Journal of Sea Research 81 (2013) 1–9

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Journal of Sea Research

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Linkages between rocky reefs and soft-bottom habitats: Effects of predation andgranulometry on sandy macrofaunal assemblages

Gustavo M. Martins a,b,⁎, João Faria a,b, Marcos Rubal a,c, Ana I. Neto a,b

a CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugalb Centro de Investigação de Recursos Naturais dos Açores, Departamento Biologia, Universidade dos Açores, 9501-801 Ponta Delgada, S. Miguel, Açores, Portugalc Department of Biology, Faculty of Sciences, University of Porto, 4150-191 Porto, Portugal

⁎ Corresponding author at: CIIMAR/CIMAR, InterdiscEnvironmental Research, University of Porto, Rua dosPortugal. Tel.: +351 296 650 101; fax: +351 296 650

E-mail address: gmartins@uac.pt (G. M. Martins).

1385-1101/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.seares.2013.03.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 October 2012Received in revised form 15 March 2013Accepted 27 March 2013Available online 6 April 2013

Keywords:Habitat connectivitySoft-bottom communitiesCommunity structureMacroinvertebratesSpatial variability

The movement of animals and materials among habitats can have an important influence on the structure ofassemblages. The nature of such linkages is, however, not always an obvious one. Here we examined theinfluence of rocky reefs on nearby sandy macrofaunal assemblages. A preliminary descriptive study confirmedthe hypothesis that sediment granulometry differs in relation to distance to rocky reefs and so did themacrofaunal assemblage composition, and relative abundance of three of the most abundant taxa, and thatthis effect was generally consistent among locations. In addition, there was a significant effect of sediment struc-ture on community structure of benthic assemblages. A subsequent manipulative experiment tested, in an inter-activeway, the effects of predation and sediment grain size in structuringmacrofaunal assemblages adjacent to arocky reef. At the scale of the assemblage (richness and composition), results showed that there was an effect ofsand granulometry and distance to the reef, while predation had no effect. Predation affected the abundance ofone of the most abundant taxa (un-identified amphipod sp.1) but this effect was consistent adjacent and awayfrom the reef. Sand granulometry had no influence on the numbers of the most abundant taxa, while the abun-dance of two of these taxa (Catapaguroides timidus and Ervilia castanea) still varied according to distance to thereef. This study adds to the wider literature by suggesting that there can be important movements of materialsand organisms from rocky reefs to adjacent soft-bottom habitats but that rock-associated predators and sandgranulometry may have a limited influence in structuring the small-bodied sediment infauna close to reefs.This study also stresses the need for caution when ascribing the importance of ecological processes based ondescriptive studies alone, even when results seem ecologically logical.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

It is general consensus that biological communities are notcontained entities but are open to themovement of individuals andma-terials among different habitat types (see Table 1 in Massol et al., 2011and references therein). The importance of such movements is some-times easily depicted such is the case, for instance, of the dependencyofmany supralittoral animals from an allochthonous source of subsidies(e.g. Lastra et al., 2008; Lewis et al., 2007; Soares et al., 1997), or theeffects of nutrient run-off originated from terrestrial or freshwater eco-systems on marine coastal production (e.g. Largier, 1993; Salen-Picardet al., 2002). The nature of these ecosystem linkages, however, cansometimes be the result of complex interactions. For example, Knightet al. (2005) showed that freshwater fish could indirectly facilitateterrestrial plant reproduction via cascading trophic interactions across

iplinary Centre of Marine andBragas 289, 4050-123 Porto,100.

rights reserved.

ecosystemboundaries, while Nakano andMurakami (2001) document-ed a reciprocal subsidy dependency of secondary production betweenterrestrial and aquatic food webs. Because ecosystem linkages canhave key impacts on biological communities, it is important that thesemovements of organisms andmaterials are understood if we are to bet-ter manage natural ecosystems.

Rocky reefs often border soft-bottom sandy areas. These two hab-itat types support very different communities of plants and animals.Yet, there is evidence of movements of materials and animals be-tween these two distinct habitats. For instance, Tuya et al. (2010)found that proximity to reefs affected the balance between predationand recruitment of benthic gastropods inhabiting seagrass beds.Rilov and Schiel (2006) found that the intensity of predation onmus-sels on intertidal rocky reefs depended on the nature of the adjacentsubtidal substratum. Where intertidal rocky reefs were bordered bysoft-bottom subtidal substrates, predation (presumably by fish)was such that all transplanted mussels were rapidly consumed ex-cept if protected by experimental cages. In addition, Unsworth etal. (2008) found influences of nearby mangroves on the numbersand types of fishes found in adjacent seagrass, showing that there

Fig. 1. Geographical location of the Azores, the island of São Miguel and the reefs sam-pled: SR — Sao Roque, LA — Lagoa, and AA — Água d'Alto.

