Changes in Coastal Benthic Algae Succession Trajectories and Assemblages Under Contrasting Nutrient and Grazer Loads

Eutrophication plays a crucial role in coastal systems, driving changes in the composition and abundance of flora and fauna with consequent effects for the entire ecosystem. Sensitive to nutrient levels, micro- and macroalgal blooms serve as valuable indicators of eutrophication. The San Antonio Bay (Northern Argentinean Patagonia, 40° 43′ S, 64° 56′ W) provides an appropriate system to study in situ eutrophication processes on coastal communities. In a multi-scale approach, using two different kind of settlement substrates (micro: polyethylene terephthalate, and macro: ceramic), the present study followed benthic algal dynamics over one year, distinguishing changes in natural succession and seasonality. Strong differences were found in the biofilm assemblages after three days, marked by tube dwelling diatoms and Cocconeis spp. under high nutrient-grazer conditions and needle like diatoms (e.g. Nitzschia spp., Tabularia spp.) under lower nutrient-grazer loads. The succession continued by the colonization of macroalgae, with a higher recruitment rate in the nutrient and grazer rich environment with a concomitant higher diversity. Our results show that under higher nutrient-grazer conditions natural benthic succession not only differs in trajectory but in its final taxa composition promoting higher biodiversity and biomass accumulation. In addition, taxa specific substrate preferences interfere with the observed eutrophication pattern, suggesting substrate dependant interrelations between the bloom forming taxa. These findings provide evidence that nutrient enrichment can not only affect an established assemblage but also affect the early succession stages, changing the succession trajectory and thus the final assemblage.


Introduction
Human activities often contribute to increased nutrient loads in coastal waters, promoting elevated growth of primary producers, leading to eutrophication close to populated or agricultural areas (e.g.Cloern 2001;Valiela et al. 1997).The process of eutrophication typically starts with a pulse of nutrients, followed by a bloom of primary producers such as phytoplankton and benthic algae (Nixon 1995), which modify the entire coastal ecosystem (Duarte 1995).Benthic algae react to variations in nutrient levels (Teichberg et al. 2008) by changing their composition and abundance (Worm et al. 1999).Furthermore, nutrients have a direct effect on algal tissue composition, changing C/N or N/P ratios, affecting higher trophic levels and altering top-down and bottom-up interactions (Burkepile and Hay 2006).
To understand benthic dynamics within coastal ecosystems, it is essential to investigate the development of benthic algal assemblages under different environmental scenarios.Generally, the succession of marine benthic communities starts within hours, with the formation of a biofilm (e.g.bacteria, fungi, diatoms, Cuba and Blake 1983;O′Toole et al. 2000), which provides the base for the settlement of early forms (e.g.filamentous algae and cyanobacteria, within days), and it is followed by the colonization of complex organisms (e.g.fleshy macroalgae; Aleem 1957; Fricke et al. 2008Fricke et al. , 2011b)).In particular, early settlers play a crucial role as they settle under certain environmental conditions (Callow et al. 2002) and either facilitate or inhibit the settlement of later species (e.g.Raimondi 1988).Biofilm and early successional forms have been observed to respond rapidly to increases in nutrients (Littler 1980).Thus, a change in nutrient concentrations during early succession can potentially structure the whole assemblage (Littler et al. 2010), and consequently alter its functional role within the benthic ecosystem (Osterling and Pihl 2001;Valiela et al. 1997).
The Argentinean Patagonian coast is composed of a mosaic of several pristine sites mixed with few places subject to locally increased anthropogenic nitrogen (N) loading (Martinetto et al. 2011;Piriz et al. 2003).The San Antonio Bay (40°43′ S, 64°56′ W, Argentina) is a good example of recent changes related to increases in human population along the coastline.This system shows high nutrient concentrations similar to those found in highly eutrophic sites, such as the central basin of the Italian Venice lagoon (Teichberg et al. 2010).The bay experiences a large daily water movement, as a consequence of the macrotidal (up to 9 m) semidiurnal regime present in the area.This considerable water flushing partially relieves the land-derived N loads, as well as the accumulation of biological products (Martinetto et al. 2010(Martinetto et al. , 2011)).Despite the large water movement, nutrient concentration pulses during low tide remain in the system long enough to support high biomass and diversity of macroalgae near the town of San Antonio Oeste (Martinetto et al. 2010).Moreover, the growth velocity of Ulva lactuca in this area is 20-25 % d -1 , which is 2 to 5-fold faster than in other eutrophic sites such as Mondego River estuary in Portugal, Venice Lagoon, and Urias estuary in Mexico (Teichberg et al. 2010).The large water movement also prevents some negative effects associated with eutrophication such as anoxia.In this case, the large macroalgae biomass supports high densities of herbivores by increasing food availability and nutritional content rather than negatively affecting the survival of organisms (Martinetto et al. 2010(Martinetto et al. , 2011)).In fact, San Antonio Bay is inhabited by a large abundance of consumers associated with areas where macroalgae blooms are common (Iribarne et al. 2003;Martinetto et al. 2010).There, herbivores can reduce macroalgae biomass up to 60 % (Martinetto et al. 2011), which is quite high compared to other eutrophic sites (e.g., Lotze and Worm 2000).Thus, it is possible that under these extreme conditions of high nutrient levels, large water movement and high levels of herbivory, benthic succession could differ from what has been reported in other eutrophic sites.
In the present study, we investigated benthic succession in two tidal channels with contrasting nutrient and grazer loads at San Antonio Bay over a one year period.Using the same arrangement of different settlement substrates, we evaluated differences between tidal channels in terms of 1) the identity of early settlers, 2) the colonization process of micro-and macroalgal assemblages through time and, 3) the final benthic algae assemblage.

