Cardoni Da
email: acardoni@mdp.edu.ar

Isacch Jp

Fanjul Me

Iribarne Oo

Relationship between anthropogenic sewage discharge, marsh structure and bird assemblages in a SW Atlantic saltmarsh

Keywords: Saltmarsh bird, Spartina alterniflora, eutrophication, coast, Bahía Blanca estuary, Argentina

One of the main effects of urbanization on coastal areas is through the discharge of sewage, which increases nutrient concentrations in the receiving environment. Salt marshes, like other coastal marine environments, are limited by nutrients, mainly nitrogen, and thus increasing nutrient loadings to a marsh may have consequences on marsh characteristics. We evaluated how the effects of nutrient enrichment in the form of sewage input, affected the vegetation structure and bird assemblages in a Spartina alterniflora salt marsh system near Bahía Blanca, Argentina (39° 01´ S -56° 25´ W). Surveys of nutrient concentration, vegetation and birds were made at three different distances from the sewage discharge source. The concentration of ammonium, phosphate, and nitrate and the percent organic matter were higher in marshes nearest to the sewage discharge source. Bird composition and abundance, and vegetation physiognomy changed along a gradient of nutrient concentration. The increased habitat complexity found near the areas of higher nutrient concentration was exploited by birds that use neighboring interior and coastal habitats, including Spartina densiflora marshes, freshwater marshes and upland shrubby habitats. Our results show that local increases of nutrient inputs directly changed the vegetation physiognomy, and indirectly the composition and abundance of bird assemblages.

Introduction

The human population tends to live near the coastline [START_REF] Wolanski | Estuarine Ecohydrology[END_REF], and thus, human activities can have greater impacts on the coastal ecosystems than on inland habitats (UNEP, 2006;[START_REF] Valiela | Global Coastal Change[END_REF]. One of the main results of coastal populations is the discharge of sewage, which involves increased nutrient concentrations in the coastal environment (GESAMP, 1990). Salt marshes, like other coastal ecosystems, have relatively low-nutrient supplies [START_REF] Valiela | Nutrient limitation in salt marsh vegetation[END_REF], and so nutrient increases can have large consequences i.e., changing the (1) structural complexity of marsh plants by enhancing biomass and stem density, (2) species composition, and (3) zonation patterns [START_REF] Levine | Nutrients, competition and plant zonation in a New England salt marsh[END_REF][START_REF] Daleo | Mycorrhizal fungi determine salt marsh plant zonation depending on nutrient supplies[END_REF]. Also, nutrient increases may lead to marsh subsidence and habitat fragmentation, depending on the balance between sediment depositions, increased plant production, and increased decomposition [START_REF] Valiela | Nutrient limitation in salt marsh vegetation[END_REF][START_REF] Harper | Eutophication of Freshwaters: Principles, problems and restoration[END_REF][START_REF] Deegan | Lessons learned: The effects of nutrient enrichment on the support of nekton by seagrass and salt marsh ecosystems[END_REF]. Specifically, in salt marshes characterized by large monospecific plant stands, nutrient enrichment may modify the plant physical structure by changing the plant growth form [START_REF] Deegan | Lessons learned: The effects of nutrient enrichment on the support of nekton by seagrass and salt marsh ecosystems[END_REF]. Nitrogen is usually the limiting nutrient controlling aboveground salt marsh vegetation production [START_REF] Mendelssohn | Nitrogen metabolism in the height forms of Spartina alterniflora in North Carolina[END_REF] and its availability increases the plant canopy and standing stocks, which is followed by higher rates of vegetation decay [START_REF] Mitsch | Wetlands[END_REF], and changes in species composition [START_REF] Craft | Response of Everglades plant communities to nitrogen and phosphorus additions[END_REF][START_REF] Vaithiyanathan | Nutrient in the Everglades: Examination along the eutrophication gradient[END_REF]. A case largely studied in the northern hemisphere is the change of the dwarf form of Spartina alterniflora into the tall form as a result of an increase in nutrients loading to a marsh [START_REF] Valiela | Nutrient limitation in salt marsh vegetation[END_REF][START_REF] Valiela | Nutrient and particulate fluxes in a salt marsh ecosystem: Tidal exchanges and inputs by precipitation and groundwater[END_REF]. This increased nitrogen loading, however may or may not lead to a net gain of the marsh depending on the balance between the change in marsh plant production and decomposition. Increases in the primary production or vegetation biomass may produce more detritus [START_REF] Harper | Eutophication of Freshwaters: Principles, problems and restoration[END_REF][START_REF] Deegan | Lessons learned: The effects of nutrient enrichment on the support of nekton by seagrass and salt marsh ecosystems[END_REF] which is important in food webs [START_REF] Deegan | Salt marsh ecosystem support of marine transient species[END_REF] and in creating peat that forms the physical structure of the marsh platform [START_REF] Friedrichs | Tidal salt marsh morphodynamics: A synthesis[END_REF]. Dead vegetation deposition ('wracks') on coastal marshes is a natural disturbance that can increase habitat heterogeneity [START_REF] Valiela | Disturbance of salt marsh vegetation by wrack mats in Great Sippewissett marsh[END_REF][START_REF] Minchinton | Disturbance by wrack facilitates spread of Phragmites australis in a coastal marsh[END_REF]. Thus when nutrients loadings to a salt marshes increase we expect greater vegetation heterogeneity because of the increased canopy structure and more frequent occurrence of wracks.

Vegetation structure, composition, and floristic characteristics are substantial factors influencing bird habitat selection, because these factors provide food, nesting sites, and cover from predators [START_REF] Macnally | The roles of floristics and physiognomy in avian community composition[END_REF][START_REF] Vickery | The management of lowland neutral grasslands in Britain: Effects of agricultural practices on birds and their food resources[END_REF][START_REF] Buchanan | The contribution of invertebrate taxa to moorland bird diets and the potential implications of land-use management[END_REF].

Individuals select habitats that are an optimal combination of resources that allows them to perform multiple activities (e.g., foraging, breeding, roosting;Hilden, 1965;[START_REF] Fretwell | On territorial behavior and other factors influencing habitat distribution in birds[END_REF][START_REF] Block | The habitat concept in ornithology: Theory and applications[END_REF][START_REF] Steele | Selection of foraging and nesting sites by Black-throated blue Warblers-their relative influence on habitat choice[END_REF]. Thus habitat structural heterogeneity is often a good predictor of bird diversity (Mac [START_REF] Arthur | On bird species diversity[END_REF][START_REF] Wiens | Pattern and process in grassland bird communities[END_REF][START_REF] Roth | Spatial heterogeneity and bird species diversity[END_REF]. This result is probably due to the role of habitat complexity in promoting niche diversification [START_REF] Wilson | Avian community organization and habitat structure[END_REF][START_REF] Roth | Spatial heterogeneity and bird species diversity[END_REF]. There are many examples of the effect of nutrient enrichment on the aquatic environment (i.e., lakes estuaries; [START_REF] Robledano | Waterbirds and nutrient enrichment in Mar Menor lagoon, a shallow coastal lake in southeast Spain[END_REF], such as those generated by algal blooms on waterbirds [START_REF] Rosa | Factors affecting waterbird abundance and species richness in an increasingly urbanized area of the Tagus estuary in Portugal[END_REF], waterbird breeding [START_REF] Rönkä | Environmental changes and population trends of breeding waterfowl in northern Baltic Sea[END_REF] and shorebirds (Raffatelli 1999;[START_REF] Lopes | Influence of macroalgal mats on abundance and distribution of dunlin Calidris alpina in estuaries: A long-term approach[END_REF]. Bird abundance is positively correlated with lake nutrient levels [START_REF] Nilsson | Breeding bird community densities and species richness in lakes[END_REF]Hoyer andCanfield, 1990, 1994;[START_REF] Mccarty | Aquatic community characteristics influence the foraging patterns of Tree Swallows[END_REF]. However, although the effects of eutrophication are a well studied phenomena worldwide [START_REF] Vitousek | Human alteration of the global nitrogen cycle: causes and consequences[END_REF][START_REF] Valiela | Global Coastal Change[END_REF], we are not aware of studies on the effects of nutrient enrichment on bird assemblages associated with salt marsh habitats.

The salt marshes of the SW Atlantic are being modified at an increasing rate [START_REF] Costa | Human impacts and threats to the conservation of South American salt marshes[END_REF], primarily by cattle grazing, fire [START_REF] Isacch | Post-fire vegetation change and bird use of a salt marsh in coastal Argentina[END_REF], and sewage discharges (Nebia and Zalba, 2007;[START_REF] Martinetto | High abundance and diversity of consumers associated to eutrophic areas in a semi-desert macrotidal coastal ecosystem in Patagonia, Argentina[END_REF]. As happens elsewhere, in this region there are sewage discharges into the salt marshes. Specifically, there is an important sewage discharge on a Spartina alterniflora salt marsh in Bahía Blanca (Argentina) where salt marshes develop characteristics of those of the SW Atlantic [START_REF] Isacch | Distribution of saltmarsh plant communities associated with environmental factors along a latitudinal gradient on the south-west Atlantic coast[END_REF]. Because increased nutrients on a salt marsh are expected to increase vegetation structure from the direct consequences of eutrophication [START_REF] Daleo | Mycorrhizal fungi determine salt marsh plant zonation depending on nutrient supplies[END_REF], we predicted that bird assemblage will also change from the indirect influences. Thus, we studied how nutrients change along a gradient from the sewage discharge source, and how this gradient is related with the vegetation structure and bird composition and abundance.