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can be important interactions between distinct habitats. A fewstudies have also documented the influence of rocky reefs on thestructure of adjacent sandy-bottom assemblages (Ambrose andAnderson, 1990; Barros et al., 2001; Davies et al., 1982; Posey andAmbrose, 1994). For instance, Barros et al. (2001) showed that thecomposition and structure of macrofaunal assemblages was general-ly different near and away from rocky reefs. Whereas a few taxashowed consistent patterns (e.g. Syllidae polychaetes being moreabundant away from the reef), the majority of taxa showed spatiallyvariable responses (within and among reefs). Similarly, Ambroseand Anderson (1990) found greater densities of the polychaetesPrionospio pygmaeus away from the reef, while densities of thetube-dwelling worm Diopatra ornata were greater near the reef.Even species judged ecologically similar (the tube-building spionidpolychaetes P. pygmaeus and Spiophanes spp.) can show oppositepatterns of abundance (Ambrose and Anderson, 1990). Furthermore,Barros et al. (2001) also noted that assemblages near reefs were spa-tially more variable than those away from the reef. These results sug-gest that the response of organisms to the presence of reefs isspecies-specific and highly variable in space.

Coarser sediment sizes are generally found close to reefs and finersediments away. Since sediment grain size is an important determi-nant of the distribution of macrofaunal taxa (Gray, 1974) this variablehas been judged important in determining the distribution ofmacrofauna in relation to the presence of reefs. For instance, Barroset al. (2004) found significant differences in assemblage structure ofbenthic macrofauna between the crests and troughs of ripple marks.The structure of ripple marks (width and height) was also affectedby the presence of reefs suggesting that reefs had an indirect effecton benthic assemblages via changes in the microtopography of thesediment.

In addition, many rocky reef associated fauna can forage overadjacent sandy areas (e.g. Frazer et al., 1991) suggesting that soft-sediment assemblages may have an important role in subsidizingthese rocky reef associated fauna (Lindquist et al., 1994). Whilesome experimental evidence suggests predation can have a strongeffect near reef edges reducing the abundance of some organisms(e.g. bivalves) (Langlois et al., 2005, 2006a) other studies showed lit-tle predation effect (Barros, 2005). On further exploring the subject,Langlois et al. (2006b) suggested a size-dependent response ofsoft-sediment assemblages to predation from rocky reef associatedfauna.While significant effects of predation had been found on largerbodied (>4 mm) organisms (Langlois et al., 2005), there were in-consistent effects on smaller-bodied organisms (Langlois et al.,2006b). It is interesting to note that in all these cases, some taxastill responded to distance from reefs over and above the effect ofpredation, suggesting that sediment and predation may have addi-tive or interactive effects on different taxa. However, there hasbeen no simultaneous test of the effects of sediment type and preda-tion in explaining differences of assemblages close and away fromrocky reefs.

In this study we first describe changes in the structure of sandymacrofaunal benthic assemblages relative to proximity to rockyreefs. We hypothesise that sandy areas close and away from rockyreefs will have distinct sediment grain sizes (coarser close to reefs)and support distinct macrofaunal assemblages. We expect that organ-ism response will be species-specific and spatially variable. As shownby Barros et al. (2001) and because the reefs increase the complexityof the environment (both physically and biologically) we alsohypothesise that assemblages adjacent to rocky reefs are more vari-able than assemblages away from the reef. Building on this study,we subsequently manipulate predation and sediment granulometryto examine their effects as the drivers of the changes observed inthe structure of sandy macrofaunal assemblages in areas adjacent toreefs. We hypothesise that for some taxa predation from rocky reefassociated fauna may be important so that their numbers should be

similar in areas away and close to rocky reefs when predators are ex-cluded; and lower adjacent to reefs when predators are not excluded(e.g. significant interaction between the factors predation and dis-tance from reef). Alternatively, if differences in the abundance oftaxa related to the distance from rocky reefs are associated with dif-ferences found in sediment granulometry close and away from thereef, then these taxa should have similar numbers in areas with sim-ilar sediment grain size irrespectively of distance to reef (e.g. signifi-cant effect of sand granulometry but not of distance). By using anorthogonal design it is also possible to examine the response ofsome taxa to the interactive effects of sediment granulometry andpredation.

2. Materials and methods

2.1. Study sites

Work was done at three locations in the south coast of São Miguel,Azores: Água d'Alto (AA), São Roque (SR) and Lagoa (LA) (Fig. 1). Ineach of these locations the rocky reef, composed of natural basalticrock, extends into a depth of approximately 15 m, which is then re-placed by sandy-bottom substrates. At AA, the reef is nearly a verticalwall and the transition between the reef and sand occurs approxi-mately 10–15 m from land. In the other two locations, SR and LA,the reef gently slopes and the transition between the reef and sandoccurs at approximately 100 and 50 m offshore, respectively.