Study Area
Field work was conducted in two tidal channels of San Antonio Bay (Fig. 1).These channels experience contrasting nutrient and grazer loads (Table 1) and have been used in previous studies to evaluate the effects of eutrophication (Martinetto et al. 2010(Martinetto et al. , 2011;;Teichberg et al. 2010).One channel runs nearby and along the town of San Antonio Oeste (hereafter SAO channel) and is characterized by high landderived nutrient loads that consequently support frequent algal blooms (Table 1, Martinetto et al. 2010Martinetto et al. , 2011)).In contrast, the second channel runs parallel to the SAO channel but is distant from human population (hereafter CONTROL channel) and shows much lower nutrient loads with algal blooms never reported (Table 1, Martinetto et al. 2010).Along with the higher nutrient loads, the SAO channel presents higher invertebrate abundance than the control channel (Martinetto et al. 2010(Martinetto et al. , 2011)).The large abundance of herbivorous, such as amphipods, snails, chitons and limpets exert a strong topdown pressure in the SAO channel (Martinetto et al. 2011).
Sampling design using contrasting natural conditions, although extensively used in ecological studies (e.g., Geertz-Hansen et al. 1993;Hauxwell et al. 2003Hauxwell et al. , 2006;;Martinetto et al. 2010;McClelland and Valiela 1998), results imperfect to draw comparisons given the lack of natural replications of treatment and control.However, by choosing this design we did a compromise between the limitations on the inference to other systems and the benefit of working under natural realistic conditions.
Both channels are subjected to similar variations in temperature and salinity (Martinetto et al. 2011).The substrate in the two channels consists of cobbles, pebbles and shells of mussels and snails where sessile invertebrates, diatoms and macroalgae grow attached.The benthic species composition differed strongly between the CONTROL and SAO channels (Martinetto et al. 2010).At the beginning of the study, the CONTROL channel was covered by few macroalgae attached to pebbles buried in the sediment; Polysiphonia was the most common macroalgal genus.The SAO channel had a high abundance of Ulvales, forming a standing bloom (see Martinetto et al. 2010).

Water Motion
In order to investigate potential differences in water motion between the different sites, clod cards (2.5×4×1.5 cm) made of plaster of Paris (Doty 1971), were exposed for three days in both channels (n=3 at each), and in an aquarium (n=2) filled with ambient seawater, which served as control treatment.
Relative difference in flow rates (C) between the different channels were calculated by C=te/me, where te refers to the measured weight loss of the clod cards deployed in the

Nutrient Analyses
To investigate differences in nutrient composition between the two channels during the study period, samples were taken for nutrient analyses at four days (16, 19, 23 and 26) in March 2013 (autumn season) during the experimental run.At each sampling day three replicates were collected from each channel at ~10 cm below low tide level, using a sterile (60 ml) syringe, extended with a plastic tube (1.5 m long).
Water samples were filtrated (Whatmann GF/F) right after taken and kept frozen (below 4 °C) in 50 ml PE bottles for later standard colorimetric measurements (Kattner 1999).Total DIN and DIP values were determined for 24 and 23 samples respectively (one DIP replicate from SAO was lost).

