1 “Effects of the ‘Prestige’ oil spill on macroalgal assemblages: large-scale 2 comparison” 3 Carla Lobón1, Consolación Fernández, Julio Arrontes, José Manuel Rico, José Luis Acuña, 4 Ricardo Anadón, Augusto Monteoliva 5 Departamento de Biología de Organismos y Sistemas, Unidad de Ecología, Universidad de Oviedo. C/ 6 Catedrático Rodrigo Uría s/n 33071 Oviedo, Spain. 7 1Corresponding 8 ABSTRACT 9 An assessment of the effects of the ‘Prestige’ oil spill on intertidal, macroalgal 10 assemblages was carried out comparing abundance data obtained before and after the 11 spill. Three zones in the North and Northwest coast of Spain were sampled, one of them 12 located at the immediate vicinity of the spill, the zone most heavily oiled. Macroalgal 13 assemblages had similar structure between years. Neither critical decrease in abundance 14 of the dominant macroalgae, nor increase in opportunistic species was found. Some 15 differences in abundance were found, but they did not show any pattern, being more 16 likely the result of the natural variability of the ecosystem. Extensive, but not intense 17 fuel deposition on the shores, and a limited use of aggressive cleanup methods are 18 suggested as possible causes for the lack of the effects in these assemblages after the 19 ‘Prestige’ oil spill. 20 Keywords: ‘Prestige’ oil spill; macroalgal assemblages; community structure; 21 species distribution; pollution; North of Spain. author: e-mail: carlalobon.uo@uniovi.es, fax: (+34) 98 510 4777 22 1 23 1. Introduction 24 In November 2002, the ‘Prestige’ oil tanker sank at ca. 130 nautical miles off the 25 Galician Coast, carrying more than 77000 tons of heavy fuel oil M-100. This fuel is 26 characterized by its low solubility and volatility, which makes it very persistent over 27 time (Markarian et al., 1993). After the wreckage, the ‘Prestige’ released more than 28 10000 tons of fuel oil, which were carried by prevailing winds and ocean currents and 29 reached extensive areas of the Cantabrian Coast, N of Spain (Montero et al., 2003; 30 García-Soto, 2004; Acuña et al., submitted). After sinking, the ‘Prestige’ gradually 31 released the rest of its fuel during ca. 4 months, causing a series of oil waves, which 32 mainly affected the Galician Coast, NW of Spain (Acuña et al., submitted). Except for 33 most oiled areas located at the "Costa da Morte" (Galicia, Fig. 1), oil deposition on the 34 Atlantic and Cantabrian Spanish coasts was extensive, but not very intense, affecting 35 mainly a 100 Km coastal zone E of Cape Peñas (Acuña et al., submitted; Fig. 1). 36 To date, moderate to negligible effects of the ‘Prestige’ oil spill have been documented 37 on benthic (Serrano et al., 2006) and planktonic (Varela et al., 2006; Bode et al., 2006; 38 Salas et al., 2006) communities, and on cell and tissue condition biomarkers in mussel, 39 hake and anchovy (Marigómez et al., 2006). This is consistent with low fuel contents 40 measured 41 http://www.ieo.es/prestige/resultados.htm) and in the water column off the Cantabrian 42 coast (IEO http://www.ieo.es/prestige/resultados.htm; González et al., 2006). Therefore, 43 the most likely target to detect any significant ecological effect should be the shoreline, 44 where much of the spilled fuel was deposited (Acuña et al., submitted). during the winter 2002-2003 in shelf sediments (IEO 2 45 Proper assessment of the ecological effects of disturbances requires baseline time-series 46 studies, documenting the situation before the impact (e.g. for application of a BACI 47 sampling design, Underwood 1992). Information of the natural variability of the 48 ecosystem is essential to differentiate and evaluate the effects of anthropogenic impacts. 