Materials and Methods

Study area

The study was performed at the Bahía Blanca estuary (39°01´S-56°25´W; Argentina), which includes one of the largest SW Atlantic salt marshes [START_REF] Isacch | Distribution of saltmarsh plant communities associated with environmental factors along a latitudinal gradient on the south-west Atlantic coast[END_REF]Fig. 1). The bay has a total area of 2300 km 2 [START_REF] Montesarchio | Dinámica sedimentaria en la denominada ría de Bahía Blanca. Contribución científica[END_REF], with 10% of that surface covered by salt marshes and 20% by mudflats [START_REF] Isacch | Distribution of saltmarsh plant communities associated with environmental factors along a latitudinal gradient on the south-west Atlantic coast[END_REF]. The salt marsh vegetation is dominated by two species; Spartina alterniflora grows in the lower marsh, and Sarcocornia perennis grows in the upper marsh. The industrial and port city of Bahía Blanca (300,000 inhabitants) is located on this coast. This city discharges its sewage into the bay relatively far from the city (Fig. 1). As in most of the bay, a large salt marsh dominated by S. alterniflora develops around the discharge point. Our observations suggested that there are strong variations in the salt marsh vegetation characteristics linked with the sewage discharge, and that they are not typical of a natural gradient. The latter conclusion is supported by a study about biomass variation of S. alterniflora marshes developed in that region [START_REF] Isacch | Generalization across space in the relationship between cord grass (Spartina alterniflora) biomass and reflectance data by using Landsat ETM+ images[END_REF], in which it was found that S. alterniflora plants reached larger sizes near the sewage discharge. Three sites were selected at different distances from the sewage to study the relationship between anthropogenic nutrient concentration, the vegetation and birds, (Fig. 1). The salt marsh growing around the sewage discharge pipe and the proximal part of the discharge focus, were considered as the area of maximum nutrient effect (Naposta marsh, 10 ha; Fig 1). A second site located approximately 200 m from that source was considered as an area of medium effect (Midway marsh, 11.5 ha; Fig. 1). A third site located at 10000 m from the main discharge source was considered as an area of low effect (Maldonado marsh, 14.5 ha; Fig 1). All marshes have similar tidal conditions, given that they are positioned to the SW, and were located at the same distance (~1500 m) from the main tidal channel. We assumed, therefore, that the three marshes are similar except for their exposure to the sewage outfall.

Nutrient levels

To evaluate if the sewage discharge caused nutrient enrichment of the soil, we collected sediment samples to determine the total organic matter content (OM), concentration of dissolved inorganic nitrogen (nitrate and ammonium), and concentration of inorganic phosphorous (i.e., phosphate) in porewater. Twenty sediment samples were obtained using a stratified random sampling design at different distances from the sewage channel: 20 m and 80 m (Naposta marsh), 150 and 220 m (Midway marsh), and 10,000 m (Maldonado marsh). Sediment samples were collected by pushing a PVC core (3 cm diameter, 8 cm length) into the sediment. Cores were immediately transported to the laboratory, where porewater was obtained by centrifugation (9000 × g, 10 min), filtered, and stored at -20°C until the analysis of dissolved nutrients. The nitrate concentration in porewater was determinated calorimetrically as nitrite after reduction by cadmium followed by diazotization [START_REF] Strickland | A practical handbook of sea water analysis[END_REF]). The concentration of ammonium was measured using the blue indophenol method [START_REF] Solórzano | Determination of ammonia in natural waters by the phenolhypochlorite method[END_REF]. The concentration of dissolved phosphate was measured using the molybdate method [START_REF] Strickland | A practical handbook of sea water analysis[END_REF]. A set of samples were dried (7 days at 60 °C) and combusted (8 h at 500 °C) to determine the total organic matter content (%) as the loss weight between dry and combusted sediment. The null hypothesis of no difference in nutrients (nitrate, ammonium and phosphate concentrations, and organic matter content) at different distances of the sewage discharge source were evaluated by using a one-way ANOVA. An a posteriori LSD Fischer test was used to identify differences when necessary [START_REF] Zar | Biostatistical analysis[END_REF]. The relationship between nutrients concentrations and distance to the sewage discharge source was assessed by simple regression analysis [START_REF] Zar | Biostatistical analysis[END_REF].

Vegetation and bird surveys

We sampled vegetation and birds along 7 randomly distributed 100 x 60 m transects in each marsh using the fixed-width strip-transect method (following [START_REF] Bibby | Bird Census Techniques[END_REF]. Transects were separated by a minimum distance of 100 m. Birds were surveyed monthly between July 2007 and June 2008. The habitats included within transects were the vegetated marsh platform and the open gaps (determined by dead vegetation deposition, i.e. wracks). The vegetation physiognomy was surveyed in each bird sampling unit (i.e., transect) during winter and summer. In each transect used to survey birds, we measured the following vegetation variables: height of the dominant strata of vegetation, plant species, percent of total and green coverage vegetation, and the number of Spartina alterniflora spikes (i.e., inflorescences). The vegetation data for each transect came from the average of two squares (1*1 m) randomly located along transect. All vegetation surveys were made in vegetated areas. We assumed that the vegetation height and cover represented the main components of the habitat structure in marsh ecosystems [START_REF] Cardoni | Indirect effects of the intertidal burrowing Crab Chasmagnathus granulatus in the habitat use of Argentina's South West Atlantic salt marsh birds[END_REF].

The null hypothesis of no difference in bird richness and abundance and vegetation characteristics (height of vegetation, percent coverage of Spartina alterniflora and spike density) among the three sites at different distances of the sewage discharge source were evaluated by using a one-way ANOVA for each sampling date. An a posteriori LSD Fischer test was used to identify differences, when necessary [START_REF] Zar | Biostatistical analysis[END_REF]. The null hypothesis of no difference in vegetation characteristics between seasons was evaluated by using a t-test [START_REF] Zar | Biostatistical analysis[END_REF]. We used simple regression analysis [START_REF] Zar | Biostatistical analysis[END_REF] to assess the relationship between nutrient concentrations and vegetation variables, and between bird and vegetation variables.

There were dead vegetation depositions of Spartina alterniflora ('wracks') in areas closes to the sewage channel. To evaluate if marshes with different nutrient levels have a different density of wracks, we compared the number of wracks among them using the Google Earth software (http://earth.google.com). The study site was covered by an image from Google Earth with high resolution (date 01-12-2007) where wracks previously geopositioned were easily recognized. Then a portion of 10 ha in the core area was analyzed for each salt marsh (Naposta, Midway and Maldonado). Wracks in each image were digitized to determine the number and the cover area. The density of wracks was then calculated in each case from the digitized images and differences among sites were evaluated using a Chi-square test [START_REF] Zar | Biostatistical analysis[END_REF].

Results

Nutrient levels

There was a nutrient gradient linked to the distance from the sewage discharge source. Ammonium, phosphate and nitrate concentrations were higher at Naposta, the marsh nearer to the sewage discharge, and lower in Midway and Maldonado marshes (ANOVA; Ammonium: F 2,16 = 16.90, P < 0.05; Phosphate: F 2,16 = 14.01, P < 0.05; Nitrate: F 2,16 = 47.91, P < 0.05; Fig. 2). The same pattern was recorded for the OM content (ANOVA; OM: F 2,16 = 4.73, P < 0.05; Fig. 3).

Nutrient concentrations decerased with increasing distances from the sewage discharge source (Ammonium: r 2 = 0.34, P < 0.05; Phosphate: r 2 = 0.30, P < 0.05; Nitrate: r 2 = 0.47, P < 0.05) and no relationship was found between distance and OM (r 2 = 0.17, P > 0.05).

Vegetation

The only species recorded in all studied sites was the cord grass Spartina alterniflora. The vegetation height and cover were higher in Naposta marsh than in the Midway and Maldonado marshes, both in winter and summer (ANOVA; Vegetation height; winter: F 2,18 = 62.85, P < 0.05; summer: F 2,18 = 18.40, P < 0.05; Vegetation cover; winter: F 2,18, = 23.79, P < 0.05; summer: F 2,18 = 10.41, P < 0.05; Fig. 4A and4B).