Settlement Substrata and Colonization Set-Up
To evaluate algal succession under contrasting levels of eutrophication, artificial substrates were set in the SAO and CON-TROL channels (21 October 2012).Since substrate characteristics play a crucial role in benthic community development and often vary with the environment, artificial settlement units (SU) are commonly used in benthic ecology for the direct comparison of different sites (Fricke et al. 2011b;Wahl et al. 2004).To study the benthic communities at different scales, we generated a bivalent substrate composed of two different materials allowing the investigation of micro-and macrophytobenthos at the same time.Based on our experience with prior settlement studies (Fricke et al. 2008(Fricke et al. , 2011a)), we constructed SU using unglazed ceramic tiles (3×6 cm) as macrosubstrate with a piece (1.5×2 cm) of polyethylene terephthalate (PET, Melinex ®) attached as microsubstrate.Thus, each SU in our study consisted of two different substrates.At each channel a total of 20 SU were exposed horizontally to the water surface~30 cm below low tide water level and 20 cm above sediment.

Sampling of Benthic Assemblages
In order to investigate successional differences under contrasting eutrophic conditions, we analyzed stepwise succession by conducting paired samplings in the two channels.The SU (n= 4 per time and channel) were destructively sampled after 4 (S1: October 25, 2012), 8 (S2: October 29, 2012), 11 (S3: November 1 st , 2012), 40 (S4: November 29, 2012) and 357 days (one year, S5: September 19, 2013).The first three samplings (S1, S2, S3, hereafter early succession) were analyzed only on microsubstrates, while the last samplings (S4 and S5, hereafter late succession) were analyzed on both micro-and macrosubstrates.For S3, two microsubstrates were lost for the SAO channel during sampling, but the remaining replicates were included in the analyses.Micro-and macrosubstrates were separated and treated independently for different analyses.Entire macrosubstrates were fixed in 4 % formaldehyde, while microsubstrates were divided in two pieces: one piece was fixed as a semipermanent slide (SLIDE) using 50 % Karo® corn-syrup and preserved with 4 % formaldehyde, and the other piece (SCAN) was fixed using 2.5 % glutaraldehyde in 0.05 M cacodilate buffer for later scanning electron microscopic analysis.The different substrates were scanned by different magnifications to investigate algae of different size classes.To investigate macroalgal growth, macrosubstrates were analyzed using a stereomicroscope (Nikon SMZ 1500, 1-3X magnification), while subsamples of microsubstrates (SLIDES) were examined under regular microscope (Nikon Eclipse 80i, 4X-100* magnification) to study smaller macroalgal stages (hereafter Bmesoalgae^).Both microscopes were equipped with an ocular grid (100 divisions) to allow quantitative measurement of individual taxa cover within each visual field (VF).In addition, subsamples of microsubstrates (SCANs) were investigated with a scanning electron microscope.

Early Succession
To determine changes in total cover, each early succession SLIDE (S1 to S3) from both channels was scanned for 25 visual fields (VFs) (20X, 8.5 mm 2 ).To quantify differences in the biofilm assemblage, we distinguished between diatoms, colored cell aggregations (Bgreen cells^), colorless detritus and filaments grown out from the cell aggregations, and macroalgal recruits.
In addition, diatom assemblages were analyzed separately.For this, each SLIDE corresponding to S1 was scanned twice, for 15 VFs (40X, 2.55 mm 2 ) to determine individual taxa cover and for 10 VFs (20X, 6.8 mm 2 ) to avoid underestimation of colony forming diatoms.As in the following time the abundance of diatoms increased strongly (see results), S2 and S3 SLIDEs were scanned for 15 VFs (20X, 5.1 mm 2 ) to capture the abundant taxa in the growing three dimensional assemblage.To identify the diatom species and potentially identify other microorganisms on the microsubstrates, two randomly chosen SCANs of S3 were prepared for scanning electronic microscopy, following the protocol described by Parodi and Cao (2003).
To compare micro-and macrosubstrate, and to determine the diatom species composition, a subsample (2×3 cm) of macrosubstrate was scraped with a sterile razor blade from the S3 SAO and CONTROL assemblages.Samples were transferred to glass vials and boiled 2 h in 30 % H 2 O 2 to remove all organic material keeping the cleaned frustules.From each subsample, a minimum of 400 valves were analyzed and species were identified.To compare diatom composition between the two substrates and techniques (percentage cover vs. cell counts), taxa were ranked by abundance.

