M Tataranni
email: mtataranni@biologia.unipi.it

F Maltagliati

A Floris

A Castelli

C Lardicci

Variance estimate and taxonomic resolution: an analysis of macrobenthic spatial patterns at different scales in a Western Mediterranean coastal lagoon

Keywords:

The effects of taxonomic resolution on the variance estimates of macrobenthic assemblages were studied at four spatial scales in a Mediterranean coastal lagoon. The assemblages exhibited significant differences at all the investigated scales; however, spatial variability was mainly associated with the smallest and the largest scales. The decrease of taxonomic resolution (from species to family) was not related to a decrease of the overall variability and similar estimates of variance components were obtained using species and family resolution levels. The ordination models derived from species and family abundances were very similar both in terms of location and dispersion effect, while further aggregation to the class level began to alter the observed spatial patterns. In future studies aimed at assessing changes in the lagoon, resources derived from the cost reductions achieved using family level could be employed to plan more frequent surveys and/or to adopt complex spatial sampling designs with a high number of replicates.

Introduction

Macrobenthic invertebrates are an essential component in soft-sediment environments playing important roles in ecosystem processes, such as dispersion, burial, nutrient cycling and energy flow [START_REF] Snelgrove | The biodiversity of macrofaunal organisms in marine sediments[END_REF]. A deep knowledge of spatial variability patterns in macrobenthic assemblages is relevant to properly characterise one of the major sources of biotic diversity in natural environments; moreover, such information represents a requirement to develop strategies of management and conservation (e.g. Lubchenco et al., 1991), as well as to advise suitable guidelines for periodical monitoring programs (e.g. [START_REF] Underwood | Experiments in ecology: their logical design and interpretation using analysis of variance[END_REF]. Soft bottom environments are usually considered homogeneous habitats; however structural analyses in marine and brackish systems have repeatedly demonstrated that patchiness of macrofaunal assemblages is a common feature at both small-medium scales, [START_REF] Thrush | Patterns in the spatial arrangements of polychaetes and bivalves in intertidal sandflats[END_REF][START_REF] Hewitt | Integrating heterogeneity across spatial scales: interactions between Atrina zelandica and benthic macrofauna[END_REF][START_REF] Noren | Spatial, temporal and interactive variability of infauna in Swedish coastal sediments[END_REF] and large scales [START_REF] Morrisey | Spatial variation in softsediment benthos[END_REF][START_REF] Edgar | Benthic macrofauna in Tasmanian estuaries: scales of distribution and relationships with environmental variables[END_REF][START_REF] Ysebaert | Spatial and temporal variation in benthic macrofauna and relationship with environmental variables in an estuarine, intertidal soft-sediment environment[END_REF]. In particular, those previous studies emphasized the lacking of a single correct scale at which assemblages can be described [START_REF] Levin | The problem of pattern and scale in ecology[END_REF], since different patterns of distribution can be obtained depending on the spatial scale of observation. The description of the distributional patterns at multiple spatial scales and identification of the most relevant ones are needed to formulate possible explanations about ecological processes, or unnatural impacts structuring ecosystems [START_REF] Underwood | Scales of spatial patterns of distribution of intertidal invertebrates[END_REF][START_REF] Underwood | Observations in ecology: you can't make progress on processes without understanding the patterns[END_REF][START_REF] Ysebaert | Large-scale spatial patterns in estuaries: estuarine macrobenthic communities in the Schelde estuary, NW Europe[END_REF]. Furthermore, such information can be useful for avoiding erroneous interpretations of spatial patterns observed at a particular scale and also to advise useful guidelines for routine environmental monitoring programs.

In order to estimate the proportion of variability associated with each examined scale and to identify the most relevant spatial scale, the hierarchical sampling approach is considered the most appropriate method [START_REF] Underwood | Experiments in ecology: their logical design and interpretation using analysis of variance[END_REF][START_REF] Hewitt | The effect of changing sampling scales on our ability to detect effects of large-scale processes on communities[END_REF]. In hierarchical designs small-scaled sampling units are nested within larger-scaled ones, allowing unconfounded statistical comparisons among each spatial scale [START_REF] Underwood | Techniques of analysis of variance in experimental marine biology and ecology[END_REF][START_REF] Kotliar | Multiple Scales of Patchiness and Patch Structure: A Hierarchical Framework for the Study of Heterogeneity[END_REF]. Nested designs have been successfully used to investigate populations and assemblages across a wide range of marine habitats and organisms. Most studies focused on intertidal and subtidal rocky shores (i.a. [START_REF] Archambault | Scales of coastal heterogeneity and benthic intertidal species richness, diversity and abundance[END_REF][START_REF] Underwood | Scales of spatial patterns of distribution of intertidal invertebrates[END_REF]Benedetti-Cecchi, 2001a;[START_REF] Fraschetti | Spatial variability and human disturbance in shallow subtidal hard substrate assemblages: a regional approach[END_REF]2005;[START_REF] Chapman | Scales of variation of gastropod densities over multiple spatial scales: comparisons of common and rare species[END_REF], while soft-bottoms have been less explored [START_REF] Morrisey | Spatial variation in softsediment benthos[END_REF][START_REF] Stark | Human impacts in soft-sediments assemblages at Casey Station, East Antarctica: spatial variation, taxonomic resolution and data transformation[END_REF][START_REF] Noren | Spatial, temporal and interactive variability of infauna in Swedish coastal sediments[END_REF]Terlizzi et al., 2008b).

The increase of soft bottom macrobenthic studies is often hidden because identifying and enumerating all organisms are time-consuming and labour-intensive processes [START_REF] Warwick | Environmental impact studies on marine communities: pragmatical considerations[END_REF][START_REF] Olsgard | Relationship between taxonomic resolution, macrobenthic community patterns and disturbance[END_REF], that requires taxonomic expertise [START_REF] Terlizzi | Taxonomic sufficiency and the increasing insufficiency of taxonomic expertise[END_REF]. In the last two decades, many studies have analysed data at several taxonomic resolutions, showing that results obtained using species or family level are very similar (i.a. [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][START_REF] Olsgard | Relationship between taxonomic resolution, macrobenthic community patterns and disturbance[END_REF][START_REF] Karakassis | Benthic disturbance due to fish farming analyzed under different levels of taxonomic resolution[END_REF][START_REF] Lampadariou | Cost/benefit analysis of a benthic monitoring programme of organic enrichment using different sampling and analysis methods[END_REF][START_REF] Wlodarska Kowalczuk | Surrogacy in natural patterns of benthic distribution and diversity: selected taxa versus lower taxonomic resolution[END_REF]. Identifying a taxonomic level higher than species that is sufficient for detecting differences in assemblage composition without losing important information is a concept termed "taxonomic sufficiency" (TS, [START_REF] Ellis | Taxonomic sufficiency in pollution assessment[END_REF].

The TS method might have some practical implications, in particular routine monitoring programs could become less expensive and faster than those conducted at the species level resolution and therefore macrobenthic assemblages could be analysed more frequently. However, at present, most studies have usually compared different levels of TS at a single spatial scale [START_REF] Vanderklift | Effect of reducing taxonomic resolution on ordinations to detect pollution -induced gradients in macrobenthic infaunal assemblages[END_REF][START_REF] Olsgard | Relationship between taxonomic resolution, macrobenthic community patterns and disturbance[END_REF][START_REF] Karakassis | Benthic disturbance due to fish farming analyzed under different levels of taxonomic resolution[END_REF][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] Wlodarska Kowalczuk | Surrogacy in natural patterns of benthic distribution and diversity: selected taxa versus lower taxonomic resolution[END_REF], while few researchers have investigated the effects of TS on the spatial distribution patterns observed at multiple scales [START_REF] Chapman | Relationships between spatial patterns of benthic assemblages in a mangrove forest using different levels of taxonomic resolution[END_REF]Anderson et al., 2005;[START_REF] Dethier | Taxonomic sufficiency in distinguishing natural spatial patterns on an estuarine shoreline[END_REF]. Moreover in these previous works, spatial variability was usually not distinguished in relation to differences in location or dispersion among groups of samples, while the ecological heterogeneity is considered a valuable feature of any habitat which can provide important information on biological assemblages [START_REF] Anderson | Distance-based tests for homogeneity of multivariate dispersion[END_REF]Terlizzi et al., 2008a).

In the present study, abundance and composition of soft bottom benthic macrofaunal assemblages in a Western Mediterranean coastal lagoon were described with particular attention to their variability across different spatial scales. The Santa Giusta lagoon can be considered as representative of small microtidal brackish environments characterizing the Mediterranean region [START_REF] Basset | Typology in Mediterranean transitional waters: new challenges and perspectives[END_REF]. Coastal lagoons are areas of considerable naturalistic interest but often are located close to urban or industrial centres, therefore they are possibly affected by direct (e.g. sewage discharge, aquaculture) or indirect (e.g. eutrophication) human activities (i.a. [START_REF] Barnes | European estuaries and lagoons, a personal overview of problems and possibilities for conservation and management[END_REF][START_REF] Lardicci | Recovery of the macrozoobenthic community after severe dystrophic crises in a Mediterranean coastal lagoon (Orbetello, Italy)[END_REF]. Given the naturalistic and economic importance of these biotopes, research that may provide appropriate quantitative data is relevant for their conservation and management.

In this study, a hierarchical sampling design including four spatial scales (ranging from meters up to thousands of metres) was used i) to estimate the relative importance and test statistical significance of macrofauna variability at different spatial scales, in order to identify the spatial scale associated with the highest variability; ii) to examine if spatial patterns are influenced mainly by changes in species composition or relative abundances, comparing results obtained from several transformations of species abundance; and iii) to analyse if lower levels of taxonomic resolution (family and class) show similar spatial patterns with respect to those obtained at species level, both in terms of location and dispersion effects.

Results will allow to increase the knowledge of macrobenthic spatial distribution in Santa Giusta lagoon and to assess the applicability of TS method for decreasing time and cost in subsequent routine surveys. The methodological approach employed in the present investigation could provide interesting practical implications for future studies, not only in this lagoon, but also in other similar brackish environments.