The vegetation height was higher at the Midway marshes than at the Maldonado marsh only during winter. Plant cover was higher at the Midway marsh than at the Maldonado marsh in the summer. The spike density was higher both in summer and winter in the Naposta and Midway marshes than in the Maldonado marsh (ANOVA; winter: F 2,18 = 9.92, P < 0.05; summer: F 2,18 = 8.95, P < 0.05; Fig. 4C).

The total and green cover of S. alterniflora were both higher in summer than winter at the three salt marshes (t-test; total cover, Naposta, t 12 = 2.95, P < 0.05; Midway, t 12 = 2.67, P < 0.05; Maldonado: t 12 = 5.18, P < 0.05; green cover, Naposta, t 12 = 10.81, P < 0.05; Midway, t 12 = 4.31, P < 0.001; Maldonado, t 12 = 8.01, P < 0.05). Vegetation height was better explained by nutrients concentrations (Phosphate, r 2 = 0.66, P < 0.05; Ammonium, r 2 = 0.70, P < 0.05; Nitrate, r 2 = 0.84, P < 0.05, Fig. 5A) than vegetation cover (Phosphate, r 2 = 0.51, P < 0.05; Ammonium, r 2 = 0.51, P < 0.05; Nitrate, r 2 = 0.66, P < 0.05).

There were 13 wracks (7.5 % of 10 ha marsh) at the Naposta marsh, 3 wracks (1.9 % of 10 ha marsh) at the Midway marsh, and none at the Maldonado marsh (Chi-Square = 227.63, df = 1, P < 0.05).

Birds

Nineteen bird species were recorded in the three sites (Table 1). The total bird species recorded for each distance were 16, 12 and 5 species in the Naposta, Midway and Maldonado marshes, respectively. Seventy-two percent of all birds, at all sites were at the Naposta marsh (the nearest area of the discharge source) and only 23 and 5 % at the Midway and at Maldonado marshes, respectively. The bird abundance was always higher in Naposta marsh than in Maldonado marsh (Table 2 and Fig. 6). The bird abundance exclusive of the Yellow-winged Blackbird also was higher in the Naposta marsh than in the Maldonado marsh for all months (Table 2 and Fig. 6). In some months it was also higher in the Naposta marsh than in the Midway marsh (Table 2 and Fig. 6).

The bird richness was always higher in the Naposta marsh (Fig. 6).

The Yellow-winged Blackbird (Agelaius thilius) was the most abundant bird species, with 72 % of the total bird abundance. However, 80 % of the Yellow-winged Blackbird abundance was recorded only in the Naposta marsh. Other bird species associated with tall grass (without considering Yellow-winged Blackbird; Table 1) were only recorded at the Naposta marsh, the site with higher values of S. alterniflora cover and height. Bird species associated with open areas (i.e., short grass and mud flats; Table 1) had a higher abundance in the Naposta marsh than in the Midway and Maldonado marshes in four of ten months, and were higher in two months in the Naposta and Midway marshes than in Maldonado marsh (Table 2). It should be noted that there were more wracks in the Naposta marsh than in the others two marshes. alterniflora height: r 2 = 0.28, P < 0.05). Bird abundance was also explained by S. alterniflora cover (r 2 = 0.53, P < 0.05). Bird richness was not correlated with vegetation cover (r 2 = 0.17, P > 0.05). Bird abundance without Yellow-winged Blackbird (species that represented the 72 % of the total bird abundance) was correlated with vegetation cover and height (Abundance without Yellow-winged Blackbird*S. alterniflora height: r 2 = 0.47, P < 0.05; Abundance without Yellow-winged Blackbird* S. alterniflora cover: r 2 = 0.41, P < 0.05).

Discussion

The concentration of ammonium, nitrate and phosphate and the percent of organic matter content were higher in marshes nearest to the sewage discharge source.

Vegetation physiognomy, bird composition and abundance changed along a gradient of nutrient concentrations in a S. alterniflora marsh. Bird species and bird abundance increased near the nutrient discharge source. These changes were associated with the increase of height and cover of the S. alterniflora. The same pattern was recorded among height and cover of S. alterniflora with nutrient concentration. This study is one of the first that evaluates the relationship between nutrient enrichment and actual effects on higher trophic levels, such as birds.

The main effect of the discharge seems to be constrained relatively near to the nutrient source, given that nutrients and vegetation structure decreased abruptly, being approximately 50 percent lower within ~250 meters of the sewage sourcewater. These patterns suggest that the S. alterniflora marsh is a 'sink' for anthropogenic nutrients (the concentration of ammonium decreased from 453 to 214 µM for, phosphate from 442 to 221 µM, nitrate from 35 to 19 µM, the OM decreased from 15 to 9%), vegetation cover declined from 85 to 75 %, and height dropped from 87 to 46 cm. These results suggest that perhaps Spartina alterniflora could be used as an ecological engineering organism to remediate eutrophicated salt marsh areas (Mitsch and Jørgensen, 1989;Marques et al., 2003).

The increase in habitat structure nearest the sewage outfall was exploited by bird species that use the neighboring interior and coastal habitats such as S. densiflora marsh, fresh marshes, upland shrubby habitats. These neighboring habitat species could take advantage of the increased refuge (e.g., species associated to dense grassland as Bay-capped Wren-Spinetail, Spartonoica maluroides; [START_REF] Cardoni | Indirect effects of the intertidal burrowing Crab Chasmagnathus granulatus in the habitat use of Argentina's South West Atlantic salt marsh birds[END_REF][START_REF] Mitchell | Impacts of marsh management on coastal-marsh bird habitats[END_REF], food (e.g., partially granivorous species as the Yellow-winged Blackbird; [START_REF] Darrieu | Ecología alimentaría del Varillero Ala Amarilla (Agelaius thilius) en ambientes ribereños de la provincia de Buenos Aires[END_REF] and nesting sites [START_REF] Mitchell | Impacts of marsh management on coastal-marsh bird habitats[END_REF]Cardoni D.A. and Isacch J.P., unpublished data). On the other hand, the open areas generated by mat deposition were used mainly by waders and shorebirds at the Midway marsh, and by passerines and shorebirds in the marshes located near the sewage discharge outfall. The eutrophication at the Bahía Blanca marshes may, therefore, be producing a bottom-up effect that directly affects vegetation, and indirectly affects organisms higher in the trophic web, such as birds.

The species using marshes changed with seasons. The Bar-winged Cinclodes (Cinclodes fuscus) and Rufous-backed Negrito (Lessonia rufa) were found during winter, while the Bay-capped Wren-Spinetail and Grassland Yellow-Finch (Sicalis luteola) were recorded in the summer. Other birds, such as the Yellow-winged Blackbird, Correndera Pipit (Anthus correndera) and Chimango Caracara (Milvago chimango) were present all year. This pattern suggests that marshes with higher nutrient levels support the seasonal or annual requirements for different bird species, which would be using the salt marsh to nest, feed and/or rest.

The southern salt marshes from the SW Atlantic (Bahía Blanca, Bahía San Blas, San Antonio Oeste; [START_REF] Isacch | Distribution of saltmarsh plant communities associated with environmental factors along a latitudinal gradient on the south-west Atlantic coast[END_REF] are dominated in the lower tidal elevation by monospecific grasslands of S. alterniflora, which have a relative short height and cover [START_REF] Isacch | Generalization across space in the relationship between cord grass (Spartina alterniflora) biomass and reflectance data by using Landsat ETM+ images[END_REF], and are used occasionally by few bird species (Maldonado marsh in this study, Table 1; Isacch J.P., pers. obs.). Vegetation structure (e.g., height and cover) is an important factor for bird habitat selection, determining the bird abundance and richness in many different ecosystems around the world (MacArthur and MacArthur, 1961;[START_REF] Wiens | Pattern and process in grassland bird communities[END_REF][START_REF] Roth | Spatial heterogeneity and bird species diversity[END_REF]. A number of bird species move from adjacent habitats into the Bahía Blanca salt marsh when the structure changes in response to the increased nutrients. This is what happens with the Pale-breasted Spinetail (Synallaxis albescens) from shrublands, Bay-capped Wren-Spinetail, and Grass Wren (Cistothorus platensis) from the S. densiflora marsh, and Many-colored Rush-Tyrant (Tachuris rubrigastra), Wren-like Rushbird (Phleocryptes melanops) and Yellow-winged Blackbird from freshwater marshes. The Yellow-winged Blackbird was also recorded in low nutrient marshes, but the number of individuals was 18 times higher in the high nutrient marshes. Increased bird abundance and richness could not only be the result of vegetation structure, but also due to increased food availability.