In each of the three locations, the reef supports a rich communityof algae, invertebrates and fishes typical of Azorean coastal waters

3G. M. Martins et al. / Journal of Sea Research 81 (2013) 1–9

(see Morton et al., 1998). In AA, encrusting coralline algae andsea-urchins dominate reefs. In the other two locations, the reef hasa rich algal community dominated by Dictyota spp. At the transitionbetween the rocky reef and sand in all locations, the benthic fish com-munity is dominated by rocky-associated invertivores species andconspicuous presences include Diplodus spp., Pagellus acarne, Chromislimbata, Abudefduf luridus, Sphoeroides marmoratus, Coris julis,Thalassoma pavo, Serranus atricauda, Scorpaena spp., Symphodus spp.and the herbivore Sarpa salpa. Our observations suggest that fishmovements in sandy areas are restricted to the vicinity of reefs andthat they do not forage more than 15 m away from the reef. This pat-tern was also noted by Langlois et al. (2005) who showed that the ef-fect of reef-associated predators was restricted to areas adjacent tothe rocky reefs. The sand-associated invertivore Bothus podas is byfar the most abundant fish but Mullus surmuletus is also commonand present both close and away from the reef. Conspicuous inverte-brate predators (e.g. crabs, star-fish) were never seen on soft-bottomareas during the study period.

2.2. Sampling design

This study was divided into two distinct phases: a preliminary de-scriptive phase where the sand-inhabiting macrofaunal assemblageswere described and a posterior manipulative phase where the effectsof predation and sand granulometry were controlled to test specifichypotheses. Underwater work was all done by SCUBA.

2.2.1. Descriptive approachSampling was performed in December 2008 when in each of the

three locations selected (AA, SR, and LA; see Fig. 1), a group of 9 rep-licate samples of sand were collected adjacent to (within 1 m fromthe reef) and away (15 m) from the reef totaling 18 samples per loca-tion. A distance smaller than 15 m from the reef was previouslyshown to provide ample space for the detection of changes in thestructure of infaunal assemblages (Barros et al., 2001). Sampleswere taken randomly at 1–5 m apart. Sampling was done using a cy-lindrical core (10 cm diameter), which was driven 10 cm into thesediment. Samples were carefully placed in zipped bags andtransported to the laboratory (see below for sample processing).

2.2.2. Manipulative approachFollowing the descriptive study, we tested specific hypotheses about

the effects of fish predation and sand granulometry in the field in July2009. The descriptive study showed that sand-associated communitiesadjacent to and away from the reefwere distinct and that this differencewas consistent in all the three locations examined (despite variabilityamong locations). Since animal sorting was extremely laborious, themanipulative study was limited to only one location: LA was selectedbecause it was closest and required the simplest logistics.

To test the hypothesis that differences in community structure ad-jacent to and away from the reef were driven by differences in sandgranulometry (see results from descriptive study), we experimentallycontrolled sediment grain size. Experimental pots (20 cm diameterand depth) were filled with either finer (b0.5 mm) or coarser(>0.5 mm) grain sizes (according to the descriptive study the frac-tion of sand between 0.25 and 0.5 mm was similarly represented onstations far and close to the reef so we chose the 0.5 mm sand for ex-perimental units because it was easier and faster to sieve the desiredamounts of sediment). For this purpose, dry sand was collected on abeach in an area rarely washed by the sea. The sand was oven-driedand then sieved into the desired granulometry. This strategy provideda standardised substratum for the colonisation of macrofauna. To testthe hypothesis that differences in community structure adjacent toand away from the reef were driven by predation from rocky reef as-sociated predators, we assigned pots to each of 3 treatments: fish ex-clusion pots, control, and an experimental control. Pots assigned to

the fish exclusion treatment were covered with plastic coated cage(1 cm mesh size, 5 cm height). Control pots were left uncoveredwhereas pots assigned to the procedural control corresponded tohalf-caged pots (half roof and sides) where potential predators(e.g. fish) were able to forage. Although our approach limits the ac-cess to experimental areas of any organism larger than 14 mm (diag-onal of a 1 cm mesh size) such as large gastropods and crustaceans,starfish, sea-urchins, these taxa were never observed on sandyareas in the studied areas suggesting that fish were the most likelypredators.

Pots were deployed accordingly to a 3-way orthogonal designwith grain size (2 levels: small and large), predation (3 levels: preda-tor exclusion, control and cage control) and distance to reef (2 levels,adjacent to and away from reef). A total of 4 replicate pots weredeployed for the combination of factors.

Pots were deployed to randomly intersperse treatments. Potswere driven into the substrate so that only a 2 cm lip was left abovethe substrate to guarantee the stability of treatments grain size. Two2 × 2 cm apertures were open on the sides of each pot to allow infau-nal migration. Pots were deployed for a period of one month afterwhich they were sampled using a core (as described above). Whilethis short period of time favoured colonisation by recruitment stagesof macrofauna, the substantial number of adult-sized animals in thesamples suggested that migration of adults from adjacent areas wasan important component of the assemblage.