Materials and Methods

Study area and sampling

The Santa Giusta lagoon (Western Mediterranean, Italy) is one of the largest coastal brackish environments of Sardinia island, it is a polyhaline basin located along the central-western coast of Sardinia. The lagoon is included in the Ramsar convention (1971) and belongs to a complex system of transitional waters of high natural and economic value. The Santa Giusta lagoon is approximately circular in shape with an area of 7.9 km 2 and a mean depth of 1 m; it is located near the town of Oristano and Santa Giusta, in the plain of Pesaria, an agricultural area that is intensively cultivated with rice. The lagoon has no natural attributes and is separated from the sea by a longshore bar, it is also connected with two inner small basins called Pauli Maiori and Pauli Figu (Figure 1). Central and peripheral canals have been dredged about 2 m deep in order to facilitate seawater flow into the lagoon. As a consequence, waters of Santa Giusta lagoon are now well mixed as regards circulation and stratification [START_REF] Sechi | Santa Giusta lagoon (Sardinia): phytoplankton and nutrients before and after waste water diversion[END_REF]. Salinity ranges from 25‰ to 42‰, with a mean annual value of 30‰ [START_REF] Sechi | Santa Giusta lagoon (Sardinia): phytoplankton and nutrients before and after waste water diversion[END_REF][START_REF] Luglié | Ecological assessment of Santa Giusta Lagoon (Sardinia, Italy)[END_REF]. There is a prevalent sandy-muddy bottom, with small patches of both macroalgae and angiosperms (e.g. Enteromorpha sp., Gracilaria sp., Ruppia cirrhosa, Zostera sp.), which are distributed all over the lagoon.

In this context, three sampling zones were randomly selected among the three habitats in the lagoon with different sediment and hydrodynamic properties [START_REF] Luglié | Ecological assessment of Santa Giusta Lagoon (Sardinia, Italy)[END_REF] (Figure 1). Specifically, the alpha zone was in the central-northern part of the lagoon and it was mostly influenced by the urban and industrial wastewaters from Oristano and Santa Giusta areas. It has been considered a low-intermediate hydrodynamic energy environment with a prevalence of clay-silty sediments [START_REF] Luglié | Ecological assessment of Santa Giusta Lagoon (Sardinia, Italy)[END_REF]. The beta zone was in the central-southern part of the lagoon and possibly influenced by the drainage from the surrounding farmlands. This zone was characterised by an intermediate-high hydrodynamic energy and sand-silty sediments [START_REF] Luglié | Ecological assessment of Santa Giusta Lagoon (Sardinia, Italy)[END_REF]. Then the gamma zone was an area near the Pesaria canal, connecting the lagoon with the sea; it was characterized mainly by sandy sediments and high hydrodynamic energy [START_REF] Luglié | Ecological assessment of Santa Giusta Lagoon (Sardinia, Italy)[END_REF], being closer to the sea (Figure 1). Samples were collected in November 2002, according to a hierarchical sampling design. Within each of the three zones, four random sites were selected and within each site, four areas were randomly chosen. Two replicate samples were taken within each area for a total of 96 samples. Spatial variability was estimated at four hierarchical scales: among zones (10 3 m apart), among sites within zones (10 2 m apart), among areas within sites (10s m apart) and among replicates (1 m apart). Since small vegetal patches are widely distributed throughout the lagoon, replicate samples were carefully placed away from vegetal patches (at least 5 m from the closest patch), in order to minimise the possible effects of background heterogeneity on macrofaunal composition. Softsediment samples were collected on bare bottom with a box-corer (10 x 17 cm 2 ), sieved through a 0.5 mm mesh and preserved in 4 % formaldehyde. All collected macrozoobenthic organisms were sorted and identified to the species level and abundances (number of specimens per taxon) were calculated. Time needed for classification and counting of diverse taxa was recorded. All analyses were performed by researchers, with low experience in taxonomic identification but supported by skilled taxonomists. The time spent to identify all organisms at the species, family and class levels was 255, 95 and 5 hours, respectively.

Statistical analyses

Permutational multivariate analysis of variance (PERMANOVA; [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF] was used to test the null hypothesis of no differences among assemblages at different spatial scales, according to a three factors (zone, site, area) nested design through 4999 permutations of residuals under a reduced model [START_REF] Anderson | Permutation tests for multi-factorial analysis of variance and regression[END_REF]. At species level, data were analysed using the Bray-Curtis dissimilarity measure on untransformed and transformed data (square-root, fourth-root, presence/absence), in order to evaluate if assemblages are mainly driven by compositional or relative abundance changes. Using stronger and stronger transformations, the emphasis of results can be shift from the most abundant species to the rarest ones; in particular, variability measured by presence/absence data reflects only compositional changes, while variation in relative abundances is more important in analyses based on other transformations or untransformed data [START_REF] Clarke | PRIMER V6: User Manual/Tutorial[END_REF]Anderson et al., 2005b). Furthermore, mean squares calculated by PERMANOVA were used to estimate multivariate variance components associated at each spatial scale, in a way analogous to univariate partitioning using ANOVA [START_REF] Searle | Variance components[END_REF]Benedetti-Cecchi, 2001b). For a better comparison, in y-axis the variability at each spatial scale was expressed as square-root of variance components; therefore, the values could be interpreted as percentages of Bray-Curtis dissimilarity (Anderson et al., 2005a). Separate analyses were performed using the square-root transformed data at species, family and class levels of taxonomic resolution. The family level was chosen because it has been often indicated as the most effective in minimizing the cost-benefit ratio [START_REF] Lardicci | Detection of stress on macrozoobenthos: evaluation of some methods in a coastal Mediterranean lagoon[END_REF][START_REF] Karakassis | Benthic disturbance due to fish farming analyzed under different levels of taxonomic resolution[END_REF][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] Lampadariou | Cost/benefit analysis of a benthic monitoring programme of organic enrichment using different sampling and analysis methods[END_REF], while the class level was chosen to assess the effectiveness of a further higher resolution. Since a significant result for a given factor from PERMANOVA could indicate that the groups differ in their location and/or dispersion, PERMDISP analyses were also performed to focus only on dispersion effects, testing the factors "zone" and "site" [START_REF] Anderson | Distance-based tests for homogeneity of multivariate dispersion[END_REF]. Analogous analyses were performed using separate data sets for the three main taxonomic groups.

The local species richness was visualised as a function of number of replicate samples in species-sample accumulation curves based on 999 permutations. To visualize multivariate patterns in assemblages across the three zones, non-metric multidimensional scaling (nMDS) ordination plots were produced. A separate plot was done for the overall species community and also for data aggregated at family and class level of resolution. All plots were done on the basis of Bray-Curtis dissimilarity matrix of square-root transformed data. To evaluate the degree of similarity among matrices obtained using different taxonomic aggregations, the RELATE routine was used to test the null hypothesis of independence of the two similarity matrices. On the other hand, in order to detect which species contributed most to dissimilarity among the three different zones, a similarity percentage (SIMPER) routine was performed (cut off 80%) [START_REF] Clarke | Non-parametric multivariate analyses of changes in community structure[END_REF]. All accumulation curves, nMDS plots, RELATE tests and SIMPER results were obtained using the PRIMER v.6 software [START_REF] Clarke | PRIMER V6: User Manual/Tutorial[END_REF].

Results

Faunal composition

A total of 23 878 individuals belonging to 83 species, 43 families, and 4 classes were collected. The time spent to identify all organisms at the species, family and class levels was 255, 95 and 5 hours, respectively.

Considering the number of individuals, crustaceans accounted for 43.5% of total abundance followed by polychaetes and molluscs representing respectively 30.0% and 26.5% of total abundance. Instead, considering the number of species, polychaetes were the most representative group (54 species) followed by crustaceans (19) and molluscs (10). The number of species per family varied widely (Figure 2), with most of families ( 29) represented by only one species. The most species-rich families were within the polychaete class (e.g. 9 Syllidae, 6 Spionidae, 4 Capitellidae, 4 Phyllodocidae and 4 Serpulidae) and to a lesser extent in the crustaceans (e.g. 3 Corophiidae and 3 Gammaridae). The majority of crustaceans (12 species) and molluscs (7 species) spanned a large part of the lagoon, being recorded in more than 12 of the 48 sampling areas; whereas most polychaetes (24 species) were restricted in less than 12 sampling areas (Figure 3). Seven species (five polychaetes and two crustaceans) were limited to a single area and they were also represented by only one individual. Five species (four polychaetes and one mollusc) were restricted to only two sampling areas and were represented by very few individuals (Figure 3). The mean abundance values for polychaetes and crustaceans were quite similar in all three sampling zones of the study site, while molluscs showed a higher variability being the most abundant taxa in gamma, but almost absent in alpha zone (Figure 4). The local species richness was higher in the alpha zone than in beta or gamma ones and cumulative samples from each zone were representative reaching an asymptote rather quickly (Figure 5). In particular, the number of species collected would be just slightly reduced, even analysing only 2 sampling sites (namely 8 areas or 16 replicates) in each zone of the lagoon (Table 1). A highly significant difference in the faunal composition of the three zones was detected by SIMPER analysis, with the greatest dissimilarity recorded for the alpha -gamma zone pair (75%), followed by the beta -gamma (69%) and the alpha -beta (68%) ones.

In alpha zone, despite the smallest number of individuals (5 550), the highest number of species (75) was recorded, with five species that accounted for 60.4% of total abundance. This group included species typical of brackish habitats, such as Monocorophium sextonae, Cymodoce truncata, Hydroides elegans and Corophium acherusicum [START_REF] Ruffo | The Amphipoda of the Mediterranean (959 pp)[END_REF]) and a species typical of sandy-muddy bottom such as Pseudopolydora antennata [START_REF] Lardicci | Recovery of the macrozoobenthic community after severe dystrophic crises in a Mediterranean coastal lagoon (Orbetello, Italy)[END_REF]. Moreover, SIMPER analyses highlighted the value of other species in determining the dissimilarity among the three zones: a polychaete, Cirriformia tentaculata, and a crustacean occurring in areas with abundant algal coverage, Pseudolirius kroyeri (Table 2). In the beta zone 60 species were found and the five most abundant ones (51.3% of total 7 601 individuals) were typical brackish species like Cymodoce truncata, Abra ovata, Monocorophium sextonae, as well as Minuspio multibranchiata and Microdeutopus anomalus, occurring where macroalgae are present [START_REF] Ruffo | The Amphipoda of the Mediterranean (959 pp)[END_REF]. Opportunistic species indicating organic enrichment [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF][START_REF] Cognetti | Adaptative strategy of brackish-water fauna in pure and polluted waters[END_REF], such as Phylo foetida and Neanthes caudata, were also characteristic of the beta zone (Table 2), as well as suspension feeders (Loripes lacteus and Cerastoderma glaucum) and grazers (Cumella limicola and Iphinoe serrata). In gamma zone 10 730 organisms from 59 species were collected and 80% of total abundance was reached with only five species: Mytilaster minimus (alone accounting for 33%), Cymodoce truncata and Tanais dulongii, all typical of brackish environments, besides Minuspio multibranchiata and Naineris laevigata.