49 In spite of the remarkable recurrence of oil spills in certain areas, including the Galician 50 coast, baseline studies with adequate replication for hypothesis testing are rarely 51 available (Teal and Howarth, 1984). During 2000 and 2002, right before the ‘Prestige’ 52 oil spill, we conducted a survey to characterize the structure of macroalgal assemblages 53 at the mid and low rocky intertidal in the northern Spanish coast (Galicia, Asturias and 54 Basque Country, Fig. 1). Hierarchical sampling allowed us to partition variance in 55 community structure among different spatial scales (from kilometers to meters). After 56 the ‘Prestige’ disaster we decided to repeat the same sampling program to evaluate the 57 effects of oil spill on these assemblages. Coincidentally, our study included locations 58 from the most affected area in Galicia to the eastern affected coast in the Basque 59 Country. Although these data lacked temporal replication before and after the spill, they 60 allowed us to test of hypothesis concerning differences in macroalgal community 61 structure after the ‘Prestige’ oil spill. 62 2. Materials and methods 63 2.1. Sampling 64 We sampled 12 localities along Galicia (Muxía, Lobeiras, and Lobadiz), West Asturias 65 (Novellana, Artedo, and Aramar), East Asturias (Rodiles, La Griega, and Vidiago), and 66 the Basque Country (Sakoneta, Zumaia, and Igeldo) coasts (Fig. 1). West and East 67 Asturias localities were sampled in August 2000 and 2003, and Galicia and Basque 3 68 Country localities in September 2002 and 2003. Six sites were randomly chosen at each 69 locality, three at "lower" (between 0.4 and 0.7 m above the Lowest Astronomical Tide) 70 and three at "upper" (between 0.9 and 1.3 m) intertidal zone. One 15 m transect parallel 71 to the coastline was sampled at each site. Five, 50x50 cm quadrats were randomly 72 placed in each transect and photographed. Algae were identified to species, or assigned 73 to higher taxonomic categories when species identification was not possible (e.g. Order 74 Ceramiales). Abundance (as percentage cover) of each taxon was estimated in the 75 laboratory by the point-contact method (Hawkins and Jones, 1992). A grid of 100 76 regularly spaced points was superimposed over the digitized pictures of the quadrats 77 and interceptions for each taxon were counted. This technique reduces sampling time at 78 the low intertidal level, but overestimates canopy species in multilayered assemblages 79 (Meese and Tomich, 1992; Dethier et al., 1993). 80 2.2 Data analysis 81 Percentage cover data were analysed using both univariate and multivariate techniques. 82 Spatio-temporal differences of most abundant taxa from each intertidal level 83 (comprising more than 75% of total cover) were analyzed. These taxa were Fucus spp. 84 (including Fucus spiralis L. and Fucus vesiculosus L., difficult to distinguish on 85 photographs), Fucus serratus L., Mastocarpus stellatus (Stack.) Guiry, Corallina 86 elongata J. Ellis & Sol., Ralfsia verrucosa Aresch., and Ceramiales (mainly Ceramium 87 spp. and Callithamnium spp.) for the "upper" level; and Bifurcaria bifurcata R. Ross, 88 Himanthalia elongata (L.) S.F. Gray, C. elongata, Ceramiales, Stypocaulon scoparium 89 (L.) Kütz., and Cladostephus spongiosus (Hudson) C. Agardh for the "lower" level. In 90 addition, species of Ulva spp. were included due to their importance as colonizers on 91 open substrata. Hypothesis of absence of spatio-temporal differences was tested using 4 92 four-way univariate analysis of variance (ANOVA). Separate analyses were done for 93 high and low tidal levels. “Time” (fixed, with 2 levels, Before and After spillage), 94 “Zone” (fixed, with 4 levels, GAL (Galicia), ASTw (West Asturias), ASTe (East 95 Asturias), and BC (Basque Country) coasts), “Locality” (random and nested in Zone, 96 with 3 levels), and “Site” (random and nested in the “Time x