Late Succession
To evaluate differences in the late successional assemblages (S4 and S5) at different scales, we analyzed the microsubstrates for filamentous stages and macroalgal germlings (mesoalgal assemblage), and the macrosubstrates for macroalgal composition (macroalgal assemblage).Due to the macroscopic colony size of the tube dwelling diatoms (TDD) we also included this taxon in the analyses for later successional stages.
For S4 and S5 each microsubstrate was scanned for 15 VFs (11.5X, 0.36 mm 2 ) and each macrosubstrate was scanned for 6 VFs (3X, 4.41 mm 2 ) to investigate for individual taxa cover.In addition, for each VF of macrosubstrate a subsample of algal material was fixed as a semi-permanent slide (SLIDE), using 50 % Karo® corn-syrup, preserved with 4 % formaldehyde and investigated under higher magnification (4-100X) for taxa identification.For S5, larger macroalgae (>1 cm) were removed from the edge of the macrosubstrate and preserved on a herbarium sheet.The coverage was calculated for every taxon.

Biodiversity Measurements
Taxa richness (S), evenness (J') and the Shannon diversity index (H') were calculated for each assemblage stage using percentage cover data (Magurran 1988).In addition, single taxon cover was added up for each assemblage and total taxon cover (COVER) was compared between channels for each stage.Taxa richness was used instead of species richness due to the lack of essential morphological features (e.g.sexual structures) in macroalgal germlings, and to the presence of organic material on preserved diatom frustules.

Statistical Analysis
Differences in nutrients (DIN and DIP), biodiversity (S, J', H) and total taxa cover between channels and among times were analyzed using a 2-way ANOVA with site and time as factors.Tukey's and Duncan's post-hoc tests were used in all cases when one of the factors or their interaction was significant.Homogeneity of variances was tested with Cochran's test.For one case (the evenness in the diatom assemblage), data could not be transformed to meet homogeneity of variance, nevertheless, ANOVA was used because it is still robust and better than other non-parametric analyses (e.g.Kruskall Wallis test, see Underwood 1997).
Differences in taxa composition between channels (SAO and CONTROL) and over time (S1, S2 and S3 for early and S4 and S5 for late succession), were evaluated using permutational multivariate analysis of variance (PERMANOVA).PERM ANOVA was based on Bray-Curtis similarity indices calculated from percentage cover prior to square root transformations in order to scale-down the importance of highly abundant taxa.

Environmental Conditions
No differences were found in the hydrodynamic environment, as clod card comparisons showed no differences between sites, but a 2.7 times elevated water motion was registered in the channels than in the aquarium control (ANOVA: F 2,7 = 6.95, p=0.01).DIN showed higher concentrations in the SAO channel (F 1,23 =11.555, p=0.004) with no significant effect of time (F 3,23 =1.291, p=0.312) nor interaction (F 3,23 = 1.253, p=0.324).The same pattern was found for DIP with higher concentrations in the SAO channel (F 1,22 =12.593, p= 0.003) and no significant effect of time (F 3,22 =2.983, p= 0.065) nor interaction (F 3,22 =3.328, p=0.058).Average nutrient concentrations were within the range reported in former studies (Table 1).