2.3. Sample processing

Samples of sediment were carefully transferred to plastic pots inthe laboratory, fixed in a seawater solution with 5% formaldehydeand stained with 0.2% Rose Bengal. Samples were elutriated throughnested 0.5 mm and 0.063 mm sieves. The content retained in the0.5 mm sieve was sorted under a dissecting microscope and themacrofauna preserved in 70% alcohol. Macrofaunawere sorted, iden-tified to the lowest practical level of taxonomic resolution, countedand stored. All the sediment was dried at 60 °C until constant weight.The dried material was then sieved using a mechanical shaker(Retsch AS200) for 4 min in a column containing sieves of 4, 2, 1,0.5, 0.25, 0.125 and 0.063 mm mesh sizes and each fractionweighted.

2.4. Data analysis

2.4.1. Descriptive studyComparison of samples adjacent to and away from the reefwas done

using analysis of variance (ANOVA) (GMav, University of Sydney). A2-way mixed ANOVA was generally applied with location (random, 3levels) and distance (fixed and orthogonal, 2 levels) as factors.Prior to analyses, data were checked for heterogeneity of variancesand transformations applied where necessary (Underwood, 1997).Student–Newman–Keuls (SNK) tests were used to compare meanswithin significant interactions. No alpha corrections were appliedbecause the tests of interest had little power and would haveresulted in an overly conservative test (Quinn and Keough, 2002).

The variables examined were, sediment grain size distribution,number of species and the abundance of each of the most abundanttaxa. For the sediment, individual samples were standardised byconverting data to percentages. For analysis, the different size classeswere then grouped in three size classes following the Wentworth(1922) scale. These classes were gravel (>2 mm), coarse sand (2–0.25 mm) and fine sand (b0.25 mm). For the fauna, only taxa whoseabundance corresponded to ≥1% of all individuals were analysed (atotal of 8 taxa). Together, the taxa analysed comprised 95% of allindividuals.

To test the hypothesis that benthic assemblages adjacent to rockyreefs were more variable than away we compared variability at two

Gravel

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Fig. 2. Percentage (mean + SE) of gravel (>2 mm), coarse (2–0.25 mm) and fine(b0.25 mm) sand in samples adjacent to and away from the reef among locations. Let-ters indicate significant differences as detected by SNK tests (see supplementary onlinedata, Tables S1 and S2). AA = Agua d'Alto, SR = Sao Roque, LA = Lagoa.

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Fig. 3. Mean (+1SE) number of species and individuals in samples taken adjacent toand away from the reef. There were no significant effects of distance (see AppendixA, Table S3). AA = Agua d'Alto, SR = Sao Roque, LA = Lagoa.

Axis 1

Axi

s 2

Fig. 4. n-MDS showing compositional (Jaccard's) differences in the assemblages adja-cent to and away from the reef (see Appendix A, Table S4). Triangles = Agua d'Alto(AA), squares = Sao Roque (SR), circles = Lagoa (LA). Filled and open symbols repre-sent samples adjacent to and away from reef, respectively. Stress = 0.24.

4 G. M. Martins et al. / Journal of Sea Research 81 (2013) 1–9

spatial scales (among locations and among cores) using analysis ofvariance to estimate the corresponding variance components separate-ly for each habitat (adjacent to and away from reef) (see Fletcher andUnderwood, 2002, for details). Differences in non-negative variancesbetween habitats were tested using two-tailed F-ratios. These analyseswere run on untransformed data.

Compositional differences in assemblages adjacent to and awayfrom the reef were tested in the same way as above but using a mul-tivariate analogous procedure: PERMANOVA (Anderson, 2001). Be-cause we were interested in changes resulting from differences inthe numbers of species, but not their relative abundance, we usedthe Jaccard's similarity measure. The Jaccard index is a standard mea-sure used to examine differences in composition and when expressedas dissimilarity, it is directly interpretable as the percentage ofunshared species between two sample units (Anderson et al., 2007)and is identical to Bray–Curtis when the latter is based on the pres-ence–absence data (Clarke and Warwick, 2001). All analyses wererun with 999 permutations.

We also sought to determine how much of the variability in thecommunity was explained by differences in sediment structure.Given the high correlation between the abundance of the differentsize classes of sand, we extracted the scores from the first principalcomponent analysis (PCA1) using log-transformed data. These scoreswere then regressed with the abundance of each taxonomic groupand with a multivariate measure of community structure. For the lat-ter, the scores from the first canonical analysis of principal coordi-nates (CAP1) (Anderson and Willis, 2003) were used. CAP was runon the square-root-transformed data and using Bray–Curtis as a sim-ilarity measure. All multivariate analyses were run on PRIMER v6(PRIMER-E Ltd., Plymouth) with PRIMER add-on (Anderson et al.,2007).

