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.

Development of a novel molecular tool for the rapid assessment of the biodiversity changes of benthic nematodes assemblages. Mares Conference Marine Ecosystems Helath and conservation

The molecular profiling system was developed using directed terminal-restriction fragment length polymorphism (dT-RFLP) to characterize soil nematode assemblages by relative abundance of feeding guilds and validation by comparison to traditional morphological method. The good performance of these molecular tools applied to soil nematodes assemblages create an opportunity to develop a novel approach for rapid assessment of the biodiversity changes of benthic nematodes assemblages of marine and estuarine sediments. The main aim of this research is to combine morphological and molecular analysis of estuarine nematodes assemblages, to establish a tool for fast assessment of the biodiversity changes within habitat recovery of Zostera noltii seagrass beds; and validate the dT-RFLP as a high-throughput tool to assess the system recovery. It was also proposed to develop a database of sequences related to individuals identified at species level to develop a new taxonomic reference system. A molecular phylogenetic analysis of the estuarine nematodes has being performed. After morphological identification, barcoding of 18S rDNA are being determined for each nematode species and the results have shown a good degree of concordance between traditional morphology-based identification and DNA sequences. The digest strategy developed for soil nematodes is not suitable for marine nematodes. Then five samples were cloned and sequenced and the sequence data was used to design a new dT-RFLP strategy to adapt this tool to marine assemblages. Several solutions were presented by DRAT and tested empirically to select the solution that cuts most efficiently, separating the different clusters. The results of quantitative PCR showed differences in nematode density between two sampling stations according the abundance of the nematode density obtained by the traditional methods. These results suggest that qPCR could be a robust tool for enumeration of nematode abundance, saving time.

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.

NEW DATA ON THE CHRONOLOGY OF THE VALE DO FORNO SEDIMENTARY SEQUENCE (LOWER TAGUS RIVER TERRACE STAIRCASE) AND ITS RELEVANCE AS FLUVIAL ARCHIVE OF THE MIDDLE PLEISTOCENE IN WESTERN IBERIA