Nutrient enrichment has several effects on the salt marsh food web. It may increase the nutritive value of plants (protein, lipids, soluble carbohydrates in S. alterniflora; [START_REF] Biudes | Changes in biomass, chemical composition and nutritive value of Spartina alterniflora due to organic pollution in the Itanhaém River Basin (SP, Brazil)[END_REF], the abundance and biomass of macroinfaunal species [START_REF] Sardá | Decadal shifts in a salt marsh macroinfaunal community in response to sustained long-term experimental nutrient enrichment[END_REF], and the insect abundance [START_REF] Bertness | Eutrophication and consumer control of New England salt marsh primary productivity[END_REF], thus causing indirect effects on the upper food web organism, such as birds and fishes [START_REF] Raffaelli | Nutrient enrichment and trophic organisation in an estuarine food web[END_REF]. Nutrient concentrations can affect birds differentially depending on the environmental characteristics or resources selected by each particular species or guild, such as benthic invertebrate-feeders (e.g., shorebirds, Correndera Pipit, Bar-winged Cinclodes), vegetation insect-feeders (e.g., Bay-capped Wren-Spinetail, Wren-like Rushbird), seed eaters (e.g., Yellow-winged Blackbird, Grassland Yellow-Finch, Great Pampa-Finch, Embernagra platensis) and generalist predators (Caracara Chimango). Our results show that salt marshes with nutrient enrichment had higher densities of S. alterniflora spikes than in salt marshes with relative low nutrient levels. The increase in the abundance of seed-eater species, especially the Yellow-winged Blackbird, may result from the increment in the spike density of S. alterniflora. Reductions in the abundance of benthic infauna were observed close to a sewage output following an increase in sewage output [START_REF] Hall | Effects of agriculture, urbanization, and climate on water quality in the northen Great Plains[END_REF]. Some benthic invertebrates, however, notably annelids, are abundant close to sources of nutrient pollution [START_REF] Green | Pond Dynamics /Aquaculture Collaborative Research Data Reports[END_REF], favouring ground feeder birds.

Increased arthropods in grass dominated habitats had been associated with increased complexity of plant architecture [START_REF] Gibson | The response of invertebrate assemblies to grazing[END_REF][START_REF] Dennis | The effects of varied grazing management on epigeal spiders, harvestmen and pseudoscorpions of Nardus stricta grassland in upland Scotland[END_REF], and the increase of vegetation height [START_REF] Morris | Responses of grassland invertebrates to management by cutting. V. Changes in Hemiptera following cessation of management[END_REF][START_REF] Dennis | The response of epigeal beetles (Col. Carabidae, Staphylinidae) to varied grazing regimes on upland Nardus stricta grasslands[END_REF] and heterogeneity [START_REF] Dennis | Distribution and abundance of small insects and arachnids in relation to structural heterogeneity of grazed, indigenous grasslands[END_REF][START_REF] Vickery | The management of lowland neutral grasslands in Britain: Effects of agricultural practices on birds and their food resources[END_REF]. Specifically, the number of herbivore insects increased when the nutritional quality of salt marsh plants improved as a result of higher nutrient loading increased [START_REF] Vince | An experimental study of the structure of herbivorous insect communities in a salt marsh[END_REF]. All of this evidence suggests that we might expect increased arthropod availability for birds with an increase of nutrient loadings to marshes. There is, therefore, an alternative possibility to explain why there are more birds in the high-nutrient marsh.

The dramatic increase of the raptor Chimango Caracara could be related to the increase of smaller birds and insects, two of the main prey of this species [START_REF] Biondi | Dieta del Chimango (Milvago chimango) durante el periodo reproductivo en el sudeste de la provincial de Buenos Aires, Argentina[END_REF]. We suspect that Chimango Caracara is taking advantage of the disturbance, since the capacity and plasticity of this species to use new habitats has been demonstrated [START_REF] Pedrana | Habitat relationships of diurnal raptors at local and landscape scales in southern temperate grasslands of Argentina[END_REF][START_REF] Bellocq | Influence of agricultural intensity and urbanization on the abundance of the raptor Chimango Caracara (Milvago chimango) in the Pampean region of Argentina[END_REF], including a remarkable ability to obtain food in novel situations [START_REF] Biondi | Experimental assessment of problem solving in Milvago chimango (Aves: Falconiformes)[END_REF].

The association between increased seed production and the increase of seed-eater birds was also recorded in other SW Atlantic salt marsh, but was explained as a result of the indirect effect of the burrowing crab (Neohelice granulata) on S. densiflora marsh [START_REF] Cardoni | Indirect effects of the intertidal burrowing Crab Chasmagnathus granulatus in the habitat use of Argentina's South West Atlantic salt marsh birds[END_REF]. This crab, because it builds caves, generates higher sediment water content and lower sediment hardness [START_REF] Bortolus | Crab-mediated phenotypic changes in Spartina densiflora Brong[END_REF][START_REF] Fanjul | Effects of the dominant SW Atlantic intertidal burrowing crab Chasmagnathus granulatus on sediment chemistry and nutrient distribution[END_REF], and increases the nitrification-denitrification rate when soil oxygenation increases [START_REF] Botto | Effect of burrowing crabs in isotope signatures of sediment, primary producers, and the food web in SW Atlantic salt marshes[END_REF][START_REF] Fanjul | Effects of the dominant SW Atlantic intertidal burrowing crab Chasmagnathus granulatus on sediment chemistry and nutrient distribution[END_REF]. The result is that plants allocate more energy to sexual reproduction when crabs are prevalent. Thus, different sources (i.e., natural and anthropogenic) of enhanced nutrient conditions (i.e., crabs caves and anthropogenic discharges) on different salt marsh plants may cause an increase of seed-eater bird species when compared with undisturbed salt marshes where insectivorous birds are the dominant trophic guild [START_REF] Isacch | Post-fire vegetation change and bird use of a salt marsh in coastal Argentina[END_REF][START_REF] Cardoni | Indirect effects of the intertidal burrowing Crab Chasmagnathus granulatus in the habitat use of Argentina's South West Atlantic salt marsh birds[END_REF].

Biomass generated by increased nutrient loading increases detrital production [START_REF] Gonzalez Trilla | Tiller dynamic and production on a SW Atlantic Spartina alterniflora marsh[END_REF] which also increases the generation of wracks. Marshes located close to the sewage showed higher number of wracks in the S. alterniflora marsh, generating higher landscape heterogeneity. Wracks, generates areas devoid of vegetation that are used by ground birds (e.g., Correndera Pipit, Bar-winged Cinclodes).

Similar to the pattern recorded for the plant physiognomy, wracks may facilitate the use of the salt marsh by species from upper open habitats. This is a small scale disturbance, which is known to be important in maintaining species diversity (e.g. [START_REF] Grime | Evidence for the existence of three primary strategies in plants and its relevance to ecological and evolutionary theory[END_REF][START_REF] Grubb | The maintenance of species-richness in plant communities: The importance of the regeneration niche[END_REF]. Indeed, one of the well known cases is the effect produced by a tree fall in the rainforest, generating gaps that increase light penetration and therefore also increases plant diversity [START_REF] Brawn | The role of disturbance in the ecology and conservation of birds[END_REF]). In our system, gaps produced by wracks generate habitat for open habitat bird species.

Salt marshes could be vulnerable if the nutrient delivery is discharged without management, given that it can ultimately degrade the structure of the marsh itself [START_REF] Levine | Nutrients, competition and plant zonation in a New England salt marsh[END_REF][START_REF] Bertness | Anthropogenic modification of New England salt marsh landscapes[END_REF][START_REF] Crain | Shifting nutrient limitation and eutrophication effects in marsh vegetation across estuarine salinity gradients[END_REF]. The sewage discharge into the Bahía Blanca estuary is being done without any treatment. In an advanced eutrophication process, the production of high amount of detritus could be generating a negative effect in marsh ecosystems [START_REF] Hall | Effects of agriculture, urbanization, and climate on water quality in the northen Great Plains[END_REF] by several processes such as decreasing the local or regional biodiversity, increased competitive advantage of invasive species, loss of nutrient retention capacity or, shifts between "clear water" macrophyte-dominated systems to turbid phytoplankton-dominated systems (see EPA, 2008 for a review). The sewage effluent may also create conditions favoring parasites on fishes and wading birds [START_REF] Coyner | Epizootiology of Eustrongylides ignotus in Florida: Distribution, density, and natural infections in intermediate hosts[END_REF][START_REF] Coyner | Influence of treated sewage on infections of Eustrongylides ignotus (Nematoda: dioctophymatoidea) in eastern mosquitotish (Gambusia holbrooki) in an urban watershed[END_REF]. However, the growth of the aboveground biomass may be limited by nitrogen, but the belowground biomass may not be. The belowground biomass accumulation is what keeps the marsh at sea level and its biomass may be limited by phosphorus (Darby and Turner, 2008a,b). If the belowground biomass is compromised, then the long-term sustainability of the marsh may be harmed [START_REF] Turner | Salt marshes and eutrophication: An unsustainable outcome[END_REF]. All the negative effects described above were not assessed in our study system, and we recognize that increases of some bird species, should be taken with caution and not as an overall beneficial effect of sewage discharges on the environment. Most bird species recorded in the eutrophicated marsh were common (Yellow-winged Blackbird) or extremely generalist species (Chimango caracara), without a specific conservation value. However, the movement of species into the eutrophicated marsh can turn in an ecological trap for ground or nearground nesting. Spartina alterniflora grows in the lower part of the intertidal, which in turn make nests vulnerable to frequent extreme floods by storms (Cardoni D.A and Isacch J.P. unpublished data).