2.4.2. Manipulative studyData were analysed using a 3-way fully orthogonal ANOVA with

sediment grain size (2 levels, fixed), predation (3 levels, fixed) anddistance (2 levels, fixed) as factors. Heterogeneity of variances wasinspected as described above. The variables examined were: percent-age of coarse (or fine) sediment, number of species, and relativeabundance of each of the 5 taxonomic groups examined. As above,analyses were restricted to groups of animals whose overall abun-dance (pooled among all samples) was≥1% of all individuals. Togeth-er, these taxa were 93% of all individuals.

3. Results

3.1. Descriptive study

3.1.1. Sediment analysisAs expected, samples were dominated by sand (b2 mm), which

corresponded, on average, to 96% of the sediment. When comparingsamples taken adjacent to and away from the reef there was a signif-icant (ANOVA P b 0.05 see Appendix A — Table S1) interaction be-tween distance and location for the three substratum size classes(gravel, coarse and fine sand). Generally, gravel tended to be slightlymore abundant adjacent to the reef and significantly more abundantat one location (Fig. 2). Coarser sand was significantly more abundantadjacent to the reef at two out of the three locations while finer sandwas significantly more abundant away from the reef also in two out ofthe three locations (Fig. 2).

PCA1 captured 68% of all the grain size variability found amongsamples. There was a strong positive correlation between PCA1 andthe proportion of sediments >0.5 mm in the samples (Appendix A— Table S2). For the smaller classes of grain size (fine sand) therewas a strong but negative correlation with PCA1. Hence, PCA1 in-creases with increasing grain size.

3.1.2. Macrofaunal assemblagesWe sampled a total of 14,690 animals distributed among 8 phyla.

The polychaete Exogone naidina Örsted, 1845, the bivalve Erviliacastanea (Montagu, 1803) and the polychaete Spio aff. filicornis (O.G.Müller, 1776) were numerically the most abundant species with atotal of 10,797, 715 and 415 individuals, respectively. Together,these three species represented 81% of the individuals in our samples.

The number of taxa did not differ with distance from reef (Fig. 3,ANOVA P > 0.05, Appendix A— Table S3). There were, however, signif-icant differences in the composition of assemblages adjacent to andaway from the reef (Fig. 4, PERMANOVA P = 0.001, Appendix A —

Table S4). Inspection of the species that were exclusive to assemblagesadjacent and away from the reef showed that a total of 28 species wasrestricted to samples taken adjacent to the reef, but only 3 were exclu-sive to samples taken away from the reef. The majority of the exclusivespecies (either adjacent to or away from the reef) was, however, rare

5G. M. Martins et al. / Journal of Sea Research 81 (2013) 1–9

(each species with b10 individuals). Only individuals belonging to thegenera Jaeridae and Protodrilus (in assemblages adjacent to the reef)were relatively abundant with a total number of 34 and 29 individualsfrom all samples combined, respectively. For the Jaeridae, however, allanimals were recorded in one single sample.

Among the most abundant taxa, we found significant differencesin the abundance adjacent to and away for 3 out of the 8 taxa exam-ined (Fig. 5, Appendix A — Table S5). The bivalve E. castanea and thepolychaete Spio filicorniswere significantly more abundant in samplesaway from the reef (ANOVA P b 0.05). In contrast, an unidentified

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Fig. 5. Mean (+1SE) number of the most abundant taxa in assemblages adjacent to and awANOVA (see Appendix A, Table S5). AA = Agua d'Alto, SR = Sao Roque, LA = Lagoa.

ostracod species (Ostracoda sp.1) was significantly more abundantin the assemblages adjacent to the reef (P b 0.05). No significant dif-ferences (ANOVA P > 0.05) were found between assemblages adja-cent to and away from the reef (Fig. 5) for any of the other taxaexamined.

At the scale of samples (regardless of location or distance fromreef), sediment grain size significantly influenced the macrofaunal as-semblage structure (Fig. 6). Surprisingly, of the 3 taxa that showedsignificant differences with distance from reef (see Fig. 5), only the bi-valve E. castanea showed a significant (negative) relationship with

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ay from reef. Letters indicate significant effects of distance from reefs as detected by

Fig. 6. Regression between the structure of macrofauna assemblages (scores of the ca-nonical analysis of principal coordinates, CAP1) and substratum structure (scores ofthe principal component analysis, PCA1).

Table 2Estimates of variance components and two-tailed F-ratios comparing patterns in spa-tial variance between assemblages adjacent to and away from reefs for the most abun-dant taxa.

Location (df 2,2) Test Core (df 8,8) Test

Taxa Away Adjacent Away Adjacent

Exogone naidina 30,822.9 36,054.0 ns 17,048.0 24,120.0 nsNematoda sp. 45.2 46.3 ns 85.0 120.0 nsErvilia castanea 42.7 13.8 ns 74.8 40.9 nsOligochaeta spp. 27.0 3.7 ns 28.0 61.1 nsSpio filicornis 35.1 2.0 ns 137.2 5.1 ***Armandia cirrhosa 0.8 2.5 ns 17.5 7.9 nsTurbellaria sp.1 1.8 17.3 ns 4.8 28.9 *Ostracoda sp.1 −0.3 −0.3 No test 3.9 21.4 *

*P b 0.05, **P b 0.01, ***P b 0.001, ns = not significant. There is no test for negativevariance components.