Scales of multivariate spatial variability and taxonomic resolution

At the taxonomic level of species, PERMANOVA showed that there was a highly significant variability at all spatial scales considered (Table 3). The greatest variability occurred at the largest spatial scale, among zones, for which the average Bray-Curtis dissimilarity was around 37%; then, the successive variation component was that among replicate samples (35% of dissimilarity), followed by less variability among areas (22%) and sites (20%) (Figure 6). Furthermore, the relative importance of different spatial scales in the hierarchy did not vary with different data transformations. Similar spatial patterns were obtained for analyses based on untransformed and transformed data (square root, fourth root, presence/absence) (Figure 6).

Highly significant variability at all spatial scales was also detected, at the family and class levels (Table 3). In addition, similar variance components, as well as the relative importance of different spatial scales, were maintained proceeding from species to family analysis (Figure 7). Instead, using the class level of resolution, the variance components decreased showing less dissimilarity among assemblages at all spatial scales analysed; moreover, proportional amount of variation changed showing the highest variability at the smallest scale, among replicate samples (Figure 7). At the species level, differences among zones were mainly due to differences in their location, since a significant dispersion effect was revealed only between alpha and gamma zones (Table 4). At the scale of site, the source of variability changed depending on the sampling zone as emerged by pairwise tests, within the alpha zone some sites were not significantly different from each other (i.e. P-values of both PERMANOVA and PERMDISP tests were not significant), in some cases sites differed in their location (i.e. P-values of PERMANOVA significant, P-values of PERMDISP not significant), in other cases sites differed both in their location and dispersion (i.e. P-values of both PERMANOVA and PERMDISP tests were significant). Similar results were obtained within the beta zone, where sites were different also because of their dispersion (i.e. P-values of PERMANOVA not significant, P-values of PERMDISP significant). Dispersion effect never contributed to differences among sites in the gamma zone (Table 4). All but two pairwise results were likewise detected using the family level of taxonomic resolution; while at the class level, the majority of results were not significant, therefore indicating different relationships compared to those obtained at finer taxonomic levels, both at zone and site spatial scales (Table 4).

Separate analyses for the three collected taxonomic groups showed a highly significant variability at all spatial scale; only the variance component for molluscs at the site scale was found not different from zero because of the greater variability at the smaller spatial scale of area (Figure 8). Such results matched the pairwise tests which showed that in very few cases sites differed, mainly in their dispersion (Table 5). For crustaceans and molluscs the greatest variability occurred at the largest spatial scale (Figure 8) and for both groups it was mainly due to differences in location among zones (Table 5). For polychaetes, the sources of the high variability at the zone scale were differences in location and dispersion (Table 5); however polychaetes showed the greatest variability among replicate samples and such spatial scale was also important for the other taxonomic groups (Figure 8). Except for molluscs, a small variability was associated with the two intermediate spatial scales (Figure 8); in particular at the site scale, there was a prevalent location effect both for polychaetes and crustaceans although differences in dispersion were also detected especially for crustaceans within the beta zone (Table 5).

The nMDS plot based on the species abundance data showed some differences among the three sampled zones. In particular, gamma samples were clearly clustered and separated from the other zones, while alpha and beta samples were partially overlapped (Figure 9a). At the family level, nMDS ordination was very similar to that obtained at the species level (Figure 9b). Further aggregation to the class level produced a different ordination pattern, with substantially higher levels of overlapping of the three zones (Figure 9c). Relationships between similarity matrices calculated for the three taxonomic levels were confirmed by RELATE results, which showed ρ = 0.960, p<0.001 between species and family levels and ρ = 0.565, p<0.001 between species and class levels.

Discussion

The first result that stands out from our work is that the benthic assemblages of Santa Giusta lagoon were extremely variable, with significant differences at all considered spatial scales, from metres up to thousands of metres. This outcome was highly consistent with results reported in studies analysing spatial variability by nested design, whatever the habitat investigated (see [START_REF] Fraschetti | Patterns of distribution of marine assemblages from rocky shores: evidence of relevant scales of variation[END_REF] for a review). In the Santa Giusta lagoon most of the variation was associated with the smallest and the largest spatial scale, thus indicating that both small-scale and large-scale processes play a major role in shaping benthic community spatial patterns. Variability among replicates at small spatial scale is usually considered a widespread feature of many different assemblages, being mainly determined by biological interactions and/or local physical factors (i.a. [START_REF] Ekman | Small-scale patterns and processes in a soft substratum, intertidal community[END_REF]Underwood and Chapman, 1986;[START_REF] Wilson | Competition and predation in marine soft sediment communities[END_REF][START_REF] Morrisey | Spatial variation in softsediment benthos[END_REF]Benedetti Cecchi et al., 2001a;[START_REF] Coleman | Small-scale spatial variability in intertidal and subtidal turfing algal assemblages and the temporal generality of these patterns[END_REF][START_REF] Rossi | Role of the nutritive value of sediment in regulating population dynamics of the deposit-feeding polychaete Streblospio shrubsolii[END_REF][START_REF] Fraschetti | Patterns of distribution of marine assemblages from rocky shores: evidence of relevant scales of variation[END_REF][START_REF] Chapman | Scales of variation of gastropod densities over multiple spatial scales: comparisons of common and rare species[END_REF]. On the other hand, differences in assemblages at large spatial scales have been mostly related to abiotic processes [START_REF] Thrush | Patterns in the spatial arrangements of polychaetes and bivalves in intertidal sandflats[END_REF][START_REF] Thrush | Spatial patterns in soft-bottom communities[END_REF]. Factors such as hydrodynamic energy, trophic status, seawater and freshwater influence, nutrients supply and confinement could differently characterise the three sampling zones of the Santa Giusta lagoon, according to the models of zonation proposed for other Mediterranean coastal lagoons (Guelorget and Perthuisot, 1982;[START_REF] Lardicci | The distribution of polychaetes along eEnvironmental gradients: an example from the Orbetello lagoon, Italy[END_REF][START_REF] Underwood | Experiments in ecology: their logical design and interpretation using analysis of variance[END_REF][START_REF] Pérez-Ruzafa | Detecting changes resulting from human pressure in a naturally quick-changing and heterogeneous environment: Spatial and temporal scales of variability in coastal lagoons[END_REF]. In addition, benthic communities could be unevenly subjected to the two main sources of anthropogenic disturbance affecting this lagoon at all the study scales. Results of this study showed that the three sampling zones were clearly distinct and characterised by typical features but analysing and explaining the effects of abiotic or biotic factors responsible of such differences were not among the explicit aims of this study.

A number of papers reported that results of statistical analyses can be greatly influenced by the choice of data transformation; in fact, the ability to detect differences along strong environmental gradients was affected more by changing the data transformation rather than the level of taxonomic identification (i.a. [START_REF] Olsgard | Relationship between taxonomic resolution, macrobenthic community patterns and disturbance[END_REF][START_REF] Karakassis | Benthic disturbance due to fish farming analyzed under different levels of taxonomic resolution[END_REF]. This is also consistent with results by [START_REF] Chapman | Relationships between spatial patterns of benthic assemblages in a mangrove forest using different levels of taxonomic resolution[END_REF] and [START_REF] Lasiak | Influence of taxonomic resolution, biological attributes and data transformations on multivariate comparisons of rocky macrofaunal assemblages[END_REF], who observed that the type of transformation altered patterns of variability within sites, which may be important for some research programmes. The choice of transformation determines the relative contribution of quantitative and qualitative intersample differences in the final outcome of all multivariate analyses. Strong transformations (fourth root, presence/absence) give little weight to differences in abundance, whereas weak (square root) or null transformations provide patterns mainly reflecting the differences of the most abundant species [START_REF] Olsgard | Relationship between taxonomic resolution, macrobenthic community patterns and disturbance[END_REF][START_REF] Karakassis | Benthic disturbance due to fish farming analyzed under different levels of taxonomic resolution[END_REF][START_REF] Clarke | PRIMER V6: User Manual/Tutorial[END_REF]. In particular, the variability estimated by analysing presence/absence data explicitly reflects the compositional changes of assemblages at different spatial or temporal scales and this can be compared with analyses based on other transformations (or untransformed data), mostly describing relative abundance differences (Anderson et al., 2005b). On this basis, some studies demonstrated that variability of benthic assemblages at larger scales is mainly "compositional", as indicated by the presence/absence analyses, while variability at medium or smaller scales is driven by changes in relative abundance, particularly by numerically dominant taxa [START_REF] Archambault | Scales of coastal heterogeneity and benthic intertidal species richness, diversity and abundance[END_REF][START_REF] Chapman | Relationships between spatial patterns of benthic assemblages in a mangrove forest using different levels of taxonomic resolution[END_REF]Anderson et al., 2005b).

Conversely, our results showed that the relative importance of examined spatial scales (from meters up to thousands of metres) was always maintained, despite different transformations used. These findings underpinned that in Santa Giusta lagoon, large scale processes shaped three distinct zones characterized by different species. At the other investigated scales, differences in species composition were possibly caused by the presence of diverse microhabitats, which created high spatial heterogeneity. For example, small vegetal patches might possibly influence the faunal composition of the surrounding bare bottoms; however, such patches are distributed all over the lagoon, thus producing a high spatial heterogeneity at the smallest spatial scale in all the three sampling zones of Santa Giusta. This outcome indicated that spatial heterogeneity is not necessarily related to the extent of the study area.

Therefore the unambiguous interpretation of results can be promoted using nested designs also in small environments, like the Santa Giusta lagoon. The multi-scale approach is recommended as a basic tool for spatial distribution analyses, especially when such information is still scarcely known in the investigated environment. In particular for future studies in the Santa Giusta lagoon, single observations should be evaluated in relation to the proper sampling zone, not being representative of the whole coastal lagoon.