In summary, our results show how local increments of nutrients generate direct and indirect changes in the salt marsh ecosystem. There are direct changes, as those caused on the vegetation physiognomy, and indirect changes in the composition and abundance of the bird assemblage.

Acknowledgments

This work was supported by grants and fellowships from ANPCYT, CONICET and Universidad Nacional de Mar del Plata to O. Iribarne. 1). The statistical data are in Table 1.

FIGURE CAPTIONS

Research highlights

Saltmarshes are increasingly affected by sewage discharge. Nutrient changes the vegetation physiognomy, increasing the habitat complexity. Bird composition and abundance increase in high nutrient saltmarshes. [START_REF] Sardá | Decadal shifts in a salt marsh macroinfaunal community in response to sustained long-term experimental nutrient enrichment[END_REF]Isacch and Martínez, 2001;Isacch et al., 2001[START_REF] Isacch | Post-fire vegetation change and bird use of a salt marsh in coastal Argentina[END_REF][START_REF] Cardoni | Indirect effects of the intertidal burrowing Crab Chasmagnathus granulatus in the habitat use of Argentina's South West Atlantic salt marsh birds[END_REF].

  Bird abundance and richness were both positively correlated with height of Spartina alterniflora (Abundance*S. alterniflora height: r 2 = 0.65, P < 0.05, Fig 5B; Richness*S.

Figure 1 :

 1 Figure 1: Top: Bahía Blanca (Argentina) during a low tide. White doted lines enclose

Figure 2 :

 2 Figure 2: The concentration of ammonium (A), phosphate (B) and nitrate (C) in

Figure 3 :

 3 Figure 3: Percentages of organic matter (OM) in sediments in three salt marshes at

Figure 4 :

 4 Figure 4: Height (A), cover (B) and spike density (C) of Spartina alterniflora in three

Figure 5 :

 5 Figure 5: Relationship between vegetation height and nitrate concentration (A), and

Figure 6 :

 6 Figure 6: Number of individuals (A), number of individuals without Yellow-winged

  of birds (total number of individuals) by species recorded in three areas with different nutrient levels in the Bahía Blanca saltmarsh, Argentina. Habitat association: TG: tall grassland; SG: short grassland; MF: mud flat. Bird-habitat association was based on studies of similar habitats in the region (Comparatore

  Results of one-way ANOVA for total bird richness and abundance, abundance without Yellow-winged Blackbird (Agelaius thilius) and abundance of species associated to open gaps (see Table1) for three areas with different nutrient levels in the Spartina alterniflora marsh from Bahía Blanca bay, Argentina. For all comparisons degrees of freedom is 2.

		Total richness		Total abundance	Abundance without	Species associated to
						Yellow-winged	open gaps
						Blackbird
		F	P	F	P	F	P	F	P
	July	7.3	0.007	32	0.00001	6.57	0.011	12.1	0.001
	August	8.3	0.003	17.2	0.00007	6.69	0.007	6.4	0.008
	September	5.5	0.01	5.7	0.01	5.10	0.018	1.7	0.2
	November	2.8	0.09	12.2	0.0005	3.15	0.067	2.8	0.09
	January	11.4	0.001	9.3	0.002	5.30	0.015	9.5	0.002
	February	10	0.001	17.6	0.00006	2.80	0.048	9.1	0.002
	March	5.6	0.01	8.6	0.002	1.50	0.25	10.2	0.1
	April	14.1	0.0001	12.4	0.0004	1.82	0.19	10.2	0.001
	June	7.5	0.004	11	0.0007	1.43	0.265	4.4	0.03

2


Luigi Musco
email: luigi.musco@unisalento.it

Barbara Mikac

Mariella Tataranni

Adriana Giangrande

Antonio Terlizzi

The use of coarser taxonomy in the detection of long-term changes in polychaete assemblages

Keywords: Taxonomic sufficiency, biomonitoring, Polychaetes, anoxia, temporal variability, taxonomy, Adriatic Sea

Taxonomic Sufficiency (TS) has been proposed as a short-cut method to quantify changes of biological assemblages in environmental monitoring. However, issues about the efficacy of taxonomic surrogates in depicting long-term temporal patterns of marine assemblages are still scant.

Here we report on the adoption of TS combined with data transformations to describe patterns of North Adriatic polychaete assemblages through 20 years. Univariate and multivariate analyses revealed large spatial-temporal variation charactering the assemblages. The efficiency to discriminate between the two research periods (1990-1994 vs. 2004-2008) was reduced when data were analyzed at family and order level leading to misinterpreting the sources of assemblage variation. Further information was lost with data transforms. Families may represent appropriate assemblages' descriptors in long-term monitoring, but using TS coupled with data transformations could lead to hazardous loss of information. We suggest that periodical analysis at fine taxonomic level should be routinely alternated to long-term monitoring based on TS in order to check its effectiveness.

INTRODUCTION

Assessing long-term changes in natural systems (i.e. recovery after mass mortality, effects of restoration or protection, climate change etc.) is one of the most intriguing challenges for quantitative ecology.

In marine environments, measuring patterns of variation in benthic assemblages can provide the most useful information for interpreting the ecological consequences of disturbances and furnish the basis for understanding environmental changes [START_REF] Underwood | Detection, interpretation, prediction and management of environmental disturbances: some roles for experimental marine ecology[END_REF][START_REF] Underwood | Towards an ecological framework for investigating pollution[END_REF]. In this context, the benthic macrofauna is particularly suitable for monitoring environmental long-term changes at the ecosystem level [START_REF] Kroncke | Long-term changes in North Sea benthos[END_REF].

Describing assemblages at the highest levels of taxonomic resolution (species) is logistically difficult due to high cost in processing samples and, above all, lack of taxonomic expertise. The development of fast and cost-effective procedures in impact assessment and monitoring studies has become a pressing issue for marine ecologists [START_REF] Bowen | Rapid Assessment of Marine Pollution (RAMP)[END_REF][START_REF] Ugland | Assessment of environmental stress with a biological index based on opportunistic species[END_REF]. In such context, Taxonomic Sufficiency (TS) [START_REF] Ellis | Taxonomic sufficiency in pollution assessment[END_REF], which implies "identifying organisms only to a level of taxonomic resolution sufficient to satisfy the objective of a study" [START_REF] Bertrand | Taxonomic surrogacy in biodiversity assessments, and the meaning of Linnaean ranks[END_REF], has been proposed as the ideal compromise procedure in environmental monitoring. TS is supported by evidences that impacts could be detected using coarser taxonomic resolution since abundance/diversity of species are usually related to abundance/diversity of the same organisms identified to supra-specific taxonomic levels (Genus, Family, Order etc.) (e.g. [START_REF] Balmford | Using higher taxon richness as a surrogate for species richness: I. Regional tests[END_REF]. It has also been suggested that higher taxa may reflect the effect of anthropogenic disturbance even better than species, being the latter more sensitive to the confounding influence of natural environmental variations [START_REF] Warwick | The level of taxonomic discrimination required to detect pollution effects on marine benthic communities[END_REF][START_REF] Vanderklift | Effect of reducing taxonomic resolution on ordinations to detect pollution-induced gradients in macrobenthic infaunal assemblages[END_REF]. In general, no significant losses of information, as well as time and costs savings, are the argumentations supporting the TS method in environmental impact assessment [START_REF] Clarke | Taxonomic sufficiency: an overview of its use in the monitoring of sublittoral benthic communities after oil spills[END_REF][START_REF] Dauvin | Benthic indicators: From subjectivity to objectivity -Where is the line?[END_REF][START_REF] Herman | On the use of meiofauna in ecological monitoring: who needs taxonomy?[END_REF][START_REF] Munari | The ecological status of Karavasta Lagoon (Albania): Closing the stable door before the horse has bolted?[END_REF].