6 G. M. Martins et al. / Journal of Sea Research 81 (2013) 1–9

grain size (Table 1). Neither the polychaete S. filicornis nor the ostra-cod taxon responded to differences in sediment granulometry at thescale of samples, suggesting that differences in their relative abundanceadjacent to and away from the reef were not caused by physical differ-ences in the sediment. Again, no influence was found between grainsize and the abundance of the remaining taxa (Table 1).

For the majority of the taxonomic groups examined, a substantialproportion of the overall variability occurred at the spatial scale ofcores (m's apart) (Table 2). At small spatial scales (core-scale) thetaxa Turbellaria sp.1 and Ostracoda sp.1 were significantly more vari-able adjacent to the reef, whereas S. filicornis was significantly morevariable away from the reef (where it was more abundant, seeabove). At the larger scale of locations, all taxa showed a similarlevel of variability adjacent and away from the reef (Table 2).

3.2. Manipulative study

3.2.1. Sediment analysis and the efficiency of experimental treatmentsAt the end of the experiment, analysis of the sediment revealed

significant differences in the percentages of coarser (and conse-quently finer) sand among treatments (Fig. 7, Appendix A — TableS6) and that these differences were consistent with the manipula-tion (e.g. greater % of coarser sand in pots assigned to coarse sandtreatment). No effects of cages (among levels of predation treat-ment)were detected (ANOVA P > 0.73), although therewas a signif-icant difference (ANOVA P b 0.001) in the relative percentages offiner and coarser sediments between treatments away and adjacentto the rocky reef (Fig. 7). This difference probably reflects some de-gree of sediment exchange between the experimental pots and thesurrounding substrate. But again, this exchange did not affect theoverall granulometry treatment. For instance, experimental potsassigned to the fine sand treatment retained a greater percentageof fine sand compared to pots assigned to the coarse sand treatment

Table 1Regression between grain size (PCA1) and the abundance of taxa. Positive and negativerelationships indicate increasing and decreasing abundances with increasing grain size,respectively. Significant relationships in bold. Taxa are ordered from the most to theleast abundant.

Taxa Slope r2 P

Exogone naidina −33.44 0.06 >0.07Nematoda +1.86 0.05 >0.09Ervilia castanea −1.88 0.08 b0.05Oligochaeta sp.1 +0.96 0.04 >0.15Spio filicornis −1.73 0.06 >0.07Armandia cirrhosa −0.49 0.04 >0.13Turbellaria sp.1 −0.27 b0.01 >0.54Ostracoda sp.1 −0.05 b0.01 >0.87

and this pattern was consistent in pots deployed adjacent and awayfrom the reef. Hence, sand manipulation was successful and the useof cages to exclude predators did not affect the sediment, suggestingthat the cages had little effect on the macrofaunal assemblagesthemselves.

3.2.2. Effects of predation, sediment grain size and distance to reefA total of 42,386 animals were counted and identified, a number

that was nearly 3 times larger than in the descriptive study. Bivalves(of which E. castanea was the dominant species) were numericallythe most abundant group accounting for 87.5% of all individuals.Malacostraca (represented by 14 species) and polychaetes (amongwhich Spio aff. filicornis was again one of the most abundant species)were the second and third most abundant groups accounting, howev-er, for only 5% and 3.5% of total individuals.

Sample richness differed significantly in the interaction betweensand granulometry and distance to the reef (Fig. 8, Appendix A — TableS7). SNK analyses revealed that richness was lower in fine sedimenttreatments deployed away from the reef, whereas fine sediment treat-ments adjacent to the reef, as well as coarse sediment (adjacent andaway from the reef), similarly supported greater numbers of taxa. Preda-tion did not affect the richness (ANOVA P > 0.5). Significant composi-tional differences (Jaccard's index) were found among assemblages asa function of distance to reef and sand granulometry (Fig. 9)(PERMANOVA P b 0.01, Appendix A — Table S8).

Among the most abundant taxa, the numbers of the paguridCatapaguroides timidus, and E. castaneawere only affected by distanceto the reef (Fig. 10, Appendix A — Table S9). Whereas C. timiduswas 2orders of magnitude more abundant in experimental treatments ad-jacent to the reef, E. castanea was 5 times more abundant awayfrom the reef (Fig. 10). Exclusion of predators significantly increasedthe abundance of amphipod sp.1 in relation to control and cage con-trol treatments (Fig. 10, Appendix A — Table S9). This effect was,however, similar both adjacent to and away from the reef. Therewas also a significant predation effect on the polychaete S. filicornis(Fig. 10, Appendix A — Table S9). However, SNK analyses (Table S9)showed that the abundance of S. filicornis was similar in the controland predator exclusion treatments (but more abundant in the cagecontrol), suggesting no effect of predator removal. For the nematodes,there was no significant effect of experimental treatments (Fig. 10,Appendix A — Table S9). None of these taxa was influenced by sandgrain.