Locality (Zone)” 97 interaction, with 3 levels) were the factors considered in the Corallina elongata, 98 Ceramiales, Ralfsia verrucosa, and Ulva spp. analyses. A different analysis, using three 99 levels for factor Zone was applied for Fucus spp., and Mastocarpus stellatus, because 100 they did not appear in the Basque Country, and for Stypocaulon scoparium, and 101 Cladostephus spongiosus, since they were not present in samples from Galicia. For 102 Himanthalia elongata, only present in Galicia and West Asturias, factor Zone had two 103 levels. Variances were homogeneous (Cochran’s test, P > 0.05) for Bifurcaria bifurcata 104 and, after arcsin transformation for Stypocaulon scoparium, and ln (x + 1) 105 transformation for the rest of the taxa. Despite heterogeneity of variances for Fucus 106 spp., Himanthalia elongata, and Corallina elongata, ANOVA was used because of its 107 robustness and its validity in case of non-significant results in large and balanced 108 designs (Underwood, 1997). When significant Time x Zone interactions were found, 109 Student-Newman-Keuls (SNK) a posteriori test was applied. 110 Multivariate analyses were used to examine changes in the community structure before 111 and after the oil spill. Percentage cover estimates of all taxa (30 and 29 in the "upper" 112 and "lower" levels respectively) were included in the analyses. A matrix of similarities 113 between each pair of samples was calculated using the Bray-Curtis similarity coefficient 114 (Bray and Curtis, 1957). Several non-metric multivariate approaches were utilized. Null 115 hypothesis of no differences in macroalgal assemblages among Times and Zones were 5 116 tested using two-way crossed analysis of similarities (ANOSIM, Clarke 1993). A 117 maximum of two factors can be included in ANOSIM, and they were Time (2 levels) 118 and Zone (4 levels). The R-statistic generated by ANOSIM ranges from –1 to 1; the 119 higher the absolute value is, the greater the dissimilarity between macroalgal 120 communities. In addition, multiple ANOSIMs for one-factor (Time) were done within 121 each level of the other factor (Zone). 122 Hierarchical clustering analysis (CLUSTER) and multidimensional scaling (MDS) were 123 used to define similarities between the macroalgal assemblages of each Zone and Time. 124 A similarity percentage analysis (SIMPER) was used to determine the contribution of 125 each taxa to Bray-Curtis dissimilarities between groups obtained from CLUSTER and 126 MDS. Multivariate analyses were performed with PRIMER statistical software package 127 (Clarke and Warwick, 1994), and univariate analyses with GMAV5 for Windows 128 (Underwood et al., 1998). 129 3. Results 130 3.1. Abundance of dominant taxa 131 Abundance of main taxa in the two tidal levels did not change in the studied zones after 132 the oil spill (Figs. 2 and 3). A west-east gradual variation of dominant taxa was 133 observed. The "upper" level was dominated by Fucaceae, mainly Fucus spiralis and 134 Fucus vesiculosus, in GAL and AST, and by Corallina elongata and Ceramiales in BC 135 (Fig. 2). Mastocarpus stellatus, abundant in GAL, gradually decreased to the east. 136 Ralfsia verrucosa, and Ulva spp. (Fig. 4) showed very low percentages in all of the 137 samples and, in most cases, Ulva spp. were epiphytes. The Time x Zone interaction was 138 significant for Ulva spp. only (Table 1A), being significantly more abundant in pre-spill 6 139 Basque Country samples than in the other Time x Zone combinations (SNK, Table 1B). 