Ragworm fatty acid profiles reveals habitat and trophic interactions with halophytes and with mercury

Marine Pollution Bulletin 64 (2012) 2528–2534

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Baseline

Edited by Bruce J. Richardson The objective of BASELINE is to publish short communications on different aspects of pollution of the marine environment. Only those papers which clearly identify the quality of the data will be considered for publication. Contributors to Baseline should refer to ‘Baseline—The New Format and Content’ (Mar. Pollut. Bull. 60, 1–2).

Ragworm fatty acid profiles reveals habitat and trophic interactions with halophytes and with mercury Ana Isabel Lillebø a,⇑, Daniel Francis Richard Cleary a, Bruna Marques a, Alberto Reis b, Teresa Lopes da Silva b, Ricardo Calado a a b

Departmento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal Unidade de Bioenergia, Laboratório Nacional de Energia e Geologia, I.P. (LNEG), Estrada do Paço do Lumiar, 22 Edifício F, 1649-038 Lisboa, Portugal

a r t i c l e

i n f o

Keywords: Fatty acid trophic markers Salt marshes Hediste diversicolor Sediment Hg

a b s t r a c t The present study aimed to assess if ragworm fatty acids (FA) profiles could be used to discriminate their spatial distribution in an historically mercury-contaminated estuarine environment, i.e., if it was possible to differentiate ragworms present in salt marsh sediments surrounding plant roots and rhizomes (rhizosediment) from adjacent unvegetated sediment. Additionally, we also tried to determine if ragworms differed in mercury content and if these values could also be used to identify the habitat they occur in. Results show that, within the same area, ragworms can be distinguished using FA profiles and that in halophyte rhizosediment ragworms display more than twice the levels of alpha-linolenic acid (18:3n-3). The ratio cis-vaccenic/oleic acids (18:1n-7/18:ln-9) in ragworms suggests higher carnivory in unvegetated sediments. Our study indicates that ragworm FA profiles can be used to identify their habitat, their trophic interaction with halophytes and reveal a spatially contrasting feeding behaviour, which also reflects mercury accumulation. Ó 2012 Elsevier Ltd. All rights reserved.

The study of trophic interactions in spatially complex ecosystems is of vital importance for understanding their functioning. In highly dynamic marine and estuarine environments it is often impossible to perform direct observations on the feeding habits displayed by organisms. To overcome this constraint researchers need to employ indirect methods to clarify trophic relationships (Würzberg et al., 2011). Biochemical tracing methodologies, namely the application of fatty acid (FA) trophic markers, have been successfully used to determine the longer-term dietary information of numerous marine and estuarine species (Dalsgaard et al., 2003; Kelly and Scheibling, 2012). The generalised use of the FA marker concept relies on the large structural diversity displayed by fatty ⇑ Corresponding author. Tel.: +351 234 370 779. E-mail address: [email protected] (A.I. Lillebø). 0025-326X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2012.08.009

acids, along with pronounced taxonomic specificity (Graeve et al., 2002; Kelly and Scheibling, 2012), and the conservative way that certain FA’s are incorporated into body tissues (Lee et al., 2006). Several studies have already assessed the FA signatures of primary producers, namely micro- and macroalgae, as well as vascular plants (see Kelly and Scheibling, 2012 for a review). Vascular plants can enter coastal benthic food webs, either by direct grazing by primary consumers or through detrital pathways. The most commonly used fatty acid tracers to identify the contribution of vascular plants in benthic food webs are: (1) long chain fatty acids (LCFA) of up to 28 carbons, commonly present in their waxy leaf cuticle (Gurr et al., 2002); and (2) the polyunsaturated fatty acids (PUFAs) 18:2n-6 (Linoleic acid, LA) and 18:3n-3 (Alpha-linolenic acid, ALA) (Dalsgaard et al., 2003). The FAs 18:2n-6 and 18:3n-3 can also be used as signature fatty acids for green algae, because