In this study, multivariate analyses revealed that community spatial patterns derived from species and family abundance data were very similar to each other. Consistent results were reported in many other works (i.a. [START_REF] Warwick | The level of taxonomic discrimination required to detect pollution effects on marine benthic communities[END_REF][START_REF] Ferraro | taxonomic level sufficient for assessing pollution impacts on the Southern California Bight macrobenthos -revisited[END_REF][START_REF] Olsgard | Relationship between taxonomic resolution, macrobenthic community patterns and disturbance[END_REF][START_REF] Lardicci | Detection of stress on macrozoobenthos: evaluation of some methods in a coastal Mediterranean lagoon[END_REF][START_REF] Mistri | Taxonomic sufficiency in lagoonal ecosystems[END_REF][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] Dethier | Taxonomic sufficiency in distinguishing natural spatial patterns on an estuarine shoreline[END_REF], demonstrating redundancy of information in large sets of benthic species data for identifying significant differences among assemblages, in both polluted and unpolluted environments. Our results showed that decreasing taxonomic resolution from species to family was not related to a strong decrease of the overall spatial variability. On the contrary, lumping species in higher taxonomic groups was usually considered leading to a probable decrease in estimates of variability as a consequence of an "averaging effect" [START_REF] Doak | The statistical inevitability of stability-diversity relationships in community ecology[END_REF][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]. Analyses based on family abundances were effective in detecting spatial patterns among the three zones of the lagoon, and they provided estimates of variance components that were not substantially different from those detected at the species level. In addition, spatial dispersion of samples was similarly described by both species and family level and this was a novel finding compared to previous works investigating the TS applicability. Such works have mainly looked for changes in the location of sample groups in multivariate space at decreasing taxonomic levels of resolution, while the effects of TS on the dispersion of sample groups were usually neglected (Terlizzi et al., 2008a). However, explicitly analysing differences in dispersion among groups is important in order to obtain more complete information as well as avoid misleading interpretation of results [START_REF] Anderson | Distance-based tests for homogeneity of multivariate dispersion[END_REF]. Thus in Santa Giusta lagoon, PERMDISP results clarified that differences in species composition concerned almost exclusively spatial differences detected at the zone scale, while spatial variability observed at site (or even area) scale was mostly due to differences in dispersion. Similar spatial patterns were found at the two lowest taxonomic levels, probably because of the high percentage of families represented by a single species as usually occurred in brackish environments [START_REF] Giangrande | Polychaetes as environmental indicatorsrevisited[END_REF]. Further aggregation at the class level showed relevant changes in observed spatial patterns; in particular, the overall spatial variability decreased reflecting a more homogenous distribution of class abundances within the lagoon. As a consequence, few significant differences were detected among levels of each investigated spatial scale. Meanwhile, the relatively higher variability among replicates probably increased because of the uneven distribution of some organisms living in small dense patches (e.g. Mytilaster minimus).

The usefulness of TS method has been evaluated and often promoted in order to streamline expensive and time consuming sampling protocols, like those employed in soft bottom macrofauna analyses (i.a. [START_REF] Olsgard | Relationship between taxonomic resolution, macrobenthic community patterns and disturbance[END_REF][START_REF] Terlizzi | Taxonomic sufficiency and the increasing insufficiency of taxonomic expertise[END_REF][START_REF] Lampadariou | Cost/benefit analysis of a benthic monitoring programme of organic enrichment using different sampling and analysis methods[END_REF][START_REF] Wlodarska Kowalczuk | Surrogacy in natural patterns of benthic distribution and diversity: selected taxa versus lower taxonomic resolution[END_REF]. However, other possibilities were also investigated. [START_REF] Lampadariou | Cost/benefit analysis of a benthic monitoring programme of organic enrichment using different sampling and analysis methods[END_REF] compared results obtained using different mesh-size and type of sampler; they indicated that small samples taken with corers and sieved at 0.5 mm provided a large proportion of benthic spatial distribution, even if data were analysed at the family level.

Other studies examined single taxonomic groups as representative of the whole assemblages, but contrasting outcomes emerged. While [START_REF] Olsgard | Polychaetes as surrogates for marine biodiversity: lower taxonomic resolution and indicator groups[END_REF] promoted polychaetes alone as useful surrogates to describe soft bottom macrofauna distribution, in other cases reduced taxonomic resolution was more effective than using a single taxonomic group (Anderson et al., 2005a;[START_REF] Wlodarska Kowalczuk | Surrogacy in natural patterns of benthic distribution and diversity: selected taxa versus lower taxonomic resolution[END_REF]. In Santa Giusta lagoon, different spatial distributions were obtained analysing separately the three main taxonomic groups and none of them reflected results obtained by the whole benthic assemblages. Therefore future studies investigating macrobenthic spatial distribution of this lagoon should prefer the TS method to analysis of a single taxonomic group.

As argued elsewhere, the relationship between time saving and taxonomic level changes from case to case [START_REF] Olsgard | Relationship between taxonomic resolution, macrobenthic community patterns and disturbance[END_REF], depends on the number of species within a single family, the taxonomical complexity of families and the availability of taxonomic expertise [START_REF] Ferraro | taxonomic level sufficient for assessing pollution impacts on the Southern California Bight macrobenthos -revisited[END_REF][START_REF] Dethier | Taxonomic sufficiency in distinguishing natural spatial patterns on an estuarine shoreline[END_REF]). However, it has been calculated that generally the cost of family level identification was 50% to 55% less than species level identification [START_REF] Ferraro | taxonomic level sufficient for assessing pollution impacts on the Southern California Bight macrobenthos -revisited[END_REF][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]. In our case, the time needed for identification at family level was 63% less than the species level identification, considering that 33% of families was represented by two or more species. However, the majority of species belonged to polychaetes and our laboratory team has significant expertise in polychaetes. Resources deriving from such cost reduction could be employed to plan more frequent surveys and/or to adopt more complex spatial sampling designs with a high number of replicates, in order to further minimize spatial variability caused by the dispersion effect. When the distribution of organisms is patchy, it is probably more important to collect many replicates at different spatial and temporal scales than to identify taxa at the finest resolution level [START_REF] Morrisey | Spatial variation in softsediment benthos[END_REF][START_REF] Chapman | Relationships between spatial patterns of benthic assemblages in a mangrove forest using different levels of taxonomic resolution[END_REF]. In Santa Giusta lagoon, our baseline detailed multiscale investigation demonstrated that at least two sites (namely 16 samples) for each zone are needed to collect the majority of species and therefore to describe correctly the spatial distribution of benthic assemblages.

Results obtained in this study have important practical consequences for investigations on the distribution of soft bottom macrofauna in brackish habitats, including those concerned with environmental monitoring. In fact, the present study can be considered as a valuable example for a rigorous approach in collecting data for ecological studies, when previous detailed knowledge is scant. The spatial variability observed at all the examined scales indicated that small-scale observations are unlikely to describe the spatial benthic distribution of the whole lagoon [START_REF] Foster | Organization of macroalgal assemblages in the North Pacific: the assumption of homogeneity and the illusion of generality[END_REF]. As a consequence, any a priori statement about composition, structure and distribution of macrobenthic communities should be avoided, even in small brackish environments usually considered as homogenous habitats. Furthermore, explicitly testing for differences in dispersion among groups has been demonstrated to obtain a more accurate interpretation of the detected spatial patterns and such an approach should be more frequently adopted in future studies. Especially for routine monitoring programs, long term data sets at the finest taxonomic level and large sampling effort are usually the preferred approach for analyses of macrobenthic assemblages. Unfortunately, there are often many practical difficulties such as reduced budgets or lack of well-trained taxonomists, and compromise solutions are unavoidable. However the present study highlighted that reasonable choices and useful advice can be obtained only if the planning of monitoring programs is proceeded by a detailed baseline study [START_REF] Terlizzi | Taxonomic sufficiency and the increasing insufficiency of taxonomic expertise[END_REF]2008a), thus avoiding any a priori decision. In particular, our results showed that in Santa Giusta lagoon, if resources are limited, analysing different spatial scales considering the whole benthic assemblages at the family level may be more important than classifying all individuals at the species level [START_REF] Kingston | Cost Effectiveness of Benthic Faunal Monitoring[END_REF][START_REF] Lampadariou | Cost/benefit analysis of a benthic monitoring programme of organic enrichment using different sampling and analysis methods[END_REF]. Although spatial patterns do not necessary remain constant over time and further analyses at several temporal scales are needed, in future routine investigations taxonomic costs can be probably reduced without losing the power to detect macrobenthic spatial patterns, both in terms of location and dispersion effect. Since the most frequent disturbance events (e.g. organic enrichment, eutrophication, chemical pollution) are likely related to changes in spatial patterns of assemblages [START_REF] Caswell | Communities in patchy environments: a model of disturbance, competition and heterogeneity[END_REF][START_REF] Warwick | Increased variability as a symptom of stress in marine communities[END_REF][START_REF] Fraschetti | Spatial variability and human disturbance in shallow subtidal hard substrate assemblages: a regional approach[END_REF][START_REF] Terlizzi | Quantifying effects of pollution on biodiversity; a case study of highly diverse molluscan assemblages in the Mediterranean[END_REF], monitoring programs, based on periodical surveys and TS, may be useful for a quick environmental assessment. Further detailed analyses, like identification at the species level, should be carry out if changes in spatial patterns are detected, in order to confirm and clarify disturbance effects on assemblages. However, other disturbance events (e.g. invasion of alien species, climate change) may act gradually and for example change the natural balance of competitive interactions among phylogenetically close species, like species of the same genus or family; in this case, disturbance effects can be detected only analysing the community at the finest taxonomic level.

The present study expanded the current knowledge of macrobenthic assemblages in Santa Giusta lagoon and emphasised the usefulness of multiscale approach to realistically describe spatial patterns of variability. In addition, our results highlighted some helpful methodological procedures, which should be promoted in order to better design future sampling designs in this lagoon, as well as in other similar brackish environments. Table 5. P-values for pairwise tests of significant variability among "zones" and among "sites" in each zone, for the three main taxonomic groups. "L+D" columns are P-values obtained by PERMANOVA, therefore indicating a "location" and/or a "dispersion" effect. "D" columns are P-values obtained by PERMDISP, therefore indicating only a "dispersion" effect. Results that are not significant at the 0.05 level are given in bold type. Note that the smallest possible P-value with 4999 permutations is 0.0002. 3)

Figure Captions

Figure 1 :Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .

 1234567 Figure 1: The Santa Giusta lagoon. The three sampling zones are delimited by dotted lines

Figure 8 .Figure 9 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .

 8923456789 Figure 8. Multivariate variance components at each of the four spatial scales for the three taxonomic group collected. The values plotted are the square root of the sizes of the variance components, obtained using mean squares from PERMANOVA performed with square root transformed data. All components were statistically significant at p<0.001, except for molluscs at the site scale which were not significant

Table 1 .

 1 The local species richness estimated by accumulation curves (Figure5) in correspondence with different number of sampling replicates in each zone of the lagoon. N: number of replicate samples analysed; Sobs: number of species observed; % Sobs: percentage of species observed in comparison with the total species number collected.

	Tables
	N	alpha	Sobs beta	gamma	alpha	% Sobs beta	gamma
	32	75	60	58	100	100	100
	24	72	58	56	96	97	97
	16	67	55	53	89	92	91
	8	57	48	46	76	80	79

Table 2 .