3.2.3. Summary of results from the manipulative studyDespite significant effects of sediment grain size on multiple vari-

ables, we did not find strong support for our hypothesis that differ-ences in sediment grain size, as influenced by the presence of arocky reef, affect benthic assemblages, as this effect was always asso-ciated with a significant effect of distance to reef. Moreover, none of

Fig. 7. Percentage (mean + SE) of coarser and finer sand in experimental treatments. For simplicity, data are arranged by significance of effects as detected by ANOVA (D — distance,P — predation, G — granulometry). Letters above bars indicate significant differences as detected by ANOVA.

7G. M. Martins et al. / Journal of Sea Research 81 (2013) 1–9

the most abundant taxa examined was influenced by sand grain size.Generally, the exclusion of predators had no significant effect on theabundance of taxa and assemblage structure except in the case of am-phipod sp.1. Still, this predator effect was similar both adjacent to andaway from the reef which is not consistent with the hypothesis thatpredator-associated reefs influence the structure of sediment infaunaclose to reefs. Differences in the assemblage structure in relation tothe distance to the reef suggest the influence of factors other thanthose manipulated influence these communities.

4. Discussion

There were differences on the structure of macrofaunal assem-blages inhabiting sandy areas adjacent to rocky reefs and areas far-ther away despite spatial variation among reefs. This pattern, inagreement with the wider literature (Ambrose and Anderson, 1990;Barros et al., 2001; Davies et al., 1982; Posey and Ambrose, 1994), fur-ther supports the idea that rocky reefs influence the structure ofmacrofaunal assemblages on adjacent soft-bottom habitats. But ex-actly how do rocky reefs influence adjacent soft-bottom areas?

Exploratory data analyses indicated that macrofaunal assemblagesin areas adjacent to the reef were more spatially variable and had agreater number of exclusive species. These results suggest an ex-change of animals generally associated with rocky reefs with nearbysandy areas. It is unclear whether this exchange is a consequence ofpassive animal transport (e.g. fall of animals or transport withdecaying algae) or if these animals actively seek to cross this barrierto exploit resources in sandy areas. Either way, this flux of animalsbetween distinct habitats may represent an important allochthonoussource of food for secondary production (Nakano and Murakami,2001), such as invertivores fishes (e.g. M. surmuletus) living abovesandy bottom. The presence of caprellid species in sandy samples,previously found living among algae on rocky reefs (Bamber andRobbins, 2009), supports the idea of animal movement betweenrocky reefs and their adjacent sandy areas.

Fig. 8. Mean (+1SE) number of species and individuals in the experimental treat-ments. Figure details as in Fig. 7.

Sediment size is well known to influence the structure ofsoft-bottom assemblages (Gray, 1974). As in Barros et al. (2004), ourdescriptive study suggests that rocky reef could influence some taxathrough changes in sand grain size as indicated by the correlation be-tween sediment size and the abundance of the bivalve E. castanea. Themanipulative study, however, showed that while important at thescale of the assemblage (e.g. richness and composition) there were noeffects of grain size on the numbers of the most abundant taxa in themanipulative experiment, including E. castanea. These results suggestreefs influence nearby benthic assemblages over and above the effectsof changes in grain size; at least for the most abundant taxa. Moreover,this discrepancy between the observational and the manipulative re-sults for E. castanea punctuate the need for careful interpretation of re-sults based solely on correlative analysis.

Predation has also been predicted to influence soft-sediment as-semblages adjacent to rocky reefs (Barros, 2005; Langlois et al.,2005). In our study, no effects of predation were detected at thescale of the assemblage (richness and assemblage composition),and among the most abundant taxa. Only one amphipod speciesseemed to be affected by predators. However, this effect was consis-tent both adjacent and away from the reef. This result does not sup-port our hypothesis that reef-associated predators influence thestructure of sediment infaunal assemblages in areas adjacent to thereef. Generally Barros (2005) did not find a significant effect of pre-dation on sediment fauna communities, although this conclusioncontrasts the results of Langlois et al. (2005) who found a strong ef-fect of rocky reef-associated predators (rock lobsters) on adjacentsoft-bottom assemblages. In a subsequent study, these authors(Langlois et al., 2006b) found inconsistencies in the effects of rockyreef associated predators on adjacent assemblages of small-bodiedorganisms and argued that the effects of predation may be restrictedto larger-bodied prey. Given that the majority of macrofauna in our

Fig. 9. n-MDS showing composition (Jaccard's index) differences in the assemblages inthe experimental treatments: adjacent samples (filled circles); away samples (open cir-cles); coarser sand (circles); finer sand (squares); predator exclusion (−P); predator nat-ural (+P); procedural control (C). Centroids were used for clarity. Stress = 0.09.