Currently, lacking a general theory on TS, reasons at the base of the effectiveness of TS are still far to be clarified [START_REF] Dethier | Taxonomic sufficiency in distinguishing natural spatial patterns on an estuarine shoreline[END_REF]. Understanding at which extent functional redundancy overlap taxonomic relatedness of species, would help to provide a general framework on the application of TS in marine systems [START_REF] Bevilacqua | Taxonomic sufficiency in the detection of natural and human-induced changes in marine assemblages: habitats and taxonomic groups compared[END_REF]. The need to broaden the array of study cases on the application of TS has been repeatedly stressed (e.g., [START_REF] Bates | Weighing the costs and benefits of reduced sampling resolution in biomonitoring studies: perspectives from the temperate rocky intertidal[END_REF][START_REF] Olsgard | Surrogates in benthic investigations. Which taxonomic unit to target?[END_REF][START_REF] Quijón | The use of coarser taxonomic resolution in studies of predation on marine sedimentary fauna[END_REF][START_REF] Terlizzi | Taxonomic sufficiency and the increasing insufficiency of taxonomic expertise[END_REF], because possible biases of TS are to be further investigated. The significance of the Linnaean ranks is being criticized so far as the treatment of same taxonomic ranks as equivalent units is suspected to allow to spurious comparisons (see [START_REF] Bertrand | Taxonomic surrogacy in biodiversity assessments, and the meaning of Linnaean ranks[END_REF]. The extent to which TS affects the results depends on the distribution of species amongst higher taxa in the original samples, that, in turn depends on habitat type and bio-geographic background [START_REF] Musco | Taxonomic structure and the effectiveness of surrogates in environmental monitoring: a lesson from polychaetes[END_REF][START_REF] Terlizzi | Beta diversity and taxonomic sufficiency: Do higher-level taxa reflect heterogeneity in species composition[END_REF]. Further, the relation among TS, and data transformation is far to be fully clarified. Several studies showed that transforming data might influence the results of analyses as well as taxonomic aggregation, suggesting that the use of TS coupled with strong transformations of data could lead to severe loss of information [START_REF] Lasiak | Influence of taxonomic resolution, biological attributes and data transformations on multivariate comparisons of rocky macrofaunal assemblages[END_REF][START_REF] Olsgard | Relationship between taxonomic resolution, macrobenthic community patterns and disturbance[END_REF]Wlodarska-Kuwalczuk and Kedra, 2007).

As far as long-term monitoring studies are concerned, the use of coarser taxonomic level, being time-efficient, could improve monitoring programs allowing the extension to additional sampling dates [START_REF] Thompson | Cost-efficient methods for marine pollution monitoring at Casey Station, east Antarctica: the choice of sieve meshsize and taxonomic resolution[END_REF][START_REF] Tataranni | Variance estimate and taxonomic resolution: An analysis of macrobenthic spatial patterns at different scales in a Western Mediterranean coastal lagoon[END_REF]. Some loss of information on community structure is considered acceptable for extensive monitoring purposes, and the detection of biodiversity changes should be admissible as well [START_REF] Bertasi | Comparing efficacy of different taxonomic resolutions and surrogates in detecting changes in soft bottom assemblages due to coastal defence structures[END_REF]. However, studying the long-term recovery pattern of polychaete assemblages of Botany Bay (NSW, Australia), [START_REF] Fraser | Long-term changes in polychaete assemblages of Botany Bay (NSW, Australia) following a dredging event[END_REF] found that the importance of differences in assemblage structure, as well as patterns of succession, could not have been examined in detail using TS. The same Authors suggest that analysis of data at various taxonomic levels, after identification of organisms to species, should be encouraged in long-term impact-assessment studies. This issue, however, is made difficult by the lack of long-term monitoring programmes based on the description of variables at species level. The present study is an attempt in this direction.

Polychaetes are widely distributed, abundant and diverse in terms of species richness and ecological requirements and have been successfully used as indicator taxon [START_REF] Giangrande | Polychaete assemblages of rocky shore along the South Adriatic coast (Mediterranean Sea)[END_REF][START_REF] Giangrande | Polychaetes as environmental indicators revisited[END_REF][START_REF] Musco | Mediterranean Syllidae (Annelida: Polychaeta) revisited: species diversity, biogeography and faunal fidelity to environmental features[END_REF][START_REF] Fraschetti | Structural and functional response of meiofauna rocky assemblages exposed to sewage pollution[END_REF][START_REF] Musco | Taxonomic structure and the effectiveness of surrogates in environmental monitoring: a lesson from polychaetes[END_REF] also in long-term monitoring studies [START_REF] Fraser | Long-term changes in polychaete assemblages of Botany Bay (NSW, Australia) following a dredging event[END_REF][START_REF] Schirosi | Benthic assemblages of Acquatina Lake (South Adriatic Sea): present state and long-term faunistic changes[END_REF]. Here, we compared the effects of taxonomic aggregation and data transformation on the analysis of multivariate patterns of variations related to the decadal changes of the polychaete assemblages, following mass mortality caused by the oxygen depletion in the Northern Adriatic Sea in 1989.

Materials and Methods

Research area

The Northern Adriatic Sea (hereafter NA) is a semi-enclosed shallow basin exposed to severe human impacts such as strong urban development, high nutrients load, commercial fisheries and different kinds of pollution [START_REF] Degobbis | Long-term changes in the northern Adriatic ecosystem related to anthropogenic eutrophication[END_REF]. Sometimes, when mucilage events coincide with specific hydrographical conditions, anoxia may affect the sea bottom. Such an event happened in the autumn 1989 and caused benthic mass mortality over an area of about 1.200 km 2 .

The study was carried out on three offshore monitoring stations of the Centre for Marine Research (Ruñer Bošković Institute, Rovinj) located within the affected area. Station SJ 005 (hereafter 005) (45°18.4' N; 13°18.0' E; 31 m depth) and station SJ 007 (007) (45°17.0' N; 13°16.0'E; 31 m depth) are situated on the profile Poreč (Croatia) -Venice-Lido (Italy), 17 and 13 NM off from the coast of Istrian peninsula (Croatia), respectively. Station SJ 107 (hereafter 107) (45°02.8 N; 13°19.0' E; 37 m depth) is situated on the profile Rovinj (Croatia) -river Po delta, 13 NM off the Istrian coast (Fig. 1). Granulometric analyses showed high similarity in sedimental composition among the three stations. Sediment is weakly sorted silty sand composed mainly by sand (ranging 66.3 -70.6 %), followed by silt and clay fraction (27.8 -32.1 %) and gravel (1.4 -1.6 %).

Field and laboratory work

Starting one year after the anoxia event, samples were taken once a year, in two phases during 1990-1994 and 2004-2008, in the period from late October to the beginning of January .

At each station 4-5 replicates were collected with 0.1 m 2 Van-Veen grab, sieved through 2 mm mesh in the first research period (1990)(1991)(1992)(1993)(1994) and 1 and 2 mm mesh in the second one (2004)(2005)(2006)(2007)(2008), and fixed in 4% buffered formaldehyde-seawater solution. As a whole, 113 samples were collected.

The 1 mm mesh size in the second period was adopted following the up-to-date recommendation for the macrobenthic fauna monitoring studies provided by [START_REF] Castelli | Manuale di metodologia di campionamento e studio del benthos marino mediterraneo[END_REF]. However, for the present study, in order to allow long-term comparisons, only 2 mm mesh size samples were used. In the laboratory the material was rinsed and macrobenthic organisms sorted and conserved in 70% ethanol. Polychaetes were determined to the species level using the stereomicroscope and light microscope. Nomenclature followed [START_REF] Castelli | Annelida Polychaeta. In: Relini G, editor. Checklist della flora e della fauna dei mari italiani (Parte I)[END_REF].

Data Analysis

Three separate analyses of variances (ANOVAs) were performed to test the null hypotheses of no differences among polychaete assemblages in number of individuals, species richness and Simpson diversity index. The experimental design included three factors: Period (Pe, a fixed factor with 2 levels), Time (T, random, with 9 levels and nested in Pe) and Station (S, random, with 3 levels and crossed to both Pe and T).

The same sampling design was used, in the multivariate context, to perform a distance-based permutational multivariate analysis of variance (PERMANOVA) [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF], which used 999 random permutations of appropriate units to test for differences in the polychaetes assemblages' structure among periods across the three stations. The analysis was based on untransformed data at species level. In order to quantify the variability associated with each examined scale, mean squares calculated by PERMANOVA were equated to the expected mean squares, like in the univariate case [START_REF] Benedetti Cecchi | Beyond BACI: optimization of environmental sampling designs through monitoring and simulation[END_REF][START_REF] Searle | Variance Components[END_REF]. For graphical comparisons, the variability at each scale was expressed as a proportion of the total variation [START_REF] Terlizzi | Scales of spatial variation in Mediterranean subtidal sessile assemblages at different depths[END_REF]. The same analyses were then repeated for the dataset lumped at genus, family and order taxonomic level.

PERMANOVAs were also carried out for calculating estimates of variance components using 2 nd root, 4 th root and presence/absence transformed data for all the taxonomic levels investigated.

Non-metric multidimensional scaling ordination plots (MDS) were produced to visualize multivariate patterns of polychaete assemblages classified at species, genus, family and order taxonomic rank. All plots were based on Bray Curtis similarity matrix of untransformed data.

A similarity percentage (SIMPER) routine was performed to detect the species most responsible for differences in the multivariate structure of assemblages between periods.

A second-stage nMDS ordination was also plotted to visualize differences among similarity matrices at different levels of taxonomic aggregation and data transformation [START_REF] Somerfield | Taxonomic levels, in marine community studies, revisited[END_REF].

All multivariate analyses were performed using the computer program PRIMER v6 [START_REF] Clarke | Taxonomic sufficiency: an overview of its use in the monitoring of sublittoral benthic communities after oil spills[END_REF], including the add-on package PERMANOVA+ [START_REF] Anderson | PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods[END_REF].