 2 Results of SIMPER analysis. Average abundance of species contributing to most of the Bray-Curtis dissimilarity between zones (cut-off value = 60%) (data square-root transformed). The highest average abundance value is in bold.

	species	Av. Transf. Abundance alpha		Av. Transf. Abundance beta		Av. Transf. Abundance gamma
	Cymodoce truncata	4.26	<	5.11	<	7.01
	Monocorophium sextonae	3.33	<	4.20	>	-
	Microdeutopus anomalus	0.74	<	4.09	>	0.15
	Abra ovata	1.31	<	3.67	>	1.86
	Loripes lacteus	1.50	<	2.73	>	1.27
	Phylo foetida	0.52	<	3.18	>	0.06
	Minuspio multibranchiata	1.57	<	3.60	<	5.28
	Cumella limicola	1.38	<	3.11	>	1.01
	Hydroides elegans	1.96	>	1.32	<	2.22
	Pseudopolydora antennata	1.67	<	2.00	>	0.75
	Neanthes caudata	1.12	<	2.85	>	0.82
	Cirriformia tentaculata	2.07	>	0.32	<	1.02
	Cerastoderma glaucum	0.33	<	1.96	>	0.37
	Corophium acherusicum	1.10	<	1.17	>	-
	Pseudolirius kroyerii	1.49	>	0.09	<	0.50
	Tapes aurea	1.28	>	0.99	<	0.50
	Iphinoe serrata	1.00	<	1.49	>	0.15
	Mytilaster minimus	0.23	<	0.70	<	9.69
	Tanais dulongii	0.27	<	0.91	<	6.83
	Nainereis laevigata	0.09	<	0.53	<	2.46
	Dynamene bidentata	0.73	>	1.04	<	2.17
	Tapes decussata	1.63	>	1.10	>	0.08
	Ophiodromus pallidus	0.31	<	0.44	<	1.46
	Podarkeopsis capensis	0.59	>	0.46	<	1.83
	Cumella limicola	1.38	<	3.11	>	1.01

Table 3 .

 3 Permutational multivariate analysis of variance based on the Bray-Curtis dissimilarity for square-root transformed data of species (83 variables), families (43 variables) and classes (4 variables) abundance. Analysis was carried out using 4999 permutations of residuals under a reduced model. Estimates of multivariate variation at each spatial scale were included.

	Taxonomic Level	Source	df	SS	MS	F	P	Variance Component
	Species	Zone	2	77637.7 38818.8 10.977	0.004	1102.6
		Site	9	31829.0	3536.6	2.182	0.002	239.5
		Area	36	58354.7	1621.0	2.071	0.002	419.2
		Residual	48	37561.7	782.5			782.5
		Total	95	205303.1				2543.8
	Family	Zone	2	69723.2 34861.6 11.409 0.0004	993.9
		Site	9	27501.4	3055.7	2.699 0.0002	240.5
		Area	36	40756.8	1132.1	1.935 0.0002	273.6
		Residual	48	28077.6	584.9			584.9
		Total	95	166059				2092.9
	Class	Zone	2	13944.7	6972.3	5.777 0.0164	180.2
		Site	9	10862.3	1206.9	3.713 0.0002	110.2
		Area	36	11703.0	325.1	1.501	0.019	54.3
		Residual	48	10395.7	216.6			216.6
		Total	95	46905.7				561.2

Table 4 .

 4 P-values for pairwise tests of significant variability among "zones" and among "sites" in each zone, for different levels of taxonomic resolution. "L+D" columns are P-values obtained by PERMANOVA, therefore indicating a "location" and/or a "dispersion" effect. "D" columns are P-values obtained by PERMDISP, therefore indicating only a "dispersion" effect. Results that are not significant at the 0.05 level are given in bold type. Note that the smallest possible P-value with 4999 permutations is 0.0002.

			Polychaetes	Crustaceans	Molluscs
	Source	Pairwise	L+D	D	L+D	D	L+D	D
	among Zones alpha -beta	0.0272	0.0290	0.0304	0.0564	0.0294	0.0600
		alpha -gamma	0.0288	0.0278	0.0244	0.0272	0.0286	0.1118
		beta -gamma	0.0274	0.9744	0.0266	0.2794	0.0290	0.0558
			Polychaetes	Crustaceans	Molluscs
	Source	Sites	L+D	D	L+D	D	L+D	D
	Sites within
	alpha zone	1 -2	0.0146	0.4206	0.0004	0.3502	0.8044	0.8878
		1 -3	0.0888	0.9636	0.1080	0.2082	0.9148	0.2124
		1 -4	0.0362	0.1118	0.0014	0.2314	0.9684	0.3426
		2 -3	0.0010	0.4020	0.0006	0.0590	0.6906	0.1934
		2 -4	0.0006	0.0062	0.0002	0.0658	0.6342	0.3874
		3 -4	0.0448	0.1234	0.2136	0.9264	0.8888	0.0720
	Sites within
	beta zone	1 -2	0.0230	0.0482	0.1104	0.1990	0.8554	0.9824
		1 -3	0.0002	0.0624	0.0374	0.6736	0.1358	0.0010
		1 -4	0.0058	0.5592	0.1298	0.0704	0.8174	0.2234
		2 -3	0.0002	0.7858	0.0002	0.0288	0.1464	0.0018
		2 -4	0.0004	0.0172	0.0166	0.0016	0.9602	0.2324
		3 -4	0.0262	0.0222	0.6834	0.0710	0.1396	0.1358
	Sites within
	gamma zone 1 -2	0.9478	0.9650	0.3334	0.1412	0.3350	0.5506
		1 -3	0.0010	0.5756	0.0046	0.2748	0.4470	0.7008
		1 -4	0.0318	0.2308	0.6134	0.0072	0.1774	0.0036
		2 -3	0.0026	0.6246	0.0264	0.8588	0.3014	0.8382
		2 -4	0.0048	0.3996	0.0294	0.2960	0.0282	0.0008
		3 -4	0.0002	0.1702	0.0150	0.2916	0.0606	0.0016

Acknowledgments

This work was funded by the Italian Ministry of Food, Agricultural and Forest Resources, the University of Pisa and the University of Sassari as part of a study on Italian coastal lagoons.

The authors gratefully acknowledge Dr. John Widdows and three anonymous reviewers for valuable comments on an early version of this manuscript.


Gustavo M Martins
email: gustavo.martins@plymouth.ac.uk

André F Amaral

Francisco M Wallenstein

Neto

INFLUENCE OF A BREAKWATER ON NEARBY ROCKY INTERTIDAL COMMUNITY STRUCTURE 2

Keywords: sea defence structure, environmental impact, benthic assemblages, waveexposure, recruitment, ACI (After-Control-Impact) design. 42 44 46 48

It is widely recognised that coastal defence structures generally affect the structure of the assemblages they support, yet their impact on adjacent systems has been largely 28 ignored. Breakwaters modify the nearby physical environment (e.g. wave-action) suggesting a local impact on biological parameters. In the present study, an ACI (After-30 Control-Impact) design was used to test the general hypothesis that the artificial sheltering of an exposed coast has a strong effect on the structure and functioning of 32 adjacent systems. The effects of a reduction in hydrodynamics were clear for a number of taxa and included the replacement of barnacles, limpets and frondose algae by an 34 increasing cover of ephemeral algae. These effects were evident both at early and late successional stages. Results suggest that the artificial sheltering of naturally exposed 36 coasts can have a strong impact promoting a shift from consumer-to producerdominated communities, which has important ecological and energetic consequences 38 for the ecosystem. 40

Introduction

Since the 1960's there has been an unprecedented rate of habitat loss, 52 modification or degradation with some European countries reporting reductions over 50% of the original coastal habitat [START_REF] Airoldi | Loss, status and trends for coastal marine habitats of 384 Europe[END_REF]. Coastal urbanisation 54 including the replacement of natural substrata by artificial ones or the deployment of artificial structures to protect the flooding of low-lying lands and to prevent coastal 56 erosion is likely to increase in the next decades due to sea-level rise or increased storm frequency [START_REF] Thompson | Rocky intertidal communities: past 506 environmental changes, present status and predictions for the next 25 years[END_REF]. There is an increasing awareness that coastal 58 urbanisation leads to important changes in the structure and functioning of marine ecosystems including the loss of keystone taxa and associated reductions of local 60 diversity (e.g. [START_REF] Benedetti-Cecchi | Predicting the consequences of anthropogenic disturbance: large-scale effects of loss of canopy algae on rocky shores[END_REF][START_REF] Mangialajo | Loss of fucoid algae along a 486 gradient of urbanisation, and structure of benthic assemblages[END_REF]. Although similarities exist between assemblages on natural and built reefs, patterns of species 62 distribution are generally modified in the latter (Bulleri et al., 2004;Bulleri and Chapman, 2004) and the assemblages are characterised by a reduced subset of species 64 that commonly occur on natural shores (e.g. [START_REF] Chapman | Paucity of mobile species on constructed seawalls: effects o 426 urbanization on biodiversity[END_REF][START_REF] Chapman | Intertidal seawalls -new features of landscape in 428 intertidal environments[END_REF]. Patterns of species recruitment to seawalls also tend to differ from that on natural 66 shores [START_REF] Bulleri | Role of recruitment in causing differences between intertidal 414 assemblages on seawalls and rocky shores[END_REF]. In addition, artificial structures can also facilitate the colonisation and spread of non-indigenous species [START_REF] Bulleri | Artificial marine structures facilitate the spread of a nonindigenous green alga, Codium fragile ssp. tomentosoides, in the north 418 Adriatic Sea[END_REF]Glasby et 68 al., 2007), acting as stepping-stones. Thus, sea defence structures seem to act as functional different habitats. 70

The above mentioned studies have generally compared the assemblage structure on artificial and natural shores while little is known on how these structures affect the 72 structure of adjacent rocky intertidal communities (but see [START_REF] Goodsell | Differences between biota in 456 anthropogenically fragmented habitats and in naturally patchy habitats[END_REF]. Work done on other systems including mangrove forests [START_REF] Kelaher | Changes in benthic assemblages near boardwalks in temperate urban mangrove forests[END_REF], fish 74 assemblages [START_REF] Clynick | Fish assemblages associated with urban structures and natural reefs in Sydney, Australia[END_REF] and soft-sediment habitats (see [START_REF] Bertasi | Effects of an 402 artificial protection structure on the sandy shore macrofaunal community: the special case of Lido di Dante (Northen Adriatic Sea)[END_REF] and references therein) has shown that the influence of artificial structures can extend 76 well beyond the very communities they support.