NEW DATA ON THE CHRONOLOGY OF THE VALE DO FORNO SEDIMENTARY SEQUENCE (LOWER TAGUS RIVER TERRACE STAIRCASE) AND ITS RELEVANCE AS FLUVIAL ARCHIVE OF THE MIDDLE PLEISTOCENE IN WESTERN IBERIA Pedro P. Cunha 1, António A. Martins 2, Jan-Pieter Buylaert 3,4, Andrew S. Murray 4, Luis Raposo 5, Paolo Mozzi 6, Martin Stokes 7 1 MARE - Marine and Environmental Sciences Centre, Department of Earth Sciences, University of Coimbra, Portugal: pcunha@dct.uc.pt 2 MARE - Marine and Environmental Sciences Centre, Dep. Geociências, University of Évora, Portugal; aam@uevora.pt 3 Centre for Nuclear Technologies, Technical University of Denmark, Risø Campus, Denmark; jabu@dtu.dk 4 Nordic Laboratory for Luminescence Dating, Aarhus University, Risø DTU, Denmark; anmu@dtu.dk 5 Museu Nacional de Arqueologia, Lisboa, Portugal; 3raposos@sapo.pt 6 Department of Geosciences, University of Padova, Italy; paolo.mozzi@unipd.it 7 School of Geography, Earth and Environmental Sciences, University of Plymouth, UK; m.stokes@plymouth.ac.uk The stratigraphic units that record the evolution of the Tagus River in Portugal (study area between Vila Velha de Ródão and Porto Alto villages; Fig. 1) have different sedimentary characteristics and lithic industries (Cunha et al., 2012): - a culminant sedimentary unit (the ancestral Tagus, before the drainage network entrenchment) – SLD13 (+142 to 262 m above river bed – a.r.b.; with probable age ca. 3,6 to 1,8 Ma), without artefacts; - T1 terrace (+84 to 180 m; ca. 1000? to 900 ka), without artefacts; - T2 terrace (+57 to 150 m; top deposits with a probable age ca. 600 ka), without artefacts; - T3 terrace (+43 to 113 m; ca. 460 to 360? ka), without artefacts; - T4 terrace (+26 to 55 m; ca. 335 a 155 ka), Lower Paleolithic (Acheulian) at basal and middle levels but early Middle Paleolithic at top levels; - T5 terrace (+5 to 34 m; 135 to 73 ka), Middle Paleolithic (Mousterian; Levallois technique); - T6 terrace (+3 to 14 m; 62 to 32 ka), late Middle Paleolithic (late Mousterian); - Carregueira Sands (aeolian sands) and colluvium (+3 a ca. 100 m; 32 to 12 ka), Upper Paleolithic to Epipaleolithic; - alluvial plain (+0 to 8 m; ca. 12 ka to present), Mesolithic and more recent industries. The differences in elevation (a.r.b.) of the several terrace staircases results from differential uplift due to active faults. Longitudinal correlation with the terrace levels indicates that a graded profile ca. 200 km long was achieved during terrace formation periods and a strong control by sea base level was determinant for terrace formation. The Neogene sedimentary units constituted the main source of sediments for the fluvial terraces (Fig. 2). Geomorphological mapping, coupled with lithostratigraphy, sedimentology and luminescence dating (quartz-OSL and K-feldspar post-IRIR290) were used in this study focused on the T4 terrace, which comprises a Lower Gravels (LG) unit and an Upper Sand (US) unit. The thick, coarse and dominantly massive gravels of the LG unit indicate deposition by a coarse bed-load braided river, with strong sediment supply, high gradient and fluvial competence, during conditions of rapidly rising sea level. Luminescence dating only provided minimum ages but it is probable that the LG unit corresponds to the earlier part of the MIS9 (ca. 335 to 325 ka), immediately postdating the incision promoted by the very low sea level (reaching ca. -140 m) during MIS10 (362 to 337 ka), a period of relatively cold climate conditions with weak vegetation cover on slopes and low sea level. Fig. 1. Main Portuguese reaches in which the Tagus River can be divided (Lower Tagus Basin): I – from the Spanish border to Arneiro (a general E–W trend, mainly consisting of polygonal segments); II – from Arneiro to Gavião (NE–SW); III – from Gavião to Arripiado (E–W); IV – from Arripiado to Vila Franca de Xira (NNE-SSW); V – from Vila Franca de Xira to the Atlantic shoreline. The faults considered to be the limit of the referred fluvial sectors are: F1 – Ponsul-Arneiro fault (WSW-ENE); F2 – Gavião fault (NW-SE); F3 – Ortiga fault (NW-SE); F4 – Vila Nova da Barquinha fault (W-E); F5 – Arripiado-Chamusca fault (NNE-SSW). 1 – estuary; 2 – terraces; 3 – faults; 4 – Tagus main channel. The main Iberian drainage basins are also represented (inset). The lower and middle parts of the US unit, comprising an alternation of clayish silts with paleosols and minor sands to the east (flood-plain deposits) and sand deposits to the west (channel belt), have a probable age of ca. 325 to 200 ka. This points to formation during MIS9 to MIS7, under conditions of high to medium sea levels and warm to mild conditions. The upper part of the US unit, dominated by sand facies and with OSL ages of ca. 200 to 154 ka, correlates with the early part of the MIS6. During this period, progradation resulted from climate deterioration and relative depletion of vegetation that promoted enhanced sediment production in the catchment, coupled with initiation of sea-level lowering that increased the longitudinal slope. The Vale do Forno and Vale da Atela archaeological sites (Alpiarça, central Portugal) document the earliest human occupation in the Lower Tagus River, well established in geomorphological and environmental terms, within the Middle Pleistocene. The Lower Palaeolithic sites were found on the T4 terrace (+26 m, a.r.b.). The oldest artefacts previously found in the LG unit, display crude bifacial forms that can be attributed to the Acheulian, with a probable age of ca. 335 to 325 ka. The T4 US unit has archaeological sites stratigraphically documenting successive phases of an evolved Acheulian, that probably date ca. 325 to 300 ka. Notably, these Lower Palaeolithic artisans were able to produce tools with different sophistication levels, simply by applying different strategies: more elaborated reduction sequences in case of bifaces and simple reduction sequences to obtain cleavers. Fig. 2. . Simplified geologic map of the Lower Tagus Cenozoic basin, adapted from the Carta Geológica de Portugal, 1/500000, 1992). The study area (comprising the Vale do Forno and Vale de Atela sites) is located on the more upstream sector of the Lower Tagus River reach IV, between Arripiado and Chamusca villages. 1 – alluvium (Holocene); 2 – terraces (Pleistocene); 3 – sands, silts and gravels (Paleogene to Pliocene); 4 – Sintra Massif (Cretaceous); 5 – limestones, marls, silts and sandstones (Mesozoic); 6 – quartzites (Ordovician); 7 – basement (Proterozoic to Palaeozoic); 8 – main fault. The main Portuguese reaches of the Tagus River are identified (I to V). The VF3 site (Milharós), containing a Final Acheulian industry, with fine and elaborated bifaces) found in a stratigraphic level located between the T4 terrace deposits and a colluvium associated with Late Pleistocene aeolian sands (32 to 12 ka), has an age younger than ca. 154 ka but much older than 32 ka. In the study area, the sedimentary units of the T4 terrace seem to record the river response to sea-level changes and climatically-driven fluctuations in sediment supply. REFERENCES Cunha P. P., Almeida N. A. C., Aubry T., Martins A. A., Murray A. S., Buylaert J.-P., Sohbati R., Raposo L., Rocha L., 2012, Records of human occupation from Pleistocene river terrace and aeolian sediments in the Arneiro depression (Lower Tejo River, central eastern Portugal). Geomorphology, vol. 165-166, pp. 78-90.