140 In agreement with the alongshore-spatial patterns mentioned above, Ceramiales, and 141 Corallina elongata were more abundant in the Basque Country (significant Zone effect 142 in ANOVA). Although significant Zone differences in Fucus spp., and Mastocarpus 143 stellatus abundance were expected (Fig. 2), ANOVA failed to detect them; possibly due 144 to the large locality effect. Abundance of Ralfsia verrucosa did not vary spatially but it 145 was large before the oil spill (significant Time effect). Fucus serratus, appeared just in 146 one West Asturias locality, where it was dominant, and decreased significantly from 147 2000 to 2003 (ANOVA, F = 17.33, p < 0.01). The Time x Locality interaction was 148 significant for C. elongata and Ceramiales, indicating differences in some localities 149 without a clear pattern and not reflected at Zone level. 150 Similar results were found at the “lower” level, where dominant taxa were Himanthalia 151 elongata in GAL, Bifurcaria bifurcata in AST, and Corallina elongata and Stypocaulon 152 scoparium in BC (Fig. 3). No significant Time x Zone interactions were detected (Table 153 2). Only Bifurcaria bifurcata, and Corallina elongata abundances showed significant 154 differences between Zones. Ceramiales increased slightly but significantly after the 155 ‘Prestige’ oil spill. Ulva spp. did not vary significantly in Zones between Times of 156 sampling (Fig. 4, Table 2). Time x Locality interaction was significant for Ceramiales 157 and Ulva spp., which increased or decreased significantly in some localities but any 158 pattern could be observed. 159 3.2. Assemblage structure 160 Significant differences in the structure of macroalgal assemblages structure in the 161 “upper” intertidal level between Times and Zones were detected by ANOSIM (Table 7 162 3A). However, minimal differences can be detected by ANOSIM when a large number 163 of replicates are included. In this case (n = 45), despite the statistical significance, we 164 examined the R statistic values to determine the degree of dissimilarity (R values near 165 to unity) or similarity (R values close to cero) between the communities compared 166 (Clarke and Warwick, 1994). The R value for Time comparison was close to zero 167 (0.081), in contrast with higher R values for Zone comparisons (Table 3A). This 168 indicates that assemblages after the ‘Prestige’ oil spill were very similar to those before, 169 but there was a great dissimilarity between zones. Analyzing each Zone separately 170 (Table 3B), differences between years were detected for ASTw, ASTe, and BC. 171 Statistical significance was again affected by the number of replicates (n = 15), and the 172 R values very close to zero indicated a similar macroalgal structure after ‘Prestige’ oil 173 spill. 174 Samples were clearly grouped according to their spatial distribution along the west-east 175 gradient, not by sampling date, in both classification analyses. The CLUSTER grouped 176 pre-spill and post-spill samples of the same zone, resulting in four groups coincident 177 with the levels of factor Zone (Fig. 5A). Same results showed MDS ordination, being 178 samples of the same zone (Before and After Prestige) closer to each other than samples 179 of different zones (Fig. 5B). This indicates that communities at each Zone were similar 180 between years. Main taxa responsible of the dissimilarity between Zones were Fucus 181 spp., Mastocarpus stellatus, and Corallina elongata (SIMPER, Table 4). The GAL 182 samples were characterized by greater abundance of Fucus spp., and M. stellatus, 183 whereas C. elongata was more abundant in BC samples. ASTw differed from ASTe by 184 the presence of Fucus serratus, and the lower abundance of Fucus spp. 8 185 Similar results were found in the “lower” level, significant differences in the macroalgal 186 assemblage structure between Zones and Times were detected by ANOSIM (Table 5B). 187 However, the R value (0.081) for Time comparison indicates that communities were 188 similar between years. Likewise, although we found significant differences between 189 years for GAL, ASTw, and ASTe, the R values indicated a similar macroalgal 190 assemblage structure (Table 5B). 