assemblage responses during seagrass beds habitat recovery following the disturbance of the traditional digging activity of bivalves harvesting.

Sediment digging is an anthropogenic activity connected to the exploitation of living resources in estuarine and marine environments. The knowledge on the functional responses of the benthic assemblages to the physical disturbance is an important baseline to understand the ecological processes of the habitat recovery and restoration and to develop tools for the management of the harvesting activities. To investigate the effects of the digging activity of the bivalves on Zostera noltii seagrass beds a manipulative field experiment was conducted that included the enzymatic activity of sediments and the associated nematode assemblages. Four plots (two undisturbed serving as control and two dug to collect bivalves - treatment) with 18 subplots were randomly located at seagrass beds in the Mira estuary at the SW coast of Portugal. Samples were randomly and unrepeatably collected from three subplots of each plot in five different occasions, before sediment digging (T0) up to six months after disturbance (T5). Microbial activity in sediments was assess by determining the extracelular enzymatic activity of six hydrolytic enzymes (sulfatase, phosphatase, b -N-acetilglucosaminidase, b-glucosidase, urease, protease) and two oxidoreductases (phenol oxidase and peroxidase). The microbial community status was also assessed through the measurement of dehydrogenase, which reflects microbial respiration. The nematode assemblages composition, biodiversity and trophic composition at different sampling occasions were also analyzed. The fluorometric and biochemical parameters analysed of the Z. noltii plants during the experimental period showed a recovery of the seagrass beds, and it was detected an increase of the enzymatic activity of the sediments after disturbance. The nematodes assemblages were similar in all sampling occasions. The seagrass beds and the nematodes assemblages associated showed a high resilience to the stress caused by the traditional bivalves digging activity. The obtained results allow the development of a management programme for the commercial fishing activity to maintain the good environmental status and minimized the secondary environmental effects on marine and estuarine habitats through the establishment of a baseline for the regulation of the harvesting frequency.

Transferability of environmental favourability models in geographic space: The case of the Iberian desman (Galemys pyrenaicus) in Portugal and Spain

Transferring distribution models between different geographical areas may be problematic, as the performance of models outside their original scope is hard to predict. A modelling procedure is needed that gets the gist of the environmental descriptors of a distribution area, without either overfitting to the training data or overestimating the species’ distribution potential.We tested the transferability power of the favourability function, a generalized linear model, on the distribution of the Iberian desman (Galemys pyrenaicus) in the Iberian territories of Portugal and Spain.We also tested the effects of two of the main potential constraints on model transferability: the analysed ranges of the predictor variables, and the completeness of the species distribution data. We modelled 10 km×10km presence/absence data from Portugal and Spain separately, extrapolated each model to the other country, and compared predictions with observations. The Spanish model, despite arguably containing more false absences, showed good predictive ability in Portugal. The Portuguese model, whose predictors ranged between only a subset of the values observed in Spain, overestimated desman distribution when transferred.We discuss possible reasons for this differential model behaviour, and highlight the importance of this kind of models for prediction and conservation applications

Geographical and environmental correlates of big and small game in Andalusia (southern Spain)

In Andalusia, southern Spain, each game estate applies its own rules and presents its results in annual hunting reports, which have been mandatory for Spanish game estates since 1989. We used the information about hunting yields, included in 32134 annual hunting reports produced during the period 1993/94 to 2001/02 by 6049 game estates, to determine the current distribution of hunting yields of big and small game species in Andalusia. Using generalised linear models and a geographic information system, we determined the most favourable municipalities to big and small game, respectively, and delimited potential areas to attain good hunting yields for big and small game at a 1-km2 resolution. Municipalities and areas favourable to big game are mainly located in the Sierra Morena and the westernmost fringe of the Betic Range, while those favourable to small game occupy the upper Guadalquivir River valley. There is a clear segregation between big and small game species according to the physiography and land uses of the territory. Big game species are typical of Mediterranean woodland areas, while the most emblematic small game species prefer agricultural areas. Our results provide a territorial ordination of hunting yields in southern Spain and have several potential applications in strategic planning for hunting activities and biodiversity conservation in Andalusia that can be extrapolated to other regions.

Land use and environmental factors affecting red-legged partridge (Alectoris rufa) hunting yields in southern Spain

The red-legged partridge is a small game species widely hunted in southern Spain. Its commercial use has important socioeconomic effects in rural areas where other agrarian uses are of marginal importance. The aims of the present work were to identify areas in Andalusia (southern Spain) where game yields for the red-legged partridge reach high values and to establish the environmental and land use factors that determine them. We analysed 32,134 annual hunting reports (HRs) produced by 6,049 game estates during the hunting seasons 1993/1994 to 2001/2002 to estimate the average hunting yields of red-legged partridge in each Andalusian municipality (n=771). We modelled the favourability for obtaining good hunting yields using stepwise logistic regression on a set of climatic, topographical, land use and vegetation variables that were available as digital coverages or tabular data applied to municipalities. Good hunting yields occur mainly in plain areas located in the Guadalquivir valley, at the bottom of Betic Range and in the Betic depressions. Favourable areas are related to highly mechanised, lowelevation areas mainly dedicated to intensive dry crops. The most favourable areas predicted by our model are mainly located in the Guadalquivir valley.