Benthic Succession
Following the benthic recruitment and further succession, the two tidal channel assemblages differed from each other.These differences were not only observable in the magnitude of recruits but also in the identity of early settlers and the composition of later successional stages.While a general more gradual shift was observed in the CONTROL channel, the SAO assemblages showed stronger over time changes in composition and diversity, which exceeded the CONTROL assemblages at later stages.Opportunistic species were responsible for these changes alternating subsequently in their presence and abundances.Below we describe in detail the alterations in the benthic assemblage successions.
Early Succession: Tube Dwelling Diatoms, Cocconeis spp.and Macroalgal Recruitment Rates In general, we observed high colonization and rapid growth of the biofilm with a significant increase in total cover over the first 8 days (S1-S2) at both channels, but higher values were registered at the SAO channel (Fig. 3, Table 3).The composition of the biofilm showed clear differences between channels (Fig. 3, Table 4).The biofilm in SAO showed more aggregated cells containing chlorophyll.A portion of the possibly Chlorophycean cells produced outgrowths, which were identified as Ulva spp. at later stages.In contrast, the biofilm in the CONTROL channel was mainly composed by diatom cells that efficiently aggregated more colorless detritus and sediments over time (Fig. 3, Table 4).
The diatom assemblage showed an increase in cover within the first 8 days (S1-S2) in both channels (Table 3).Although no differences were found in species richness and diversity, we observed a lower evenness and higher variance in the CONTROL channel (Fig. 4, Table 3).Furthermore, the composition of the diatom assemblages differed strongly between channels depending on the successional stage (Fig. 3, Table 4).The diatom assemblage in the SAO channel was dominated by Cocconeis scutellum var.scutellum, C. euglypta and tube dwelling diatoms (TDDs) (Fig. 2, Table 4).A strong increase in these taxa caused a change in the assemblage composition in the SAO channel over the first week of succession (S1-S2, Table 4).In contrast, the diatom assemblage in the CON-TROL channel was mainly dominated by the group of needle like diatoms (e.g.Nitzschia spp., Tabularia spp.) and Licmophora (Fig. 3).Overall, differences in the species present resulted in distinct microstructure in the two channels, recognizable in the individual laying (Nitzschia spp.) and radiate attached members of needle like diatoms (e.g.Tabularia sp.), giving a sprinkled appearance to the CONTROL channel assemblage (Fig. 2 a).In contrast, the erect mucilaginous tubes of TDDs (Fig. 2b-c) and plaster-forming C. scutellum and C. euglypta (Fig. 2d) dominated the structure of the SAO channel assemblage.
Comparing the different substrates, we found differences in the diatom assemblage after 11 days.The diversity seemed to be slightly increased by the macrosubstrate, as we found 12 diatom taxa on the microsubstrate in both channels and 14 and 13 diatom taxa on the macrosubstrate in SAO and CONTROL channels respectively.In the SAO channel, the most dominant taxa on the microsusbtrate were C. scutellum, followed by TDD and C. euglypta, whereas the most dominant taxa of the macrosubstrate were found to be the TDDs, followed by the chain forming taxa Grammatophora and Melosira.
After colonization by Chlorophycean and Phaeophycean cells, rapid recruitment of the red algae Ceramiales, including members of the Ceramiaceae and Rhodomelaceae, was observed in the assemblages of both channels.Germlings were present after three days within the CONTROL assemblage (mean (± SE) CONTROL: 0.5 ± 1 recruits/sample, Ceramiales).Following succession, the recruitment number increased in the SAO channel from 1.25 ± 0.96 recruits/ sample (40 % Ceramiales, 40 % Ceramiacae and 20 % Rhodomelacea) after 6 days to 2.5±2.12recruits/sample after 11 days (60 % Ceramiales and 40 % Rhodomelaceae).In Table 2 Average percentage cover per taxa in the assemblages at the CONTROL and SAO channels over different times of succession (S1: 3 days, S2: 6 days, S3: 11 days, S4: 40 days, and S5: 357 days=1 yr) grown on microsubstrates (1.5 x 2 cm, polyethylene terephthalate) and (underlined) macrosubstrates (3 x 6 cm, unglazed ceramic tiles).x indicates average cover<1 %.LF indicates