191 Both, CLUSTER and MDS ordination grouped samples according to the levels of factor 192 Zone. Pre-spill and post-spill samples of the same Zone were not separate, indicating a 193 similar macroalgal structure (Fig. 6). The SIMPER analysis (Table 6) revealed that 194 Bifurcaria bifurcata, Himanthalia elongata, and Corallina elongata were the main 195 contributors to Zone differences. Himanthalia elongata explained most of the 196 dissimilarity between GAL and the other Zones. Corallina elongata and, secondly, 197 Stypocaulon scoparium, were the most important macroalgae in BC. ASTw 198 communities were characterized by the presence of Himanthalia elongata, and a higher 199 abundance of Bifurcaria bifurcata than in ASTe communities, where Stypocaulon 200 scoparium was very abundant. 201 4. Discussion 202 Along the coast of northern Spain, from West to East, there is a gradual replacement of 203 cold-temperate species (Fucus spp., Himanthalia elongata, Mastocarpus stellatus, and 204 Bifurcaria bifurcata) by warm-temperate ones (Corallina elongata, Stypocaulon 205 scoparium, and Cladostephus spongiosus). This transition has been previously 206 documented (Fischer-Piette, 1957; Anadón and Niell, 1981; Anadón, 1983; Arrontes 207 1993), and is probably related to a summer upwelling centred on the westernmost Spain, 9 208 Galician coast (Fraga et al., 1982; Botas et al., 1992), which influence decreases 209 towards the East. 210 (Topinka and Tucker, 1981; Hawkins and Southward, 1992; Peterson, 2001; Peterson 211 et al., 2003). But none of these effects were observed. No relevant changes were found 212 neither in the structure of the assemblage nor in the biomass of dominant macroalgal 213 species. 214 The structure of the studied assemblages did not change noticeably (the same dominant 215 macroalgae and a similar number of species). Some differences in macroalgal 216 abundance were found in various localities; however they did not exhibit a clear spatial 217 trend. Some taxa increase in ones and decrease in others. Although lacking a time series 218 prior to the oil spill, the magnitude of the changes in abundance of some macroalgae 219 found in some localities could be considered within the range of natural variability, and 220 these changes would render statistical significant differences between years (see 221 Mathieson et al. 1976 for an example in Fucaceae). 222 When localities were grouped by zones, no significant reduction of the main macroalgae 223 after the ‘Prestige’ oil spill was found. Only Ulva spp. in the “upper” intertidal level of 224 the Basque Country decreased significantly after the disaster. However, this trend is 225 opposite to the expected result, because these opportunistic algae rapidly colonize the 226 newly available surface after the removal of the canopy-forming algae by intense oil 227 deposition (Houghton et al., 1996; Southward and Southward, 1978; Floc’h and Diouris, 228 1980; and Kingston et al., 1997, respectively) but no destruction of the algal canopy was 229 observed in any of the localities of this study. 10 230 What may be the reason for the lack of significant effects of the ‘Prestige’ oil spill on 231 macroalgal assemblages? The degree and persistence of damage from oil spills depends 232 on several factors like type of fuel, quantity and duration of the spill, oceanographic and 233 meteorological conditions, and the type of clean-up treatments used (Clark and Finley, 234 1977). In this case, the tanker sank carrying most of its cargo far from the coast, 235 releasing the fuel in several pulses which impacted a large area of the coast. This 236 generated extensive but not intense fuel deposition (Acuña et al., submitted) but the 237 most likely causes for the absence of severe impacts were fuel dilution due to intense 238 winter mixing and advection during the wreckage period (García-Soto, 2004; Acuña et 239 al., submitted) and limited use of aggressive cleanup methods, that sometimes cause 240 more damage to organisms than fuel itself, delaying recovery of the ecosystem for 241 several years (Southward and Southward, 1978; Houghton et al., 1996). 