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.

Geographical gradients in Argentinean terrestrial mammal species richness and their environmental correlates.

We analysed the main geographical trends of terrestrial mammal species richness (SR) in Argentina, assessing how broad-scale environmental variation (defined by climatic and topographic variables) and the spatial form of the country (defined by spatial filters based on spatial eigenvector mapping (SEVM)) influence the kinds and the numbers of mammal species along these geographical trends. We also evaluated if there are pure geographical trends not accounted for by the environmental or spatial factors. The environmental variables and spatial filters that simultaneously correlated with the geographical variables and SR were considered potential causes of the geographic trends. We performed partial correlations between SR and the geographical variables, maintaining the selected explanatory variables statistically constant, to determine if SR was fully explained by them or if a significant residual geographic pattern remained. All groups and subgroups presented a latitudinal gradient not attributable to the spatial form of the country. Most of these trends were not explained by climate.We used a variation partitioning procedure to quantify the pure geographic trend (PGT) that remained unaccounted for. The PGT was larger for latitudinal than for longitudinal gradients. This suggests that historical or purely geographical causes may also be relevant drivers of these geographical gradients in mammal diversity.

Favourable areas for expansion and reintroduction of Iberian lynx accounting for distribution trends and genetic diversity of the European rabbit

Although on a local scale Iberian lynx distribution is determined by the availability of prey rabbits, recent modelling analyses have uncovered broad-scale disagreements between these two species’ distribution trends. These analyses showed also that the lynx had become restricted to only a fraction of the rabbit’s genetic diversity, and that this could be jeopardising its survival in the face of environmental hazards and uncertainty. In the present paper, a follow-up was carried out through the building of lynx and rabbit distribution models based on the most recent Spanish mammal atlas. Environmental favourability values for lynx and rabbit were positively correlated within the lynx’s current distribution area, but they were negatively correlated within the total Spanish area where lynx used to occur in the 1980’s. Environmental favourability for rabbits was significantly higher where lynx maintains reproductive populations than where it recently disappeared, indicating that rabbit favourability plays an important role and can be a good predictor of lynx persistence. The lynx and rabbit models were extrapolated to predict favourable areas for both species in Spain as well as in Portugal, on the original scale of the distribution data (10x10 km) and on a 100 times finer spatial resolution (1x1 km). The lynx and rabbit models were also combined through fuzzy logic to forecast the potential for lynx occurrence incorporating information on favourable areas for its main prey. Several areas are proposed as favourable for lynx expansion or re-introduction,

Predictors of red fox (Vulpes vulpes) helminth parasite diversity in the provinces of Spain

We analysed the viscera of 321 red foxes collected over the last 30 years in 34 of the 47 provinces of peninsular Spain, and identified their helminth parasites. We measured parasite diversity in each sampled province using four diversity indices: Species richness, Marg a l e f’s species richness index, Shannon’s species diversity index, and inverse Simpson’s index. In order to find geographical, environmental, and/or human-related predictors of fox parasite diversity, we recorded 45 variables related to topography, climate, lithology, habitat heterogeneity, land use, spatial situation, human activity, sampling effort, and fox presence probability (obtained after environmental modelling of fox distribution). We then performed a stepwise linear regression of each diversity index on these variables, to find a minimal subset of statistically significant variables that account for the variation in each diversity index. We found that most parasite diversity indices increase with the mean distance to urban centres, or in other words, foxes in more rural provinces have a more diverse helminth fauna. Sampling effort and fox presence probability (probably related to fox density) also appeared as conditioning variables for some indices, as well as soil permeability (related with water availability). We then extrapolated the models to predict these fox parasite diversity indices in non-sampled provinces and have a view of their geographical trends.

Relative importance of environment, human activity and spatial situation in determining the distribution of terrestrial mammal diversity in Argentina

We recorded the number of terrestrial mammal species in each Argentinian province, and the number of species belonging to particular groups (Marsupialia, Placentaria, and among the latter, Xenarthra, Carnivora, Ungulates and Rodentia). We performed multiple regressions of each group’s SR on environmental, human and spatial variables, to determine the amounts of variation explained by these factors. We then used a variance partitioning procedure to specify which proportion of the variation in SR is explained by each of the three factors exclusively and which proportions are attributable to interactions between factors.

Spatial, environmental and human influences on the distribution of otter (Lutra lutra) in the Spanish provinces

In a previous survey of otters ( Lutra lutra L. 1758) in Spain, different causes were invoked to explain the frequency of the species in each province. To find common causes of the distribution of the otter in Spain, we recorded a number of spatial, environmental and human variables in each Spanish province. We then performed a stepwise linear multiple regression of the proportion of positive sites of otter in the Spanish provinces separately on each of the three groups of variables. Geographic longitude, January air humidity, soil permeability and highway density were the variables selected. A linear regression of the proportion of otter presence on these variables explained 62.4% of the variance. We then used the selected variables in a partial regression analysis to specify which proportions of the variation are explained exclusively by spatial, environmental and human factors, and which proportions are attributable to interactions between these components. Pure environmental effects accounted for only 5.5% of the variation, while pure spatial and pure human effects explained 18% and 9.7%, respectively. Shared variation among the components totalled 29.2%, of which 10.9% was explained by the interaction between environmental and spatial factors. Human factors explained globally less variance than spatial and environmental ones, but the pure human influence was higher than the pure environmental one. We concluded that most of the variation in the proportion of occurrences of otter in Spanish provinces is spatially structured, and that environmental factors have more influence on otter presence than human ones; however, the human influence on otter distribution is less structured in space, and thus can be more disruptive. This effect of large infrastructures on wild populations must be taken into account when planning large-scale conservation policies

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.