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green algae and terrestrial plants share common ancestors (Raven et al., 1992). In this way, the use of single FA’s as trophic tracers must always be performed with caution. Salt marshes are a good example of spatially complex ecosystems where intricate trophic interactions with vascular plants are known to occur in benthic food webs. Found predominantly in low-energy environments along sheltered coastlines with temperate climates, these key ecotones provide essential ecosystem services. They are also among the most productive of the biosphere, and although marsh composition and aboveground primary production (APP) varies according to marsh maturity, the majority of salt marshes provide abundant macrodetritus, i.e., aboveground senescent plant tissues, that can be exported to coastal waters (Valiela et al., 2000; Lefeuvre et al., 2003). The belowground primary production (BPP) is much less available for export by tides, and remains in the sediment as organic matter, where it is either consumed by micro and macro organisms or together with inorganic sedimentation drives salt marsh accretion (Mattheus et al., 2010; Chmura, in press). Salt marsh ecosystem functions and services are well recognised (Barbier et al., 2011); primary producers in these ecosystems play crucial roles in nutrient dynamics with adjacent ecosystems and buffer excess nutrients and contaminants from land-based sources (Sousa et al., 2011). Salt marsh plants are able to reduce metal bioavailability through phytoaccumulation, phytostabilization or metal rhizofiltration (Ghosh and Singh, 2005). Previous studies in contaminated salt marshes have shown that the accumulation of metals occurs mainly in the belowground biomass and that aboveground macrodetritus should not be a significant source of metals to adjacent marsh areas (Almeida et al., 2006; Castro et al., 2006). Mercury (Hg) is included in the list of high priority environmental pollutants and is one of the most hazardous contaminants present in coastal environments (Pereira et al., 2009 and references

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therein). Due to the deleterious effects of Hg on ecosystem functioning, namely through transference within the food web, Hg-contaminated salt marshes resulting from historical contamination are a cause of concern. This is the case with the Laranjo basin, a shallow area of approximately 2 km2, located in the Ria de Aveiro coastal lagoon (40°380 N, 8°440 W), that is contaminated with mercury (Pereira et al., 2009). In Laranjo, with the exception of the area closest to the point source, Juncus maritimus Lam. and Bolboschoenus maritimus L. Palla (formerly known as Scirpus maritimus L.) are the most abundant salt marsh halophytes covering, respectively, 30% and 50% of the Hg-contaminated salt marsh area (Válega et al., 2008). J. maritimus is widely distributed in Europe, West Africa and North Asia, occurring on permanently wet soils along littoral salt marshes (Menéndez, 2008). B. maritimus is also widely distributed in European and North American marshes (Peláez et al., 1998) where it forms dense monospecific stands in shallow brackish marshes during spring and summer. In the Laranjo salt marsh, the APP and BPP of both halophytes corresponds to 92% and 8%, respectively (Marques et al., 2011). The APP of J. maritimus and B. maritimus (hereafter referred to as Juncus and Bolboschoenus) was similar, respectively 1166 and 1100 g DW m2 y1, as well as the BPP, respectively 107 and 93 g DW m2 y1 (Marques et al., 2011). In the below-ground biomass, the median concentration of Hg at 5–15 cm depth was respectively 0.90 and 1.33 ng Hg mg1, with no clear seasonal trend; this is thirty to forty times lower than in halophyte rhizosediment (Marques et al., 2011). Knowing the fate of Hg in contaminated salt marshes is crucial to understanding the repercussions that this kind of contaminant may have within the ecosystem. The ragworm Hediste diversicolor (O.F. Müller, 1776) (previously known as Nereis diversicolor) is a burrowing species and therefore in close contact with sediment, the belowground primary production (BPP) and the Hg-contaminated detritus pool. H. diversicolor is one of the most widely distributed marine polychaetes in the NE

Fig. 1. The location of Ria de Aveiro coastal lagoon with the location of the Laranjo salt marsh, historically contaminated by mercury.