242 Acknowledgements 243 We would like to thank D. Álvarez, I. Martínez, J.L. Menéndez, J. Oliveros, I. Sánchez, 244 and L. S. Pato, for their assistance during sampling. We also thank M. López-Álvarez 245 and I. Sánchez for their comments on an early version of this paper. Financial support 246 during the first stage of the study came from the Spanish Ministry of Education and 247 Science (CICYT Project MAR1999-1162). Carla Lobón was financially supported by a 248 fellowship from the Ministry of Education for the improvement of higher education 249 (MEC-03-EA2003-0066). 250 References 11 251 Acuña, J.L., Anadón, R., Puente, A., Fernández, C., Vera, M.L., Rico, J.L., Arrontes, J., 252 & Juanes, J, submitted. Large scale fuel deposition patterns in northern Spanish shores 253 following the ‘Prestige's’ oil spill. 254 Anadón, R. 1983. Zonación en la costa asturiana: variación longitudinal de las 255 comunidades de macrófitos en diferentes niveles de marea. Investigación Pesquera 47, 256 125-141. 257 258 259 260 Anadón, R., & Niell, F.X. 1981. Distribución longitudinal de macrófitos en la costa asturiana (N de España). Investigación Pesquera 45, 143-156. Arrontes, J. 1993. Nature of the distributional boundary of Fucus serratus on the north shore of Spain. 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GMAV5 for Windows. 351 Institute of Marine Ecology, University of Sydney, Australia. 352 Varela, M., Bode, A., Lorenzo, J., Álvarez-Ossorio, M.T., Miranda, , A., Patrocinio, T., 353 Anadón, R., Viesca, L., Rodríguez, N., Valdés, L., et al. 2006. The effect of the 354 ‘Prestige’ oil spill on the plankton of the N–NW Spanish coast. Marine Pollution 355 Bulletin 53, 272-286. 356 16 357 FIGURES 358 Figure 1. Geographic situation of sampling localities in the study area (N and NW 359 Spanish coast). The line shows the ‘Prestige’ course since it started to leak oil on 360 November 13th () until the sinking on 19th (). 1 = Muxía; 2 = Lobeiras; 3 = Lobadiz; 361 4 = Novellana; 5 = Artedo; 6 = Aramar; 7 = Rodiles; 8 = La Griega; 9 = Vidiago; 10 = 362 Sakoneta; 11 = Zumaia; 12 = Igeldo. 363 Figure 2. Abundance (% cover, mean SE) of the most abundant taxa in the high 364 intertidal level in the four zones sampled before (light grey bar) and after (dark grey 365 bar) ‘Prestige’ oil spill. GAL = Galicia; ASTw = West Asturias; ASTe = East Asturias; 366 BC = Basque Country. 367 Figure 3. Abundance (% cover, mean SE) of the most abundant taxa in the low 368 intertidal level in the four zones sampled before (light grey bar) and after (dark grey 369 bar) ‘Prestige’ oil spill (abbreviations as in Fig. 2). 370 Figure 4. Abundance (% cover, mean SE) of Ulva spp. in the four zones sampled 371 before (light grey bar) and after (dark grey bar) ‘Prestige’ oil spill (abbreviations as in 372 Fig. 2). 373 Figure 5. (A) MDS ordination plot and (B) dendrogram of CLUSTER analysis of the 374 macroalgal assemblages of the “upper” intertidal level in each Zone and Time 375 (abbreviations as in Fig. 2; * samples taken after Prestige). 376 Figure 6. (A) MDS ordination plot and (B) dendrogram of CLUSTER analysis of the 377 macroalgal assemblages of the “lower” intertidal level in each Zone and Time 378 (abbreviations as in Fig. 2; * samples taken after Prestige). 