life form following Cattaneo (1990) for diatoms: A=forms that grow oppressed on the substratum, St= comprised forms forming stalks or cushion like aggregations to attach on the substrate, Td = comprises colony forming forms living in mucilaginous partly branching tubes, M=comprising all motile pennate forms, and D=comprising suspended or trapped centric forms; and following Steneck and Dethier (1994) for meso-and macroalgae: si= siphonal, single cell tube, ff=fine filamentous uniseriate, main axis one cell thick, f=filamentous main axis multiseriate, fo=foliose single or bilayered, forming sheet, sac = saccate, foliose, inflated, bilayeredthallus, cor = corticated, multiple cell layers with partly different growth directions, corfol=corticated foliose multiseriate, cru= crust forming, calc crus = calcerouscrustous, partly calcified crust forming, artcalc=articulated calcerouscalcified upright growing forms.Nano diatoms=comprises all micro diatoms, indistinguishable in their valve shapes at scanning At later successional stages clear differences between the assemblages grown on different settlement substrates were observed.The composition of mesoalgal assemblages (grown on microsubstrates) and macroalgal assemblages (grown on macrosubstrate) differed in terms of the presence and abundance of different taxa.For the mesoalgal assemblage 17 different taxa were distinguished: 1 Bacillariophyta, 4 Phaeophyceae, 5 Chlorophyta, 6 Rhodophyta, 1 Cyanobacteria.For the macroalgal assemblages we distinguished 15 different taxa: 1 Bacillariophyta, 5 Phaeophyceae, 5 Chlorophyta, 3 Rhodophyta, 1 Cyanobacteria (Table 2).The discrepancies in taxa numbers were partly due to the presence of minute taxa like crustose Ulvales, crustose Ectocarpales, Hydrolithon, and Anotrichium, and early life stages of Ulva (germlings) and Porphyra, which required higher magnification for identification.Next to these cryptic taxa found on the microsubstrates, other taxa were detected and were   2).
The macroalgal assemblage, grown on the macrosubstrate, differed strongly in cover and species richness (S) between the two channels, being higher in SAO, with an increase in diversity values within the SAO channel at the end of the study (Fig. 6, Table 3).In contrast, evenness (J), increased with time at both channels (Fig. 6), indicating a similar change in the relative abundance of different taxa through succession.For the mesoalgal assemblages, grown on the microsubstrates, no differences were found between the SAO and CONTROL channels in S, J', and H' indexes, or in cover, although the cover significantly increased in both channels at the end of the study (after one year) (Fig. 5, Table 3).
Overall, both meso-and macroalgal assemblages differed between channels in their composition (Table 4).These differences were mainly caused by crust forming Ulvales (e.g.Ulvella lens) and crustose Ectocarpales (e.g.Myrionema sp.) in the mesoalgal assemblage, and by foliose Ulva and Cladophora in the macroalgal assemblage (Fig. 2, Table 4).For the mesoalgal assemblages, differences between channels at the end of the study (Table 4) were due principally to the increase of crustose Ulvales (e.g.Ulvella lens) in the CONTROL and crustose Ectocarpales (e.g.Myrionema sp.) in the SAO assemblage (Fig. 5).For the macroalgal assemblages taxa composition differed between channels throughout the study period (Table 4).Differences were mainly driven by the Ulvales (Foliose Ulva and Turbulose Ulvales), which sequentially bloomed in the SAO channel.Thus, after 40 days, the SAO assemblage was mainly composed of tubular Ulvales (Ulva prolifera, U. flexuosa, Blidingia sp.), whereas the CON-TROL channel showed a high abundance of the Cladophora.After a year, the number of tubular Ulvales decreased in the SAO channel accompanied by a decline in percentage cover (Fig. 6), and an increase in diversity (Fig. 7).
The SAO assemblage strongly changed its composition over time while differences in the CONTROL assemblage decreased at the end of the study (Fig. 7, Table 2).Despite the lack of differences in the analyzed assemblages, differences became evident when observing the edge of the macrosubstrates, characterized by four different grown out macroalgae.Thus, Ulva lactuca and Polysiphonia aff.argentinica dominated the SAO channel, whereas Scytosiphon sp. and Punctaria sp. were more abundant in the CONTROL channel (Fig. 6).