Results

Faunal composition

A total of 5680 polychaete specimens belonging to 173 species, 115 genera, 35 families and 14 orders were collected: among them, 2941 individuals (belonging to 94 species, 67 genera, 29 families and 12 orders) were collected during the first period (1990)(1991)(1992)(1993)(1994), and 2739 (130 species, 95 genera, 35 families and 14 orders) during the second one (2004)(2005)(2006)(2007)(2008).

As far as the species abundances are concerned, during the first period Owenia fusiformis ranked first (21.2 % of the individuals) followed by Pectinaria auricoma (15.4 %), Eunice vittata (13.1 %) and Nothria conchilega (10 %). No other species exceeded the 5 % of the total abundance. During the second period O. fusiformis remained the most abundant species (10.7 %), but 3 species showed similar abundances, namely Ampharete acutifrons (10.1 %) (not recorded during 1990-1994), Notomastus latericeus (9.9 %) and P. auricoma (9.7 %). Among the other species only Galathowenia oculata (6.6 %) exceeded the 5 % of the total abundance. As a whole, 39 singletons (i.e. species represented by a single individual) were recorded, among them 12 during the first period (1 at station 005, 4 at 007, 7 at 107) and 17 during the second one (6 at station 005, 9 at 007, 12 at 107). Considering the genera, 24 were singletons, among them 9 during the first period (3 at 007, 6 at 107) and 15 during the second one (1 at station 005, 7 at 007, 7 at 107). No singleton families and orders were observed.

The distribution of individuals was variable during the first period (1990)(1991)(1992)(1993)(1994) both considering the three stations and the four sampling times (Fig. 2); a lesser degree of variation was visible during the second period (2004)(2005)(2006)(2007)(2008). The species richness (Fig. 2) was generally the lowest at the station 5 during the first period and particularly variable during time 2 (period 1) at stations 7 and 107; during the second period, the species richness showed a temporal trend variable within each station but quite similar among stations. The Simpson diversity index (Fig. 2) showed similar trends at all stations; this index appeared more variable, tending to increase from time 1 to time 4 during the first period, while it appeared more homogeneous among times and stations during the second one.

The ANOVA test (Table 1) confirmed the high degree of spatial and temporal variation of the polychaete abundance at the 3 stations during the two analyzed periods; particularly, the significance of the T(Pe) × St interaction term in the analysis of the number of individuals, revealed that the polychaete abundance varied among times depending on the considered station without a clear pattern between the two considered periods. The test for the species richness indicated significant differences among times and stations, but no difference between periods. The analyses of Simpson diversity revealed significant differences between the polychaete assemblages sampled during 1990-1994 and those sampled during 2004-2008; the test also indicated significant differences among times and stations.

Multivariate Analyses

The results of the MDS analyses carried out on the untransformed abundance species matrix and on matrices derived from the aggregation of species distributions to genus, family and order levels are shown in Figure 3. The plot relative to the polychaete assemblages analyzed at species level (Fig. 3a) showed that centroids representing the 5 times for each of the 3 station sampled during 2004-2008 (full triangles on the right) formed a group apart from those representing the 4 times for the same stations for the period 1990-1994, thus confirming differences between periods already observed for the univariate indices (Table 1). Almost identical positions of the centroids in the plot were observed using the genera as surrogates of the species (Fig. 3b). The multivariate pattern remained clearly visible when the polychaete assemblage was analyzed at family (Fig. 3c) and order level (Fig. 3d), although the separation between periods became less evident.

The species contributing to the 70% dissimilarity between the two periods (Average dissimilarity 79.82), and their average abundance in both periods are shown in Table 2 (SIMPER test). The species mostly contributing to differentiate the two periods were also the species dominating the assemblages (see 3.1). Interestingly, among the co-familial species only those belonging to Flabelligeridae (2 species) and Pectinariidae (2) showed similar trend of average abundance between the two periods, while species belonging to Onuphidae (3), Oweniidae (2), Spionidae (2) and Ampharetidae (2) showed opposite temporal trends.

Table 3 shows the outcomes of the PERMANOVA tests carried out on the original, untransformed species matrix and on matrices derived from the aggregation of species distributions to genus, family and order level. A large degree of variation characterized the polychaete assemblages at all the considered taxonomic levels. The four tests indicate the significance of the T(Pe) × St interaction term revealing that, whatever the analyzed taxonomic level, the polychaete assemblages varied among times depending on the considered station without a clear pattern, even between periods.

Considering the estimate of the variance components (Table 3) however, some difference characterized the analyzed datasets: apart from the residuals, the major source of variation belonged to the factor period for the assemblages analyzed at species and genus level, while for the same assemblages analyzed at family and order level the major source of variation belonged to the T(Pe) × St interaction term. Also, the total variation appeared to decrease with the increasing taxonomic level; this pattern was particularly evident when the assemblages were analyzed at family and order level.

Figure 4 compares the relative estimates of the variance components of the untransformed datasets (species, genus, family and order) (Fig. 4a), to those obtained after square-root (Fig. 4b), fourth-root (Fig. 4c) and presence/absence (Fig. 4d) data transform. In every case, the largest variation was among replicate units (i.e. Residual term), while, apart from the analysis of the untransformed datasets at family and order level, the second largest source of variation was always associated to the factor period whatever the taxonomic level and the data transformation used.

The ordination of similarity matrices in the second-stage nMDS plot (Fig. 5) showed a typical 'fan' pattern, with a vertical and horizontal spread of points at increasing data transformation and taxonomic aggregation respectively, indicating independent effects of transformations and taxonomic resolution. The effect of taxonomic resolution was particularly evident moving from genus to family level, while the effect of data transformation was already noticeable using the weakest 2 nd root transformation because the similarity matrix based on untransformed data fall apart from those obtained by data transformation independently from the taxonomic level considered. In general, within the matrices relative to the same taxonomic rank, those derived from each transformation tend to cluster apart from the similarity matrix obtained by untransformed data.

Discussion

As well summarized by the univariate indices, a large and significant degree of variation characterizes the analyzed polychaete assemblages both spatially, at the three stations, and temporally, considering the two analyzed time scales.

Differences between the two periods were visible by the nMDS plot relative to the species showing separation among the centroids representing the assemblages sampled during the 1990-1994 post anoxic period and those sampled two decades later (2004)(2005)(2006)(2007)(2008). This seems to confirm temporal changes in the polychaete assemblages possibly due to recovery from anoxia. Also in another area of the NA, [START_REF] Stachowitsch | Long-term changes in the benthos of the Northern Adriatic Sea[END_REF] observed that the recovery of the macrobenthic community after the mass mortality is not reached even ten years after the anoxic event, suggesting that dramatic events might influence assemblages for decades.

PERMANOVA tests confirmed the high spatial-temporal variation as a proper characteristic of the analyzed polychaete assemblages. The main results of differences in assemblages between periods were consistent whatever the considered taxonomic level. However, the describing efficiency of surrogates showed differences concerning the capability to properly describe the assemblages' intrinsic variability. This should not be underrated due to the importance of variability in describing the analyzed assemblages.

Particularly, when untransformed data were considered, the use of families and orders as species surrogates led to misinterpreting the sources of assemblage variation and to noticeable decreasing the estimate of the total variation thus allowing considering the two period and the three stations more similar to each other that they really are. In general, it might be expected that using TS (i.e. a lesser number of variables) leads to reduce the explicated variability, but this ''averaging effect" [START_REF] De Biasi | Analysis of macrobenthic communities at different taxonomic levels: an example from an estuarine environment in the Ligurian Sea NW Mediterranean[END_REF][START_REF] Doak | The statistical inevitability of stability-diversity relationships in community ecology[END_REF] is not necessarily the rule (see [START_REF] Tataranni | Variance estimate and taxonomic resolution: An analysis of macrobenthic spatial patterns at different scales in a Western Mediterranean coastal lagoon[END_REF]. The question is to determine the extent to which this reduction is acceptable, that is, the amount of variability that can be lost without loosing the capability to highlight differences among assemblages. If the pattern of community change is marked, interpretable results are possible independent of both the examined component of the benthos and the taxonomic level at which the analyses are carried out [START_REF] Somerfield | Taxonomic levels, in marine community studies, revisited[END_REF]. Anyway, evidences suggest that using TS might cause a significant loss of information when differences among assemblages are not robust enough [START_REF] Musco | Taxonomic structure and the effectiveness of surrogates in environmental monitoring: a lesson from polychaetes[END_REF]. The decrease of explicated variability might also result from not concordant distribution patterns of taxonomically related species that show differential response to the stressor [START_REF] Musco | Taxonomic structure and the effectiveness of surrogates in environmental monitoring: a lesson from polychaetes[END_REF]. Several co-familial species showed opposite abundance patterns between the two periods (see 3.2, results of SIMPER). The loss of information observed in the MDS plots based on family and order taxonomic ranks should be carefully considered. The presence of dominant species belonging to the same genus (or family in this case) showing different responses to a perturbation is considered one of the most important factor limiting the efficiency of the TS method [START_REF] Clarke | Taxonomic sufficiency: an overview of its use in the monitoring of sublittoral benthic communities after oil spills[END_REF].