17 379 Figure 1. Lobón et al. 380 381 18 382 Figure 2. Lobón et al. 383 384 19 385 Figure 3. Lobón et al. 386 387 20 388 Figure 4. Lobón et al. 389 390 21 391 Figure 5. Lobón et al. 392 393 22 394 Figure 6. Lobón et al. 395 396 23 397 Table 1. Summary of the four-way ANOVA (A) results of taxa abundance in the 398 “upper” intertidal level (n = 5) showing F-values. In order to obtain homogeneity of 399 variances, variables were ln(X+1) transformed. Variances were still heterogeneous for 400 Fucus spp. (Cochran’s test, P < 0.05). SNK tests (B) of the significant Time x Zone 401 interaction (GAL = Galicia; ASTw = West Asturias; ASTe = East Asturias; BC = 402 Basque Country). *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant. A. ANOVA Source of variation Time = T Zone = Z TxZ Locality = L(Z) T x L(Z) Site = S(T x L(Z)) Residual Transformation Cochran's Test F df F versus 1 T x L(Z) 2 L(Z) 2 T x L(Z) 6 S(T x L(Z)) 6 S(T x L(Z)) 36 Residual 216 Fucus spp. M. stellatus ns 2,51 0,96ns 0,72ns 12,27*** 2,67ns 3,14ns 0,97ns 10,66*** 1,73ns 4,38*** 1,21ns 2,97*** none 0,128* ln(x+1) 0,095ns F Source of variation Time = T Zone = Z TxZ Locality = L(Z) T x L(Z) Site = S(T x L(Z)) Residual Transformation Cochran's Test 403 df F versus C. elongata Ceramiales R. verrucosa Ulvaceae 8,2* 1 T x L(Z) 0,05ns 0,13ns 0,0ns ns ns 3 L(Z) 4,56* 2,12 3,97 3,2ns 3 T x L(Z) 4,4* 2,26ns 0,35ns 0,14ns 7,45*** 8 S(T x L(Z)) 29,8*** 10,08*** 1,72ns 8 S(T x L(Z)) 3,83** 4,26*** 2,35* 2,08ns 2,14*** 48 Residual 4,39*** 4,78*** 4,45*** 288 ln(x+1) none ln(x+1) ln(x+1) 0,065ns 0,076ns 0,069ns 0,059ns B. SNK of "Time x Zone" interaction in Ulvaceae Time Before GAL = ASTw = ASTe < BC Zone GAL Before = After After GAL = ASTw = ASTe = BC ATSw Before = After ASTe Before = After BC Before > After 404 24 405 Table 2. Summary of the four-way ANOVA results of taxa cover in the “lower” 406 intertidal level (n = 5) showing F-values. In order to obtain homogeneity of variances, 407 C. spongiosus, Ceramiales, and Ulva spp. were ln(x + 1), whereas S. scoparium was 408 arcsin(%) transformed. Variances were still heterogeneous for H. elongata, and C. 409 elongata (Cochran’s test, P < 0.01). *P < 0.05; **P < 0.01; ***P < 0.001; 410 significant. ANOVA F Source of variation df F versus H.elongata Time = T T x L(Z) 2,87ns Zone = Z L(Z) 0,52ns TxZ T x L(Z) 0,01ns Locality = L(Z) S(T x L(Z)) 20,67*** T x L(Z) S(T x L(Z)) 1,46ns Site = S(T x L(Z)) Residual 3,83*** Residual 144 Transformation none Cochran's Test 0,122** 411 Source of variation df F versus Ceramiales Time = T T x L(Z) 7,31* Zone = Z L(Z) 2,53ns TxZ T x L(Z) 0,8ns Locality = L(Z) S(T x L(Z)) 6,48*** T x L(Z) S(T x L(Z)) 3,04** Site = S(T x L(Z)) Residual 3,7*** Residual 288 Transformation ln(x+1) Cochran's Test 0,05ns ns not F df S.scoparium C.spongiosus 1 0,95ns 4,37ns ns 2 1,49 5,13ns 2 3,11ns 0,21ns 9,37*** 3,28* 6 ns 6 1,28 0,41ns 11,24*** 4,6*** 36 216 arcsin(%) ln(x+1) ns 0,088 0,072ns F B.bifurcata C.elongata Ulvaceae 0,02ns 5,14* 0,13ns 4,35* 0,64ns 4,38*** 1,17ns 21,6*** 1,57ns 4,68*** 1,85ns 1,68** none none 0,208** 0,07 ns 1,92ns 3,33ns 3,06ns 2,7* 2,76* 4,59*** ln(x+1) 0,074ns 412 25 413 Table 3. Two-way ANOSIM (A) testing differences in the “upper” intertidal level 414 macroalgal assemblages among Times and Zones. The Bonferroni adjusted probability 415 of type I error for multiple comparisons in the pairwise test was /6 = 0.008. ANOSIM 416 (B) for one factor, Time, was done within each level of the other factor, Zone 417 (abbreviations as in Table 1). In all analyses, 999 permutations, a random sample from a 418 large number, were used. A. Two-way crossed ANOSIM Global Test Time R 0,081 P 0,001 Global Test Pairwise Test ASTw, ASTe ASTw, BC ASTw, GAL ASTe, BC ASTe, GAL BC, GAL B. One-way ANOSIM Time Global Test - GAL - ASTw Time Global Test - ASTe Time Global Test Time Global Test - BC 0,454 0,001 0,221 