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Atlantic and one of the few of economic value. It exhibits an omnivorous type of feeding mode by swallowing sediment (Luis and Passos, 1995) and is a potential prey for crabs, birds and fish. H. diversicolor occupies a key position in the trophic chain with respect to Hg accumulation and transference to higher trophic levels (Coelho et al., 2008). The present study aimed to identify whether signature fatty acids of salt marsh vascular plants (halophytes) present in the belowground biomass were also present in ragworms occupying mudflats colonised either by Juncus or Bolboschoenus, and if ragworm fatty acid profiles could be used to discriminate their spatial distribution in an estuarine environment (ragworms from an adjacent area with no vascular plants were used as a reference site). Additionally, our study also tried to determine whether ragworms present in mudflats colonised by vascular plants in an estuarine area contaminated with mercury (Hg) displayed contrasting levels of this contaminant and if these values could also be used to identify the habitat they colonise. Sediment samples and ragworms were collected during the winter (January 2011) respectively, from an area colonised by Juncus, an area colonised by Bolboschoenus, and an adjacent unvegetated area (Fig. 1). This season was chosen to reduce the potential interference of microphytobenthos on the pool of fatty acids recorded for halophytes. All sampling locations were located in the same salt marsh bank and were less than 20 m apart. Nine sediment corers were collected per area using a steel corer (Ø 7 and 15 cm depth) and stored separately in polyethylene plastic bags. At least 20 adult ragworms (to account for within-site variability) were collected per area with small gardening shovels and placed in 50 ml PP/HDPE sample containers for transportation. In situ sediment temperature and pH were measured in triplicate per area with the field set: WTW pH 330i/set and SenTixÒ 41 probe (WTW Company). In the laboratory, sediment cores were pulled together, to form three different sediment samples, each composited of three cores. The rhizosediment samples from the areas colonised by Juncus and by Bolboschoenus were carefully separated from the belowground plant material and homogenised. The sediment samples from the adjacent unvegetated area was cleaned of shells and homogenised. Afterwards, all sediment samples were freeze-dried at 50 °C and 0.06 bar, and stored at 32 °C for later Hg and biochemical analyses. Sediment homogenised sub-samples were analysed for the percentage of fine particle (<63 mm) content. The belowground plant material was carefully rinsed with demineralised water, freeze-dried and homogenised with a glass mortar and pestle, and stored as described for sediment samples. Since H. diversicolor is a sediment-ingesting species, the collected organisms were left to depurate overnight for gut cleansing. Afterwards, ragworms were freeze-dried (in three composed samples with 20 adult specimens from each sampling area), homogenised and stored for Hg and biochemical analyses as previously described. Fatty acid extraction and preparation of methyl esters were carried out according to Lepage and Roy (1986) modified by Cohen et al. (1988). Freeze-dried samples (100 mg) were transmethylated with 5 ml of methanol/acetyl chloride (95:5 v/v). The mixture was sealed in a light-protected Teflon-lined vial under nitrogen atmosphere and heated at 80 °C for 1 h. The vial contents were then cooled, diluted with 1 ml water, and extracted with 2 ml of n-heptane. The heptane layer was dried over Na2SO4, evaporated to dryness under a nitrogen atmosphere and redissolved in heptane, which contained the methyl esters. The methyl esters were then analysed by gas–liquid chromatography, on a Varian CP-3800 gas chromatograph with a flame ionization detector (Varian Inc., Palo Alto, CA, USA). Separation was carried out on a 0.32 mm  30 m fused silica capillary column (film 0.32 lm) Supelcowax 10 (SUPELCO, Bellafonte, PA, USA) with helium as the carrier gas at a flow