Interestingly, weak data transformation was sufficient to allow perfect correspondence in the estimate of variance components among species and taxonomic surrogates. This clearly indicates that abundance is a key factor characterizing the polychaete assemblages. The importance of abundance was further confirmed by the 2 nd stage MDS plot, which however indicated that the similarity among matrices rapidly declined after data transformations, independently from the analyzed taxonomic level. This also recommends care in the choice of data transformations.

This study indicates that untransformed data at family level (the most common taxonomic surrogate in environmental monitoring) may represent appropriate assemblages' descriptors in long-term monitoring studies [START_REF] Bevilacqua | Low sensitiveness of taxonomic distinctness indices to human impacts: Evidences across marine benthic organisms and habitat types[END_REF][START_REF] Lasiak | Influence of taxonomic resolution, biological attributes and data transformations on multivariate comparisons of rocky macrofaunal assemblages[END_REF][START_REF] Olsgard | Relationship between taxonomic resolution, macrobenthic community patterns and disturbance[END_REF]Wlodarska-Kuwalczuk and Kedra, 2007). Our results are in accordance with [START_REF] Bevilacqua | Taxonomic sufficiency in the detection of natural and human-induced changes in marine assemblages: habitats and taxonomic groups compared[END_REF] suggesting that, in the absence of previous acknowledgements, untransformed data should be analyzed, since it is often difficult to recognize a priori how environmental variations will affect assemblages' structure (e.g., influencing rare or abundant species).

Our data also suggest that, before applying TS and perform approximations of the faunal features of an area, the species-level information should be achieved [START_REF] Fraser | Long-term changes in polychaete assemblages of Botany Bay (NSW, Australia) following a dredging event[END_REF][START_REF] Gage | Deep-sea benthic community and environmental impact assessment at the Atlantic Frontier[END_REF]Musco et al., 2009;[START_REF] Olsgard | Surrogates in benthic investigations. Which taxonomic unit to target?[END_REF][START_REF] Terlizzi | Taxonomic sufficiency and the increasing insufficiency of taxonomic expertise[END_REF]. Moreover, as argued by [START_REF] Tataranni | Variance estimate and taxonomic resolution: An analysis of macrobenthic spatial patterns at different scales in a Western Mediterranean coastal lagoon[END_REF], our data support that periodical analysis at fine taxonomic level should be alternated to long-term routine monitoring based on TS, since shifts in dominance between taxonomically related species may occur gradually, altering competitive interactions. This would be particularly alarming if changes in species dominance result from events that are detected only by analyses at the finest taxonomic level, such as alien invasions (see [START_REF] Zenetos | Additions to the annotated list of marine alien biota in the Mediterranean with special emphasis on Foraminifera and Parasites[END_REF] or meridionalization of the NA fauna [START_REF] Bianchi | Biodiversity issues for the forthcoming tropical Mediterranean Sea[END_REF][START_REF] Boero | Climate warming and related changes in Mediterranean marine biota[END_REF][START_REF] Mikac | Faunal and biogeographic analysis of Syllidae (Polychaeta) from Rovinj (Croatia, northern Adriatic Sea)[END_REF][START_REF] Musco | Mediterranean Syllidae (Annelida: Polychaeta) revisited: species diversity, biogeography and faunal fidelity to environmental features[END_REF].

Changes in community composition over the long trend make up the history of ecosystems and are inherently "unpredictable" [START_REF] Boero | Episodic events: Their relevance to ecology and evolution[END_REF][START_REF] Boero | Recent innovations in marine biology[END_REF], see also [START_REF] Doak | Understanding and predicting ecological dynamics: Are major surprises inevitable?[END_REF]. Programming environmental long-term monitoring exclusively based on TS implicitly "predicts" families (higher taxa) as locally fixed unchangeable entities, but this prevents from appreciating important changes within the families, or even genera. For instance, among the worst invasive algae of the Mediterranean, Caulerpa racemosa and C. taxifolia, rank among the 100 worst aliens [START_REF] Streftaris | Alien Marine Species in the Mediterranean -the 100 'Worst Invasives' and their Impact[END_REF], but their presence would pass unnoticed if the genus Caulerpa (represented in the Mediterranean by the common C. prolifera) would not be investigated at the species level. The case of the genus Caulerpa shows that systems evolve (i.e. change) in time and TS is clearly a not good tool to detect this change. The variability of each term was expressed as a proportion of the total variation

Figure 1 .

 1 Figure 1. Study area and position of the three investigated stations

Figure 5 .

 5 Figure 5. 'second stage' MDS ordination of resemblance matrices derived from species (S), genus (G), family (F) and order (O) untransformed abundance data and several transformations (2rt: square root; 4rt: fourth root; PA: presence/absence)

Table 1 .

 1 Results of ANOVA analyses for number of individuals, species richness and Simpson diversity testing for differences in polychaete assemblages. P = period; St = station; T = time. Significant P-values are given in italics.

			Number of individuals	Species richness		Simpson diversity
	Source	df	MS	F	P	MS	F	P	MS	F	P
	Pe	1	12687 2.66	0.111	228.78 1.83	0.214	0.1751 5.27	0.030
	St	2	1112.3 1.06	0.395	163.05 11.07 0.002	0.0219 7.52	0.005
	T(Pe)	7	2929.9 2.74	0.046	86.01 5.86	0.003	0.0281 9.70	0.001
	Pe × St	2	2299.7 2.19	0.136	49.031 3.33	0.067	0.0063 2.16	0.166
	T(Pe) × St	14	1069.2 3.49	0.001	14.665 0.85	0.630	0.0029 0.95	0.530
	Res	86	306.47			17.344			0.0031

Table 2 .

 2 Results of SIMPER analysis. Average abundance (Av. Abund.) and percentage contribution (Contrib. %) to the average dissimilarity between periods for species reaching the cumulative contribution of 70% are reported. Species are divided in relation to the family and order taxonomical levels, which are listed alphabetically

	Period 1	Period 2

Table 3 .

 3 PERMANOVA analyses based on the Bray-Curtis dissimilarity for untransformed abundance data at species (173 variables), genera (115), family (35) and order (14) taxonomic level. The analyses were carried out using 999 permutations of residuals under a reduced model. Estimates of variance components (VC) for each analysed source of variation were also included. P = period; St = station; T = time

			Species				Genera
	Source	df	MS	F	P(perm) VC	MS	F	P(perm) VC
	Pe	1	56099	3.81 0.006	798.78	54286	3.98 0.007	783.09
	St	2	11372	3.98 0.001	234.55	11143	4.21 0.001	234.02
	T(Pe)	7	6965.8 2.41 0.001	325.09	6644.1 2.48 0.001	316.35
	Pe × St	2	8643.2 3.02 0.001	318.75	7791.2 2.94 0.001	283.36
	T(Pe) × St	14	2893.4 2.07 0.001	358.36	2681.2 2.08 0.001	333.44
	Res	86	1395.5			1395.	1287.4		1287.4
	Tot	112				3431.03			3237.66
			Families				Orders
	Source	df	MS	F	P(perm) VC	MS	F	P(perm) VC
	Pe	1	25183	2.23 0.031	278.61	15721	2.06 0.039	164.61
	St	2	9606.8 4.13 0.001	200.61	6880.3 4.12 0.001	143.51
	T(Pe)	7	6019.1 2.55 0.001	292.32	3490	2.06 0.002	143.09
	P × St	2	6440.4 2.77 0.001	226.74	5009.8 2.99 0.002	183.95
	T(Pe) × St	14	2357.2 2.37 0.001	325.69	1697.8 3.02 0.001	271.64
	Res	86	995.83			995.83	562.38		562.38
	Tot	112				2319.08			1469.18

Acknowledgements

Thanks to Nando Boero for invaluable comments. The results of this investigation as well as the work of BM were carried out within the projects "Mechanisms of long-term changes in the Adriatic Sea ecosystem" (project no. 0098111) and "Biodiversity of benthic communities in the Adriatic: natural and human impacts" (project no. 098-0982705-2732) both funded by the Ministry of Science, Education and Sports of the Republic of Croatia. The work of LM, AT and AG has been carried out within the MARBEF Network of Excellence which is funded in the Community's Sixth Framework Programme (Contract no. GOCE-CT-2003-505446), the E.U. Integrated Project SESAME and the CMCC (Centro Euro-Mediterraneo per i Cambiamenti Climatici).

Research highlights

• Polychaetes families detect decadal changes in the multivariate assemblages structure

• TS and data transformation may underrate patterns of spatial assemblages variability

• TS-based long-term monitoring must include periodical fine taxonomic analyses