0,354 0,26 0,746 0,267 0,899 R 0,014 0,073 0,156 0,072 0,001 0,001 0,001 0,001 0,001 0,001 P 0,139 0,007 0,001 0,005 Zone 419 420 26 421 Table 4. Summary of SIMPER analysis results of the “upper” intertidal level macroalgal 422 contributions to dissimilarity between the four Zones (abbreviations as in Table 1) 423 obtained by CLUSTER and MDS techniques. X = average abundance, = average 424 dissimilarity between groups, SD = standard deviation of the , % = percentage 425 contribution of each taxon to the overall dissimilarity between two groups. Only main 426 contributors to the overall dissimilarity between groups are shown. GAL-ASTw = 77,4 Taxa Fucus spp. M. stellatus C. elongata F. serratus Ceramiales XGAL XASTw /SD 51,5 19,9 1,41 27,3 10,3 1,09 3,9 16,6 0,76 0,0 19,2 0,61 1,4 8,9 0,63 % 29,8 17,7 11,0 12,4 6,0 ASTw-ASTe = 75,55 % 30,8 20,5 4,9 6,1 ASTw-BC =75,3 GAL-BC = 91,2 XGAL XBC 51,5 27,3 3,9 1,4 0,0 0,0 44,9 22,1 /SD 1,46 1,03 1,87 1,57 % 28,2 15,0 22,7 11,5 ASTe-BC = 79,3 XASTw XASTe /SD % XASTw XBC /SD % XASTe Fucus spp. 19,9 44,7 1,33 27,5 19,9 0,0 0,65 13,2 44,7 M. stellatus 10,3 1,7 0,56 7,2 10,3 0,0 0,51 6,8 C. elongata 16,6 5,5 0,8 11,4 16,6 44,9 1,66 21,5 5,5 F. serratus 19,2 0,0 0,61 12,7 19,2 0,0 0,61 12,8 Ceramiales 8,9 8,0 0,9 8,0 8,9 22,1 0,88 6,1 8,0 Bold text indicates best discriminating taxon between groups (major /SD ratio). Taxa 427 GAL-ASTe = 65,2 XGAL XASTe /SD 51,5 44,7 1,39 27,3 1,7 1,04 3,9 5,5 0,96 1,4 8,0 0,88 XBC 0,0 44,9 22,1 /SD 1,32 1,81 1,4 % 28,2 25,1 11,1 428 27 429 Table 5. Two-way ANOSIM (A) testing differences in “lower” intertidal level 430 macroalgal assemblages among Times and Zones. The Bonferroni adjusted probability 431 of type I error for multiple comparisons in the pairwise test was /6 = 0.008. ANOSIM 432 (B) for one factor, Time, was done within each level of the other factor, Zone 433 (abbreviations as in Table 1). In all analyses, 999 permutations, a random sample from a 434 large number, were used. A. Two-way crossed ANOSIM Global Test Time R 0,081 P 0,001 Global Test Pairwise Test ASTw, ASTe ASTw, BC ASTw, GAL ASTe, BC ASTe, GAL BC, GAL B. One-way ANOSIM Time Global Test - GAL - ASTw Time Global Test Time Global Test - ASTe Time Global Test - BC 0,503 0,001 0,243 0,613 0,374 0,472 0,682 0,696 R 0,06 0,08 0,171 0,018 0,001 0,001 0,001 0,001 0,001 0,001 P 0,011 0,007 0,001 0,11 Zone 435 436 28 437 Table 6. Summary of SIMPER analysis results of “lower” intertidal level macroalgal 438 contributions to dissimilarity between the four zones (abbreviations as in Table 1) 439 obtained by CLUSTER and MDS techniques. X = average abundance, = average 440 dissimilarity between groups, SD = standard deviation of the , % = percentage 441 contribution of each taxon to the overall dissimilarity between two groups. Only main 442 contributors to the overall dissimilarity between groups are shown. Taxa GAL-ASTw = 76,9 XGAL XASTw /SD % GAL-ASTe = 85,9 XGAL XASTe /SD % GAL-BC = 86,2 XGAL XBC /SD % B. bifurcata H. elongata C. elongata Ceramiales S. scoparium 11,7 38,9 9,6 2,3 - 11,7 38,9 9,6 2,3 0,3 11,7 38,9 9,6 2,3 0,3 Taxa 443 48,3 20,4 4,0 4,6 - 1,47 28,2 1,25 26,1 1,08 5,9 0,81 3,3 - ASTw-ASTe = 68,9 31,1 0,0 5,3 7,1 19,1 1,19 17,6 1,16 22,6 1,07 5,0 1,04 4,0 1,05 11,0 ASTw-BC =84,9 7,3 0,0 34,2 9,6 26,7 0,71 9,2 1,16 22,6 1,17 16,1 1,17 5,0 0,92 15,4 ASTe-BC = 73,5 XASTw XASTe /SD % XASTw XBC /SD % XASTe B. bifurcata 48,3 31,1 1,42 26,7 48,3 7,3 1,49 26,5 31,1 H. elongata 20,4 0,0 0,59 14,8 20,4 0,0 0,59 12,0 C. elongata 4,0 5,3 0,83 4,2 4,0 34,2 1,25 18,4 5,3 Ceramiales 4,6 7,1 1,09 5,5 4,6 9,6 1,19 5,1 7,1 S. scoparium 3,7 19,1 1,02 12,8 3,7 26,7 0,96 15,3 19,1 Bold text indicates best discriminating taxa between groups (major /SD ratio). XBC 7,3 34,2 9,6 26,7 /SD 1,18 1,21 0,66 1,19 % 20,7 20,5 6,3 18,2 444 29