Breakwaters are deployed for reducing wave action and preventing coastal 78 erosion. Breakwaters also modify near-shore water circulation [START_REF] Zyserman | Analysis of far-field 532 erosion induced by low-crested rubble-mound structures[END_REF] and sediment transport [START_REF] Thomalla | Beach response to shore-parallel breakwaters at Sea 504 Palling, Norfolk, UK[END_REF][START_REF] Cuadrado | Tidal and longshore sediment 444 transport associated to a coastal structure[END_REF]. Changes 80 in the physical environment produced by the deployment of breakwaters will likely affect the structure of assemblages under their influence. For instance, wave-action 82 conditions have recurrently been shown to affect the distribution and abundance of intertidal organisms (e.g. [START_REF] Underwood | The effects of tidal height, wave-exposure, 522 seasonality and rock-pools on grazing and the distribution of intertidal macroalgae in New SouthWales[END_REF], organism settlement (e.g. 84 [START_REF] Abelson | Settlement of marine organisms in flow[END_REF]Denny, 1997), mortality (e.g. McQuaid and[START_REF] Mcquaid | Effect of wave exposure on growth and mortality 492 rates of the mussel Perna perna: bottom-up regulation of intertidal populations[END_REF], and biological interactions (e.g. Jonsson et al., 2006). Sedimentation has also been shown to influence 86 rocky intertidal community structure of intertidal assemblages (reviewed by [START_REF] Airoldi | The effects of sedimentation on rocky coast assemblages. 382[END_REF]. Specifically, sedimentation increases disturbance leading to a reduction in the 88 vertical structure of algal assemblages and thus the replacement of frondose by turfforming algae [START_REF] Airoldi | Roles of disturbance, sediment stress, and substratum retention on 378 spatial dominance in algal turf[END_REF]. In addition, sedimentation interferes with grazing 90 gastropod activities and thus reducing their abundance [START_REF] Airoldi | Negative effects of sediment deposition on grazing 386 activity and survival of the limpet Patella vulgata[END_REF].

In the present study we compared the rocky intertidal structure of assemblages 92 sheltered by an existing breakwater to that of control areas. Specific hypothesis were that the increased sediment deposition in the area sheltered by the breakwater will lead 94 to a reduction in the abundance of frondose algae promoting the proliferation of turfforming species lower on the shore and a reduction in the abundance of grazing 96 gastropods upper on the shore. Chthamalus stellatus, a barnacle which dominates on wave-exposed shores [START_REF] Jenkins | Larval habitat selection, not larval supply, determines settlement patterns and adult distribution in two chthamalid barnacles[END_REF] is predicted to decrease in cover in response to the 98 reduction in wave-action and to abrasion by the increased sedimentation in the sheltered area. In addition, changes in the water-circulation caused by the presence of the breakwater are hypothesised to affect macroalgae and/or invertebrate recruitment [START_REF] Abelson | Settlement of marine organisms in flow[END_REF]. However, and except for the recruitment of Chthamalus 102 stellatus, there is no directional hypothesis as to how early successional assemblages will differ between the impacted and control locations. 104

Materials and methods 106

Study site and community

The present study was done on a moderate to highly exposed rocky platform on 108 the south coast of Pico, Azores (Fig. 1). Unlike most shores on the remainder of the Azores archipelago, Lajes do Pico is a large and gentle sloping reef with a variety of 110 habitats [START_REF] Morton | Coastal ecology of the Azores[END_REF] that support a diverse community of plants and animals (for details on the composition and community structure of this reef see Morton et al., 112 1998;Ávila et al., 2005;[START_REF] Martins | Rocky intertidal community structure in oceanic islands: scales of spatial variability[END_REF]. The uniqueness of this area has contributed to its classification as SCI (site of community interest) and SAC (special 114 area of conservation). The main economic activity of the small village of Lajes do Pico is agriculture and, to a smaller extent, fishing and tourism (whale-watching). Extractive 116 activities on the coastal area include angling, crabbing and harvesting of octopus and limpets. The rocky platform is divided in two by a small lagoon in which the inner part 118 is used as an anchorage point harbouring small fishing and recreational boats. Due to high wave action in winter, an already existing, but small, breakwater was extended 120 ~100 m offshore towards the north in order to protect the entrance of the lagoon. The breakwater runs parallel to the northern rock platform at approximately 50 m away from 122 the shore and is made of large, regularly sized concrete blocks that rise approximately 4 m above seawater level. The construction resulted in the artificial sheltering of the 124 northern area of the rocky platform, locally denominated as Lajido, previously exposed to open swell. Besides the presence of the breakwater, there are no apparent differences 126 (e.g. geomorphology) between the northern and southern rock platforms and the impact of the small harbour is expected to be small. The study began one year following the 128 completion of the breakwater. During the course of study, occasional construction work was done in the inner part of the lagoon close to the harbour. Although the harbour is 130 ~200 m away this may have led to pulse increases in sediment load. The southern platform is protected from such disturbances due to the ground elevation separating the 132 two areas. 134

Sampling design

In order to examine the effects of the breakwater on the adjacent algal and 136 animal assemblages, a beyond-BACI design (see [START_REF] Underwood | Beyond BACI: experimental designs for detecting human environmental impacts on temporal variations in natural populations[END_REF][START_REF] Underwood | Beyond BACI: the detection of environmental impacts on 512 populations in the real, but variable world[END_REF][START_REF] Underwood | The mechanisms of spatially replicated sampling programmes to detect environmental impact in a variable world[END_REF]Underwood, and 1994 for revision) for revision) was not possible due to the lack of a priori data. However, beyond-138 BACI designs can be modified and applied to situations where no before data exist and have been widely used (e.g. [START_REF] Chapman | Variability at different spatial scales between a subtidal assemblage exposed to the discharge of sewage and 434 two control assemblages[END_REF][START_REF] Terlizzi | Multivariate and univariate asymmetrical analyses in environmental impact assessment: a case study of Mediterranean subtidal sessile assemblages[END_REF]. These designs 140 allow the determination of consistent differences between the disturbed and several control locations although it is generally not possible to attribute causation to a 142 particular event (see [START_REF] Terlizzi | Multivariate and univariate asymmetrical analyses in environmental impact assessment: a case study of Mediterranean subtidal sessile assemblages[END_REF], Glasby, 1997 for discussion).

In the present study, we used an asymmetrical ACI design to compare the 144 abundance of conspicuous eulittoral taxa in a putatively disturbed location (a rocky platform artificially sheltered by a breakwater) and three control locations. All locations 146 (the disturbed and controls) are on the same rocky platform and at least 150 m apart from each other. The disturbed location is located in the northern part of this platform 148 whereas all control locations were located south to this. Although the non-interspersion of control locations introduces an issue of spatial confounding which may affect the interpretation of results [START_REF] Underwood | On beyond BACI: sampling designs that might reliably detect 518 environmental disturbances[END_REF], this situation was compulsory as north of the disturbed location the rocky platform gives rise to a vertical wall falling directly into 152 the water. As mentioned above, this specific rocky platform is unique on the Azorean shores, which are characterised by steep slopes. Since the slope of the shore can 154 significantly affect the processes structuring intertidal communities [START_REF] Knott | Epibiota on 480 vertical and on horizontal surfaces on natural reefs and on artificial structures[END_REF] these were not considered appropriate controls to the disturbed location (see Underwood 156 (1994) for the desirable characteristics that control locations must have). Analyses of spatial correlation (data not shown) showed that the abundance of organisms was only 158 weekly correlated with the location of controls and hence suggesting that no discernible horizontal gradients were operating along this platform, which could interfere with the 160 interpretation of results.

At each location (disturbed and controls), sampling of mature assemblages was 162 done by means of 10 random quadrats (25 x 25 cm) located at least 1 m apart at the lower algal dominated shore (~0.6 m above Chart Datum) and upper animal dominated 164 shore (~1.8 m above CD). Sampling was done at 4 different times of the year (July '06, October '06, January '07, March '07) to provide an estimate of temporal variability in 166 the abundance of organisms. Quadrats were not permanent and were randomly selected on each sampling occasion so as to guarantee independence of data. The percentage 168 cover of algal species was estimated from 36 uniformly distributed points and expressed as percentage cover. An arbitrary value of 0.5 percentage cover was given to species 170 whose position did not match any of the intersections. Barnacle cover was estimated by giving a score from 0 (absent) to 4 (100% cover) in each of the 25 sub-quadrats (Dethier 172 et al., 1993), while mobile animals (limpets and littorinids) were counted and expressed as density per quadrat. Other animals present (e.g. chitons, whelks) were rare in the 174 sampled areas and, although their presence was recorded, they were not further analysed. Sampling of algae was done at the highest taxonomic resolution achievable in 176 the field. This included the identification of several organisms to species level, although others were identified to genera. Algae were later grouped into distinct 178 morphological/functional groups which included: leathery and corticated erect algae (e.g. Fucoids, Stypocaulon scoparium; hereafter referred to as frondose algae), coarsely 180 branched algae (e.g. Osmundea spp., articulated coralline algae; hereafter referred to as turf-forming algae), filamentous algae (e.g. Ceramium spp.), ephemeral algae (e.g. 182 Enteromorpha (Ulva) spp., Rivularia sp.) and crustose algae (e.g. Nemoderma sp., encrusting coralline algae). 184

The influence of the breakwater on organism recruitment was examined by means of 10 x 10 cm recruitment plates (n = 5 per combination of location and tidal 186 height). The selected locations and shore height were as described above. Plates were preferred over experimental clearings since these constitute suitable colonisation 188 substrates for a variety of organisms (at least in the short-term) and eliminate the influence of non-evident processes that could affect the interpretation of results between 190 the impacted and control locations [START_REF] Chapman | Scales of variation of gastropod densities over 430 multiple spatial scales: comparison of common and rare species[END_REF]. Hence, if recruitment in the impacted location differs from that of control locations, this is most 192 likely due to the presence of the breakwater and consequent changes in the local physical environment (e.g. water circulation). The plates were made of basaltic rock, the 194 natural substratum in the area, perforated in the centre and screwed to the shore with stainless steel screws at least 1 m apart. In order to provide temporal replication, after 196 first deployment in July '07, plates were retrieved and replaced every three months. A total of three sets of plates were thus available for analysis corresponding to the periods 198 of colonisation of July '06 -October '06, October '06 -January '07 and January '07 -April '07. The percentage cover of algae on recruitment plates was done under a dissecting microscope by overlaying a translucent sheet with 50 uniformly-spaced points and recording the species underneath. A space of 1 cm was left between the 202 edges of the plate and the beginning of the quantification points as well as to the central hole (used to attach the plate to the shore) to reduce edge effects. Barnacles were 204 counted and expressed as density per 100 cm 2 . Four sub-samples of 1 cm 2 were used where these were too abundant to be counted and the mean of the 4 sub-samples 206 standardized to 100 cm 2 . Other animals present were either too rare to be analysed (e.g. bryozoans) or were mobile (limpets and littorinids) and thus it was not possible to be 208 sure in ascribing their presence on plates to recruitment.