Discussion
Our results show that natural benthic succession under different nutrient and grazing conditions not only differs in trajectory but in the final taxonomic composition.Under high nutrient-grazing conditions, the benthic assemblage was dominated by tube dwelling diatoms (TDDs) and Cocconeis spp.
at early successional stages while under lower nutrient-grazer loads motile needle-like diatoms (e.g. of the genus Nitzschia, Tabularia) were the most dominant species.Succession continued with macroalgal colonization, e.g., Ceramiales, which showed a higher recruitment rate in the nutrient rich environment.After 40 days, differences were mainly due to the presence of turbulose Ulvales blooming at SAO channel, and a more diverse macroalgal assemblage in the CONTROL channel.Following up the succession, the assemblages in terms of biodiversity did not change in the control channel while in the SAO channel biodiversity increase exceeding that found in the control.These findings suggest that nutrient enrichment can not only alter an established assemblage, as has been extensively described in the literature, but also affect the benthic assemblage from the very early succession stages, changing the succession trajectory and concomitantly, the resulting final assemblage.Below we discuss the possible mechanisms that could drive the observed differences at different scales.
Early settlers already differed between channels after three days.Despite an observed similar increase in cover, differences were found in the diatom assemblages, with TDD and Cocconeis spp. in the SAO and needle-like diatoms in the CONTROL channel.Because the two channels were similar in terms of light, current, tide, and substrate, the differences found could be the result of the more than two fold higher nutrient concentration in the SAO channel.In fact, the observed formation of mucilaginous diatom tubes (TDDs), which bloomed in the eutrophic SAO assemblage, resembled a pattern observed in the North Sea (Hillebrand et al. 2000), where the tube forming Berkeleya rutilans strongly increased under artificial N and phosphorus (P) enrichment.This study supports our findings suggesting that the difference found is probably due to the difference in nutrient availability between channels.
The interpretations of the differences in the abundances of other diatom taxa are less obvious, as other taxa observed in the less polluted CONTROL channel, like the pennate genera Nitzschia and Tabularia, were also commonly found in nutrient-rich habitats (Hillebrand et al. 2000;Michels-Estrada 1998), and are even classified as highly tolerant to organic pollution (Kelly and Whitton 1995).In general, nutrient uptake rates of diatoms strongly depend upon characteristic nanostructures (Mitchell et al. 2013).The observed discrepancies in nutrient tolerant taxa might therefore underline the need of better species knowledge to identify suitable indicators for water quality measurements in the area.
The presence of crustacean tubes, already attached to the different substrates in both channels after a short time, would indicate high grazing (Fricke pers. obs.).In fact, the oxygenated nutrient rich SAO channel provides a unique environment favoring the development of abundant invertebrate fauna (Martinetto et al. 2010(Martinetto et al. , 2011) ) which might strongly affect the In general, members of the genus Cocconeis have been reported as common early colonists after disturbances in several studies (e.g.Patrick 1976;Jones 1978;Robinson and Rushforth 1987).Interestingly, Cocconeis was only sparsely observed in the CONTROL channel.This difference may suggest that factors other than nutrient concentration or grazer abundance play an important role.Microstructures play a crucial role in diatom attachment (e.g.Wu et al. 2013), which consequently alter the benthic assemblage composition at later successional stages (Schneck et al. 2011).Accordingly, in our study we observed strong substrate specific differences, with Cocconeis dominating in the smoother microsubstrate surfaces, where its firmly attachment mode presents a greater resistance to the drag forces of the flow (Gari and Corigliano 2007).Potential differences in substrate availabilities and qualities might be responsible for the observed differences in Cocconeis abundances between channels.In fact, the SAO channel has a closer connection to the adjacent town of San Antonio Oeste and thus it exhibits a higher availability and diversity of substrates derived from anthropogenic activities (e.g.pieces of concrete, bricks, etc).
Close macroalgae-diatom interactions have been observed in other studies.Diatoms play a crucial role for macroalgal settlement (Davis 2009), and thus might facilitate or inhibit their settlement (Connell and Slatyer 1977).Mucilaginous diatom tubes (TDD) can alter the microstructure and threedimensionality of the early benthic assemblages, through production of extracellular polymers that can facilitate settlement (Lam et al. 2005) and support a variety of epiphytic species (Round et al. 1990).However, given the opportunistic life style of tube dwelling diatoms characterized by seasonal blooms (Minzuno 1989), it is unclear how far this taxon facilitates benthic succession (e.g.supports epiphytic macroalgae over time).However, the composition of epiphytic diatoms depends on the host identity (Al-Handal and Wulff 2008) and is related to different interspecific interactions such as grazing pressure and sloughing (Liess et al. 2009).Thus, it is not clear if there is a closer connection between the observed abundant and bloom forming taxa of macroalgae and the diatoms (e.g.Ulvales, Cocconeis and tube dwelling diatoms), which consequently would affect not only the benthic but also the planktonic system of the research area.Macroalgae, in turn, provide suitable substrate to different diatom groups (Cejudo-Figueiras et al. 2010).Thus, the abundant and sediment free thalli of macroalgae reported in the SAO channel (Martinetto et al. 2010) might play an important role as suitable substrate for diatom settlement as has been found in other studies (e.g.Cejudo-Figueiras et al. 2010).Therefore, it is possible that micro-macroalgal interactions might impact development of the different macroalgal assemblages in the two channels.Our results indicate that the succession of benthic algal assemblages follows different trajectories, resulting different taxonomic compositions in the two channels.As moderate successional and seasonal changes were observed in the CON-TROL channel, strong alterations in the eutrophic SAO channel were mainly driven by the formation of different micro-and macroalgal blooms.Interestingly, substrate characteristics provided by the first settlers significantly interfered with the observed assemblage patterns, changing the habitat diversity (βdiversity, Whittaker 1972).Thus, the observed decrease in tubular shaped bloom formers in the SAO channel led to a significant increase in the biodiversity in the macroalgal assemblage, whereas no changes were found in the mesoalgal assemblages.This difference might be related to a specific pattern, as grown-out thalli of foliose Ulvales were exclusively found at the borders of settlement tiles.The observed spatial discrepancy might be explained by spore settlement preferences (Callow et al. 2002), inter-specific relations like settlement inhibition (Connell and Slatyer 1977) and grazing pressure (Kamermans et al. 2002), or more likely by increasing vulnerability to shearing forces, removing grown individuals, as we commonly found loose algal material drifting in the area.As a consequence, the newly bare substrate allows the colonization and development of new taxa, provoking a restructuring in the SAO assemblage on the settlement tile that interestingly resembled the CONTROL assemblage at the end of the study.In addition, other substrate specific differences of Chaetomorpha and Dictyota and the sparse presence of Sphacelaria and Derbesia shaped the benthic pattern of the area.Overall, the observed differences between different substrates (habitats, therefore βdiversity, Whittaker 1972) may play a crucial role in the formation and duration of benthic algal blooms in eutrophic areas, and might become crucial in environmental assessment as well as in the evaluation of future pollution scenarios.
The benthic assemblages established after one year differ between channels.In the SAO channel, the assemblage was characterized by the presence of species typically associated with eutrophic systems, like the foliose Ulvales, but also by high diversity, which is not usually associated with eutrophication.The frequent tidal flushing seems not only to allow the development of a diverse grazing fauna (Martinetto et al. 2010), but also to prevent anoxic events in this system (Martinetto et al. 2011), which otherwise would favor the growth of anaerobic bacteria and lead to the degradation of benthic organisms as commonly observed in many eutrophic systems (Diaz and Rosenberg 2013).