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.

The role of anglers' perceptions and habits in biological invasions: perspectives from the Iberian Peninsula

Anglers are a recognized vector for invasive fauna, with both intentional and accidental introductions reported worldwide. * The purpose of this study was to investigate the Iberian freshwater angler's habits and perceptions related to biological invasions, using an international and bilingual survey in Spain and Portugal. * The results showed that anglers from both countries cover great distances to fish, and that they commonly visit several places and fish during consecutive days, representing a major risk for invasions. Yet, anglers' activity patterns throughout the year are not constant: the summer months involve more intense activity and, hence, entail higher risk. * In both countries, the preferred fish species were invasive, and their introductions were reported more often than native species, with motivations being country-dependent. * The use of live bait was not frequent, but sometimes invasive species were used and the release of unused bait in the water was reported very frequently. * Most anglers recognize that introductions have environmental impacts and that anglers have an active role in intentional introductions. However, only a minority is aware of the angler's role in accidental transportation of invasive species. * These findings on anglers' behaviour patterns and perceptions may be used to model invasion risks and also improve monitoring and awareness programmes led by government agencies.

The role of angler’s perceptions and habits in biological invasions: Perspectives from Iberian Peninsula

1. Anglers are a recognized vector for invasive fauna, with both intentional and accidental introductions reported worldwide. 2. The purpose of this study 2. The purpose of this study was to investigate the Iberian freshwater angler’s habits and perceptions related to biological invasions, using an international and bilingual survey in Spain and Portugal. 3. The results showed that anglers from both countries cover great distances to fish, and that they commonly visit several places and fish during consecutive days, representing a major risk for invasions. Yet, anglers’ activity patterns throughout the year are not constant: the summer months involve more intense activity and, hence, entail higher risk. 4. In both countries, the preferred fish species were invasive, and their introductions were reported more often than native species, with motivations being country-dependent. 5. The use of live bait was not frequent, but sometimes invasive species were used and the release of unused bait in the water was reported very frequently. 6. Most anglers recognize that introductions have environmental impacts and that anglers have an active role in intentional introductions. However, only a minority is aware of the angler’s role in accidental transportation of invasive species. 7. These findings on anglers’ behaviour patterns and perceptions may be used to model invasion risks and also improve monitoring and awareness programmes led by government agencies.