Plaster discs were used to examine the effect of the breakwater on the 210 hydrodynamics of the adjacent reef. This was done by screwing three replicate plaster discs (~5 m apart) of known weight per site to mid-shore tidal heights. The loss of mass 212 (dry weight) after one high tide was used as a surrogate for water motion or wave action (e.g. [START_REF] Lindegarth | Comparing categorical and continuous ecological 484 analyses: effects of "wave exposure" on rocky shores[END_REF]. Wave action was estimated three times during the 214 study -in July '06, October '06 and April '07. Although the typical large small-scale variation in water-motion suggests that the number of plaster discs used is small, the 216 small size of error bars (see Fig. 6) indicates replication was enough to provide a precise estimate of this variable. We also aimed at quantifying the level of sedimentation by 218 deploying sediment traps in July '07 [START_REF] Airoldi | The effects of sedimentation on rocky coast assemblages. 382[END_REF] but this methodology proved rather unfruitful in the intertidal since the majority of the sediment traps were lost after 5 days, 220 probably due to the high resistance of the containers to wave action. As an alternative, the abundance of sediment was estimated as percentage cover while sampling for 222 mature assemblages in subsequent sampling events. 224

Data analysis

To test for differences in the abundance of the different taxa between the 226 disturbed and control locations, data were analysed using 2-way asymmetrical ANOVAs with time and location as random factors and orthogonal to each other. A 228 general model of the analysis as well as the calculation of the expected mean squares and the consequent determination of denominators for each test was adapted from 230 [START_REF] Terlizzi | Multivariate and univariate asymmetrical analyses in environmental impact assessment: a case study of Mediterranean subtidal sessile assemblages[END_REF] and is presented in Table 1. Generally, the factor location was partitioned into two components: one contrasting the impacted and control locations (I 232 vs. C's) and the other contrasting control locations among each other (C's). This was also true for the time x location interaction and for the residuals associated with 234 variability among quadrats respectively within I and C's (see [START_REF] Terlizzi | Multivariate and univariate asymmetrical analyses in environmental impact assessment: a case study of Mediterranean subtidal sessile assemblages[END_REF] for a detailed description of these procedures). Prior to analysis, Cochran's test was used to 236 check for problems of heteroscedascity and data were transformed where appropriate [START_REF] Underwood | Experiments in ecology: their logical design and interpretation 520 using analysis of variance[END_REF]. Where heterogeneity of variances persisted, data were analysed 238 untransformed but a more conservative P-value was used (Underwood, 1997, Blockley and[START_REF] Blockley | Recruitment determines differences between 408 assemblages on shaded or unshaded seawalls[END_REF]. 240

Results 242

3.1. Mature assemblages Patterns of distribution in the disturbed and control locations were variable among the 244 different taxa examined. As expected, on the lower shore algal dominated assemblage (Fig. 2,Table 2) the cover of frondose algae was lower in the disturbed location and this 246 was consistent in time despite variability among control locations. Crustose algae also occurred at lower abundances in the disturbed location although this was temporally 248 variable. The space released by the reduction in cover of frondose and crustose algae in the disturbed location was occupied by ephemeral algae, which increased in cover although its cover varied with time. Turf-forming and filamentous algae showed no clear variation being similarly distributed across the rocky platform. 252

On the upper animal dominated shore (Fig. 3, Table 3), both the cover of barnacles and the number of limpets were consistently lower in the disturbed location. 254 Again, ephemerals seemed to take advantage from the release in competition and were consistently more abundant in the upper shore of the disturbed location. Although the 256 abundance of littorinids in the disturbed locations tended to be lower than the mean abundance among control locations, this was not statistically significant. 258

Early successional assemblages 260

Recruitment of organisms to lower shore settlement plates was also variable and depended on taxa (Fig. 4, Table 4). Recruitment to panels was dominated by ephemeral 262 species and this was similar in the disturbed and control locations. The colonisation of turf-forming algae was temporally variable among control locations although no clear 264 difference was detected between the disturbed and its overall mean abundance in control locations. In contrast, the abundance of filamentous algae was consistently 266 lower in the disturbed location. The abundance of crustose algae also differed between the disturbed and control locations, but this was variable in time. The barnacle 268

Chthamalus stellatus also recruited to lower shore plates, albeit at lower abundances compared to that recorded on upper shore plates. However, although recruitment tended 270 to be lower in the disturbed location, this was not statistically significant.

On the upper shore recruitment plates (Fig. 5, Table 5), the only discernible algal 272 recruitment was that of ephemeral species, which were present at larger abundances in the disturbed location during most of the study period. Filamentous and crustose algae 274 were sporadically recorded but were too rare to be analysed. In contrast, barnacle recruitment was virtually null in the disturbed location, whilst reaching a mean number 276 of 146  33 (mean  1SE) recruits per plate among control locations during the peak of recruitment. 278

Physical environment 280

As expected, plaster dissolution was 2-fold greater in control sites than in the area sheltered by the breakwater (Fig. 6, Table 6) and this was consistent in time 282 revealing that the breakwater exerts a putative impact on this part of the rocky platform.

Accumulation of sediment occurred lower on the shore in the impacted location trapped 284 inside mats of turf-forming algae although this was temporally variable. A peak of sediment accumulation was recorded in October '06 with a mean percentage cover of 286 19.7 ± 4.4 (mean ± SE), whilst lower abundances were recorded in January '07 (3.9% ± 1.6) and April '07 (6.7% ± 3.7). No sedimentation was recorded on the lower shore of 288 control sites, or on the upper shores of either the disturbed or control locations. 290

Discussion

Generally, the area sheltered by the breakwater supported a structurally and 292 functionally different assemblage than that recorded in control locations and this was especially evident in the upper eulittoral. At this tidal height, ephemeral algae replaced 294 the dominant space occupier, the barnacle Chthamalus stellatus, in the disturbed area and persisted throughout the year. The shift between animal dominated and algal 296 dominated assemblages along a decreasing gradient of wave action is common to temperate regions worldwide even though the processes that structure these 298 communities can differ among locations (see Jenkins et al., 2008 and references therein). In European coasts, much experimental work has convincingly shown that grazing by patellid limpets limits the growth of macroalgae in wave-beaten shores and that their removal leads to a change in dominance between barnacles and mussels, and 302 macroalgae (e.g. [START_REF] Hawkins | The influence of Patella grazing on the fucoid-barnacle mosaic on moderately exposed rocky shores[END_REF][START_REF] Jenkins | Regional scale differences in the determinism of grazing effects in the rocky intertidal[END_REF][START_REF] Arrontes | Effect of grazing by limpets on mid-shore species 390 assemblages in northern Spain[END_REF][START_REF] Coleman | A continental scale evaluation of the role of limpet grazing on rocky 442 shores[END_REF]. Similarly, the reduced abundance of patellid limpet in the sheltered area 304 probably led to the divergence in structure of assemblages found between the disturbed and control locations. Such shift in dominance at the upper eulittoral zone has important 306 ecological and energetic consequences by altering a net consumer ecosystem into one dominated by producers. 308

At the lower algal dominated shore, important differences in the assemblage structure were also clear with ephemeral algae similarly replacing frondose algae in the 310 disturbed location. The hydrodynamic forces generated by the breaking of waves in the intertidal are known to limit the size and survival of larger plants so that these generally 312 become restricted to more sheltered conditions [START_REF] Blanchette | Size and survival of intertidal plants in response to wave action: 406 a case study with Fucus gardneri[END_REF]. However, this was not the case. While our attempts to estimate the intensity of sedimentation were not 314 particularly successful, they do suggest that sediment tended to accumulate in the disturbed area and this is known to negatively affect the colonisation of space by erect 316 algae, while having no discernible effect on the spatial dominance of algal turfs [START_REF] Airoldi | Roles of disturbance, sediment stress, and substratum retention on 378 spatial dominance in algal turf[END_REF]. 318

Significant differences between the disturbed and control locations were also evident during the early successional stages of assemblage development. During the 320 peak of barnacle settlement, these recruited in abundance on upper shore plates in the control locations but not in the sheltered area, where ephemeral algae composed the 322 bulk of the early successional assemblage. In the lower shore, recruitment of filamentous algae was also lower in the disturbed location than on the adjacent exposed 324 controls. As for adult populations, exposure to wave-action is known to influence the recruitment and survival of a number of intertidal animals and plants. For instance, low 326 energy waves were shown to remove up to 99% of recently settled zygotes of Ascophyllum nodosum, and thus explaining its inability to colonise exposed locations 328 [START_REF] Vadas | Recruitment of Ascophyllum nodosum: 526 wave action as a source of mortality[END_REF]. Although populations of the barnacle Chthamalus stellatus predominate on wave-exposed shores (e.g. [START_REF] Jenkins | Larval habitat selection, not larval supply, determines settlement patterns and adult distribution in two chthamalid barnacles[END_REF], their ability to colonise 330 sheltered shores does not seem limited by the abundance of larvae, which actually tend to concentrate on sheltered bays [START_REF] Jenkins | Barnacle larval supply to sheltered rocky shores: a limiting factor?[END_REF], but by post-settlement 332 processes that limit the ability of barnacles to settle [START_REF] Jenkins | Barnacle larval supply to sheltered rocky shores: a limiting factor?[END_REF] or cause the mortality of recently settled animals (e.g. the sweeping of frondose algae, 334 [START_REF] Jenkins | Settlement and post-settlement interactions between Semibalanus balanoides (L.) (Crustacea: Cirripedia) and 474 three species of fucoid canopy algae[END_REF]. In the present study, barnacle settlement on plates in the sheltered location was probably limited by competition for space with ephemeral algae. 336

Alternatively, both the recruitment of barnacles and filamentous algae in the sheltered areas may have been driven by the breakwater-induced variations in water-motion, 338 which likely influence the dispersion of propagules and nutrients [START_REF] Mullineaux | Initial contact, exploration and attachment of barnacle (Balanus amphitrite) cyprids settling in flow[END_REF][START_REF] Abelson | Settlement of marine organisms in flow[END_REF]. 340

Although the non-interspersion of controls and the disturbed location is a potential confounding factor [START_REF] Underwood | On beyond BACI: sampling designs that might reliably detect 518 environmental disturbances[END_REF], the range of responses of individual 342 taxa, or the assemblage as a whole, to the sheltering by the breakwater could easily be interpreted in light of the vast literature examining the influence of wave-action as a 344 structuring process. In addition, the lack of spatial correlation in the abundance of the various taxa among control locations, indicating that no major gradients are affecting 346 the horizontal distribution of organisms on this rocky platform, suggests that results have not been affected by the segregation of controls in relation to the impacted 348 location.