Conclusion
In conclusion, our work suggests that even at the very early succession stages, nutrients play an important role in driving succession of benthic species, and along with other factors (e.g.grazers, availability of spores and early settlers) establish the succession pathways and the final composition of the algal assemblage.In addition, settlement substrate seems to play a crucial role by supporting favoring taxa specific characteristics and leading to different assemblages under equal environmental conditions.These findings might play a crucial role for the management of such eutrophied systems.In fact the traditional approach using the presence of certain indicator species might be extended by integrate information on substrate composition of the area and/or vice versa be simplified by using comparable monitoring substrates and fast settling indicators, like specific diatoms and macroalgal at early stages (e.g.germlings).
Daily oxygenation through the macrotidal cycle and the herbivory pressure exhibited in this area may lead to a more diverse assemblage in the nutrient-rich area avoiding the typical domination by few taxa typically described in nutrientrich environments.

Fig. 1
Fig. 1 Map of San Antonio Bay showing the flooded area during high tide in light grey and the underwater area during low tide in white.The SAO channel passes through the town of San Antonio Oeste while the CONTROL channel is situated farther away from human activities

Fig. 3
Fig. 3 Early succession at CONTROL and SAO channels during the first eleven days of succession (S1=4 days, S2=8 days, S3=11 days).a Changes in total biofilm percentage cover composed by diatoms, cells containing chlorophyll (Chl cells) and filamentous algae (filaments), aggregating different amounts of detritus and sediment (detritus).Superimposed letters indicate differences identified by ANOVA.b Changes in taxa composition and percentage cover of diatom assemblage at CONTROL (white bars) and SAO (black bars) channels.

Fig. 4 Fig. 5
Fig. 4 Changes during early succession in species richness (dots), Evenness J' (squares) and Shannon diversity H'(diamonds) during early succession of benthic assemblages grown in the CONTROL

Fig. 6
Fig. 6 Macroalgal assemblages at CONTROL and SAO channels during late succession (S4=40 days and S5=365 days).a Changes in total percentage cover.Superimposed letters indicate differences identified by ANOVA.Graph shows mean (±SE).b Changes in taxa composition and percentage cover of macroalgal assemblage at CONTROL (white bars) and SAO (black bars) channels.Only individual taxa percentage cover>5 % in a treatment is shown.Graph shows mean (±SE).c Non-metric multi-dimensional scaling (nMDS) plot

Fig. 7
Fig. 7 Biodiversity changes during later succession.Graphs showing changes in Species richness (S), Evenness (J') and Shannon diversity H' in the Meso-and Macroalgal assemblages grown on micro-and macrosubstrates in the CONTROL (white) and SAO (black) channels over 40 (S4) and 365 days (S5).Different letters indicate differences between the different treatments.Data shows means (±SE)

Table 3
ANOVA results for differences in percent cover, total number (COVER/ Number), species richness (S) evenness (J') and diversity (H') between assemblages exposed to contrasting nutrient conditions (SAO, CONTROL) over different times (S1-S5).Significant differences are marked in bold.Conclusions indicate results of Tukey and Duncan's post-hoc test.Italics indicate square root transformation of data,* indicates no homogeneity of data could be achieved by transformation

Table 4
PERMANOVA results showing differences in taxa composition during a) early succession and b) late succession of different algal assemblages (Biofilm, Diatoms, Mesoalgae and Macroalgae) in the CONTROL and SAO channels.Results of SIMPER (similarity percentage analysis) are given, showing percentage contribution of single or grouped taxa to total dissimilarity between channels or times.Only taxa causing major differences (cut-off level 60 %) are presented.The channel or succession stage showing higher abundance of taxa is in parenthesis.Bold values are statistically significant.Asterisk indicates lower replicate number for 40 days old control Mesoalgal assemblages (n=2)