rate of 1.3 ml min1. The column temperature was programmed at an initial temperature of 200 °C for 10 min, then increased at 4 °C min1 to 240 °C and held there for 16 min. Injector and detector temperatures were 250 and 280 °C, respectively, and the split ratio was 1:100. Peak identification was carried out using known standards (GC 462, Nu-Chek-Prep, Elysian, USA). Peak areas were determined using the Varian software and FA 21:0 was used as an internal standard. The detection limit for the FAs analysed was 0.01 lg mg1 dry weight. Unvegetated sediment, rhizosediment and ragworm samples were analysed for total mercury by thermal decomposition atomic absorption spectrometry with gold amalgamation, using a LECO AMA-254 (Advanced Mercury Analyzer). All mercury quantifications were done in triplicate and blank procedures always being run in parallel. In order to assess the accuracy and precision of the analytical methodology, analyses of certified reference materials were carried out (PACS-2 harbour sediment; TORT-2 lobster hepatopancreas) in parallel with samples and procedure blanks. Certified and measured values were in line with recoveries between 93% and 106% and 91–92%, respectively. A square matrix containing the fatty acid composition of sampled plant roots, sediment and ragworms was imported into R (http://www.r-project.org/; last checked 2012-08-02), log10(x + 1) transformed and a distance matrix constructed using the Euclidean index with the vegdist() function in the vegan package in R (Oksanen et al., 2008). Variation in the fatty acid composition of plant roots, sediment and polychaetes was assessed with Principal Coordinates Analysis (PCO) using the cmdscale() function in R with the Euclidean distance matrix as input. Variation between the roots of different plants, sediments and polychaetes was tested for significance using the adonis() function in vegan. The adonis() function performs an analysis of variance with distance matrices using permutations that partitions distance matrices among sources of variation. In the adonis analysis, the Euclidean distance matrix of fatty acid composition was the response variable with belowground plant material, sediment and ragworms the independent variables. The number of permutations was set at 999; all other arguments used the default values set in the function. In addition to testing for significant differences in the composition of fatty acids, we also tested for differences in the contribution of total SAT (saturated fatty acids), MUFA (monounsaturated fatty acids), PUFA (excluding HUFA, highly unsaturated fatty acids) and HUFA in the total pool of fatty acids. Because the distributions of all of these deviated significantly from normal expectations (shapiro test, P > 0.05), we used adonis analyses. The response variable consisted of the Euclidean distance matrix for SAT, MUFA, PUFA and HUFA. For each response variable, we tested for significant differences in concentration among the isolation source (namely, rhizosediment, sediment or ragworms). In addition to this, we also tested for significant differences in the concentrations of SAT, MUFA, PUFA and HUFA in ragworms sampled in different habitats (namely, Juncus sediment, Bolboschoenus sediment or unvegetated sediment).

Table 1 Physicochemical parameters in the Hg-contaminated area colonised by Juncus and by Bolboschoenus and in the adjacent unvegetated area (no plant): pH and percentage of fine particles (<63 mm) (mean value ± STD, N = 3). Different letters in values displayed in the same column represent significant differences at P < 0.05.

Juncus Bolboschoenus No plant

pH

% Fine particles (<63 mm)

6.4 ± 0.2a 6.5 ± 0.1a 7.2 ± 0.0b

51.3 ± 8.1a 44.5 ± 4.7a,b 32.2 ± 5.5b

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Table 2 Fatty acid profiles (% of total fatty acids) of belowground biomass (B_biomass) of salt marsh plants Juncus and Bolboschoenus, sediment samples of mud flats colonised by each of these plants (rhizosediment) and without vegetation (no plant) and ragworms H. diversicolor collected in mud flats colonised by J. maritimus, or Bolboschoenus or without vegetation. Only the most representative fatty acids are presented. Different letters in values displayed in the same row and with the same shade represent significant differences at P < 0.05. Values are average of three replicates ± standard deviation. ND – fatty acid not detected.