Anecdotal evidence suggests the assemblage that developed as a consequence of the artificial sheltering is similar to that of sheltered shores in warmer-temperate regions 352 [START_REF] Airoldi | Distribution and morphological variation of low-shore algal turfs[END_REF][START_REF] Boaventura | Intertidal rocky shores communities of the continental Portuguese coast: analysis of distribution 412 patterns[END_REF]. However, caution is needed as there is evidence that the impacts of natural and artificial rocky reefs on the structure of marine 354 assemblages can differ, while being similar in the way they may affect the environment (e.g. wave exposure) [START_REF] Barros | The influence of rocky reefs on structure of benthic macrofauna in nearby soft-sediments[END_REF][START_REF] Knott | Epibiota on 480 vertical and on horizontal surfaces on natural reefs and on artificial structures[END_REF][START_REF] Vaselli | Hard coastal-defence structures as habitats for native and exotic rocky-bottom species[END_REF]. 356

Conclusion 358

The present study has shown that the construction of breakwaters can have a strong effect on the structure and functioning of adjacent rocky intertidal communities. 360

The artificial sheltering of naturally exposed shores is itself a strong impact and here resulted in the development of a structurally and functionally divergent assemblage. 362

This study adds to the existing literature by showing that anthropogenic activities such as the construction of artificial structures can have important ecological as well as 364 energetic consequences for the functioning of the local ecosystem.

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Fig. 1 .

 1 Fig. 1. Azores archipelago; Pico island; and the study area showing the built breakwater

  Figure1

  Figure 4

Table 1 .

 1 Asymmetrical analysis of variance comparing the disturbed (I) and control (C's) locations, where: a = times of sampling, b = all locations (the disturbed and 3 controls), n = number of replicates. Both time and location are random and orthogonal with each other. tested over the Residual if T x L and C's can be eliminated from the model; tested over T x L if C's can be eliminated but not T x L; tested over C's if this term cannot be eliminated from the model. These tests assume equivalence among  2

	Source of variation	df	Expected mean square
	Time = T		a-1	 2	e + n 2	TxL + bn 2	T
	Location = L	b-1	 2	e + n 2	TxL + an 2	L
	e I vs. C's	1	 2	e-I + n 2	TxIvs.C's + an 2	L + an 2	Ivs.C's
	d C's	b-2	 2	e-C's + n 2	TxC's + an 2	C's
	c T x L		(a-1)(b-1)	 2	e + n 2	TxL
	b T x I vs. C's	(a-1)	 2	e-I + n 2	TxL + n 2	TxIvs.C's
	a T x C's	(a-1)(b-2)	 2	e-C's + n 2	TxC's
	Residual		ab(n-1)	 2	e
	Res I	a(n-1)	 2	e-I
	Res C's	a(b-1)(n-1)	 2	e-C's
	a tested over Res C's
					T x I vs. C's,  2	T x C's and  2	T x L , and
	among  2	L and  2 C's.
	Adapted from Terlizzi et al., (2005)

b tested over the Residual if T x C's can be eliminated from the model (not significant at  = 0.25,

[START_REF] Underwood | Experiments in ecology: their logical design and interpretation 520 using analysis of variance[END_REF]

; tested over T x C's otherwise. These tests assume equivalence among  2 T x L and  2 T x C's (see

[START_REF] Glasby | Analysing data from post-impact studies using asymmetrical 450 analysis of variance: A case study of epibiota on marinas[END_REF]

). c tested over the Residual d tested over T x C's e

Table 2 .

 2 Asymmetrical analysis of variance comparing the abundance of conspicuous lower shore taxa in the disturbed (I) and control (C's) locations.

			Frondose algae	Turf-forming algae	Crustose algae	Filamentous algae	Ephemeral algae
	Source	df	MS	F	MS	F	MS	F	MS	F	MS	F
	T	3	0.17		1.49E 3		1.72		2.00		2.13
	L	3	45.97		153E 3		0.66		0.12		2.49
	I vs. C's	1	126.77 22.76*	3.55E 2 0.17	1.64		0.11	0.43	7.16
	C's	2	5.57	9.24*	2.11E 3 3.41	0.17	1.76	0.13	0.46	0.15	2.13
	T x L	9	0.44	0.45	1.07E 3 3.01***	0.32	1.80	0.24	1.53	0.45	2.98**
	T x I vs. C's	3	0.12	0.13	1.98E 3 3.20	0.78	4.32**	0.15	0.51	1.22	8.01***
	T x C's	6	0.60	0.46	6.20E 2 1.42	0.10	0.52	0.29	1.68	0.07	0.44
	Residual	144	0.99		3.57E 2		0.18		0.16		0.15
	Res I	36	0.05		1.19E 2		0.17		0.12		0.13
	Res C's	108	1.30		4.36E 2		0.02		0.17		0.16
	Transformation		Ln (x + 1)			x 0.1		x 0.1		x 0.1
	Cochran's test		C = 0.17		C = 0.27**	C = 0.17		C = 0.15		C = 0.13
	Significant terms in bold: * p  0.05, ** p  0.01, *** p  0.001
	E = x10

Table 3 .

 3 Asymmetrical analysis of variance comparing the abundance of conspicuous upper shore taxa in the disturbed (I) and control (C's) locations.

			Barnacles	Limpets		Littorinids	Ephemeral algae
	Source	df	MS	F	MS	F	MS	F	MS	F
	T	3	1.25E 2		0.60		16.99		0.14
	L	3	2.69E 3		1.30		31.93		6.78
	I vs. C's	1	7.80E 3 61.01*	3.54	9.27*	87.94 4.58	19.97 68.73***
	C's	2	1.28E 2 2.17	0.18	0.40	3.93		0.18	0.67
	T x L	9	4.01E 1 0.21	0.38	2.24*	19.21 7.58***	0.29	1.41
	T x I vs. C's	3	2.67E 0 0.01	0.25	0.57	18.58 0.95	0.31	1.53
	T x C's	6	5.88E 1 0.24	0.45	1.99	19.52 6.10***	0.28	1.15
	Residual	144	1.94E 2		0.17		2.53		0.21
	Res I	36	2.83E 1		0.01		0.54		0.10
	Res C's	108	2.50E 2		0.22		3.20		0.24
	Transformation				Ln (x + 1)	Sq-rt		x 0.1
	Cochran's		C = 0.16		C = 0.16		C = 0.15		C = 0.09
	Significant terms in bold: p-values as in Table 2.

Table 4 .

 4 Asymmetrical analysis of variance comparing the abundance of conspicuous lower shore taxa on recruitment plates in the disturbed (I) and control (C's) locations.

			Ephemeral algae	Filamentous algae	Crustose algae	Turf-forming algae	Barnacles
	Source	df	MS	F	MS	F	MS	F	MS	F	MS	F
	T	2	4.50E 2		8.44		1.77E 2		2.10		1.05E 3
	L	3	2.41E 3		2.69		4.42E 2		5.40		5.00E 2
	I vs. C's	1	1.92E 3 0.73	5.83	6.09*	4.80E 2		4.49	0.74	4.54E 2 1.13
	C's	2	2.65E 3 3.99	1.13	0.62	4.23E 2 2.37	5.86	0.75	5.22E 2 1.22
	T x L	6	5.15E 2 1.19	1.23	1.28	6.28E 2 3.02*	6.07	14.08***	4.00E 2 1.81
	T x I vs. C's	2	2.18E 2 0.33	0.06	0.03	1.53E 3 7.34**	2.67	0.34	3.50E 2 0.82
	T x C's	4	6.64E 2 1.59	1.81	1.73	1.79E 2 1.21	7.76	16.04***	4.26E 2 1.44
	Residual	48	4.31E 2		0.96		2.08E 2		0.43		2.21E 2
	Res I's	12	4.74E 2		0.69		3.88E 2		0.27		0
	Res C's	36	4.17E 2		1.05		1.48E 2		0.48		2.95e 2
	Transformation				Ln (x + 1)			Ln (x + 1)
	Cochran's		C = 0.21		C = 0.23		C = 0.43**	C = 0.25		C = 0.67**
	Significant terms in bold: p-values as in Table 2.

Table 5 .

 5 Asymmetrical analysis of variance comparing the abundance of conspicuous upper shore taxa on recruitment plates in the disturbed (I) and control (C's) locations.

			Ephemeral algae	Barnacles
	Source	df	MS	F	MS	F
	T	2	2.31E 3		52.13
	L	3	3.40E 3		20.24
	I vs. C's	1	8.54E 3		55.14
	C's	2	8.30E 2	1.67	2.80	1.48
	T x L	6	8.38E 2	2.43*	5.80	7.81***
	T x I vs. C's 2	1.52E 3	4.41*	13.61	7.17*
	T x C's	4	4.97E 2	1.15	1.90	2.03
	Residual	48	3.45E 2		0.74
	Res I	12	9.04E 1		0.17
	Res C's	36	4.30E 2		0.93
	Transformation				Ln (x + 1)
	Cochran's		C = 0.22		C = 0.26

Significant terms in bold: p-values as in Table

2

.

Table 6 .

 6 Asymmetrical analysis of variance comparing plaster dissolution (g of dry weight) in the disturbed (I) and control (C's) locations.

	Source	df	MS	F
	T	2	0.30
	L	3	76.89
	I vs. C's	1	222.17	61.17***
	C's	2	4.26	1.26
	T x L	6	2.28	0.63
	T x I vs. C's 2	0.09	0.02
	T x C's	4	3.38	0.54
	Residual	24	3.63
	Res I	6	1.92
	Res C's	12	6.31
	Transformation
	Cochran's		C = 0.36

Significant terms in bold: p-values as in Table

2

.

Acknowledgements

The present study was supported by Secretaria Regional do Ambiente e do Mar 368 and Universidade dos Açores. The Câmara Municipal das Lajes do Pico and Escola Básica Integrada e Secundária das Lajes do Pico provided logistic support. G.M. 370 Martins and F.M. Wallenstein were supported by PhD grants from FCT (SFRH/BD/22009/2005 and SFRH/BD/27466/2006, respectively). A.F. Amaral was 372

supported by Secretaria Regional do Ambiente e do Mar.