8 G. M. Martins et al. / Journal of Sea Research 81 (2013) 1–9

study were small (b4 mm), this size-dependent model suggested byLanglois et al. (2006b) could explain the limited effect of predationwe observed. Another possible explanation is that rates of turnoverfor small-bodied soft-bottom sediment fauna may be so fast that itbecomes difficult to detect any predation effects. The large numberof individuals that colonised the experimental pots after onemonth of deployment nonetheless supports high turnover ratesand productivity in these communities.

The structure ofmacrofaunal assemblages in experimental pots gen-erally varied with distance to rocky reefs as suggested by differences inthe richness and composition as well as the abundance of two of themost abundant species (E. castanea and C. timidus). These results indi-cate that the influence of rocky reefs on nearby sandy assemblageswas important above and beyond the effects of predation. Other factorsmust therefore influence these communities. It is worth noting thatdecaying algae (sometimes in large amounts) tended to accumulate inareas adjacent to the reef. Detritus was shown to influence the typesand relative abundance of species in the sediment via changes in the or-ganic content and physical patchiness (e.g. Rossi and Underwood,2002) suggesting it could add to differences in assemblage structure

Fig. 10. Mean (+1SE) number of the most abundant taxa in experimental treatments. Lett(Appendix A — Table S9). Figure details as in Fig. 7.

with distance with rocky reefs. The presence of decaying macroalgaecan affect benthic assemblages shifting their trophic structure fromsuspension-feeders to detritivores (Norkko and Bonsdorff, 1996). Re-sults of the descriptive study support findings by Norkko andBonsdorff (1996) with significant greater abundance of detritivores(Ostracoda) close to the reefs and higher abundance of the filter-feeding E. castanea on samples away from the reef. Moreover, the highsmall-scale variability found close to the reef could represent the re-sponse of detritivores to the patchy distribution of decaying algae.

In the descriptive study, reefs were spatially replicated (3 loca-tions) and results showed that distance to reef had a pervasive andconsistent effect on assemblage structure over and above spatial var-iability. Sorting, identification, and counting sedimentary fauna is ex-tremely time-consuming, and given the consistent effects of distanceto reef in the descriptive study, we decided to restrict the manipula-tive study to only one location. It should be noted however that thisdecision limits the extrapolation of results to other locations andmust be interpreted with care.

It is also important to note that the response of some taxa variedbetween the descriptive and manipulative studies, perhaps because

ers above bars indicate significant effects of distance from reefs as detected by ANOVA

9G. M. Martins et al. / Journal of Sea Research 81 (2013) 1–9

the studies were done at different times of year with potential differ-ences in abundance of taxa. For instance, the bivalve E. castanea wasan order of magnitude more abundant during the manipulativestudy, whereas the polychaete E. naidina was an order of magnitudemore abundant during the descriptive study. All of these argumentssuggest that this ecosystem is extremely variable and future experi-ments should be replicated not only in space but also in time.

In summary, rocky reefs influence the structure of nearby sandymacrofaunal assemblages. Predation and sand grain size had little in-fluence, at least for the small-bodied taxa we examined. Even withmanipulations of sand grain size and predation, distance to reef stillaffected the structure of these assemblages, indicating other process-es must affect sediment infauna. For instance, passive transport ofmatter (decaying algae) and associated organisms (rocky reef associ-ated macrofauna) likely influences soft-bottom communities adjacentto reefs, although this effect is yet to be confirmed experimentally.Our results also suggest that seasonal variation in animal recruitmentmay be important in this system.

Linkages between adjacent habitats can clearly impact biologicalcommunities. From a conservation perspective, failure to detectsuch linkages can negatively influence our ability to evaluate thefull biodiversity of a habitat. Moreover, understanding the functionalconsequences of such linkages, as well as their effect on the abun-dance, movement and growth of marine organisms is essential forthe management of habitats which must consider the wider and het-erogeneous landscape.

Acknowledgements

During this study, GMM was supported by a post-doctoral grantawarded by Fundação para a Ciência e Tecnologia, (FCT, Portugal)(SFRH/BDP/63040/2009). During the last part of this studyMRwas sup-ported by a post-doctoral grant awarded by FCT (SFRH/BDP/81567/2011). Field assistance was provided by Afonso Prestes, João Brumand Nuno Álvaro. We also thank Sérgio Ávila for mollusc identificationand the Geosciences Department from the University of Azores for pro-viding the equipment and facilities for sediment analyses. This researchwas partially supported by the European Regional DevelopmentFund (ERDF) through the COMPETE — Operational CompetitivenessProgramme and national funds through FCT — Foundation for Sci-ence and Technology, under the project “PEst-C/MAR/LA0015/2011”. Two anonymous reviewers provided insightful comments inthis manuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.seares.2013.03.007.

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