SFA, saturated fatty acids: 10:0, 12:0, 14:0, 16:0, 18:0, 20:0, 22:0. MUFA, monounsaturated fatty acids: 14:1n-5, 16:1n-7, 18:1n-9, 18:1n-7, 20:1n-9, 22:1n-9, 24:1n-9. PUFA, Polyunsaturated fatty acids: 18:2n-6; 18:3n-6; 18:3n-3; 20:2n-6; 20:3n-6; 20:3n-3, 22:2n-6. HUFA, Highly unsaturated fatty acids: 20:4n-6; 20:5n-3; 22:4n-6, 22:5n-3, 22:6n-3. P A Although PUFA are defined as all FA with P2 double bonds, in the present study HUFA (FA with P4 double bonds) are not considered within PUFA.

The average in situ sediment temperature at time of sampling ranged between 11.8 and 13.3 °C. Sediment physicochemical parameters determined in situ and in the laboratory (mean value ± STD, N = 3) are summarised in Table 1. Compared to the unvegetated sediment, Juncus and Bolboschoenus rhizosediments had significantly lower pH values (respectively, P < 0.05 and P < 0.001). The percentage of fine particles (<63 mm) in the unvegetated sediment was significantly lower (P < 0.05) than in Juncus rhizosediment, but did not differ significantly from Bolboschoenus rhizosediment. The fatty acid profiles of belowground plant material, sediment and ragworms are summarised in Table 2. The most abundant FA for belowground plant material was 18:2n-6 (LA) for Juncus and Bolboschoenus, although it was more abundant in the Juncus samples. 18:3n-3 (ALA, Fig. 2) was more abundant in Juncus than Bolboschoenus, rhizosediment, and was completely absent from unvegetated sediment. There were significant differences in the concentrations of SAT (F2,21 = 241.03, P < 0.001, R2 = 0.958), MUFA (F2,21 = 20.76, P < 0.001, R2 = 0.660), PUFA (F2,21 = 45.10, P < 0.001, R2 = 0.811) and HUFA (F2,21 = 77.62, P < 0.001, R2 = 0.881) among sources of isolation, i.e., roots, sediment or ragworms (Fig. 2). In addition to this, there were also significant differences in the concentrations of SAT (F2,6 = 19.60, P = 0.025, R2 = 0.867), MUFA (F2,6 = 6.70, P = 0.006, R2 = 0.691), PUFA (F2,6 = 6.60, P = 0.015, R2 = 0.687) and HUFA (F2,6 = 35.40, P = 0.018, R2 = 0.922) in ragworms from different habitats, namely, Juncus, Bolboschoenus or unvegetated sediment (Fig. 2). The major differences in fatty acid composition were related to differences among sources of isolation (ragworms, roots or sediment) with sediment characterised by high concentrations of saturated fatty acids (SFA), roots by high concentrations of 22:1n-9, and ragworms by high concentrations of 20:4n-6, 20:1n-12, 20:1n-9, 22:3, 20:5n-3 and 24:-0 (Fig. 3a). The differences in composition among sources of isolation were highly significant (F2,6 = 130.34, P < 0.001, R2 = 0.925). There were also significant differences (F2,6 = 55.08, P = 0.005, R2 = 0.948) in the fatty acid composition of sediment from different habitats (namely, Juncus,

Bolboschoenus or unvegetated sediment). The difference in composition among habitats was primarily related to the absence of 16:1n-7 and 18:1n-7 from sediment samples collected from the unvegetated sediment and absence of 20:3n-3 from the vegetated areas; the distinction between the unvegated sediment and vegetated (Bolboschoenus or Juncus) rhizosediment is highlighted in Fig. 3. Samples collected from Juncus rhizosediment displayed significantly higher (P < 0.001) levels of 18:0 (stearic acid) than those collected from Bolboschoenus There was a marginally non-significant difference in fatty acid composition between Juncus and Bolboschoenus roots (F1,4 = 95.66, P = 0.10, R2 = 0.960). The variation between roots of both species was primarily related to higher concentrations of 18:0 in Bolboschoenus and higher concentrations of 24:0, 22:6n-3 and 20:5n-3 in Juncus. There was a significant difference in the composition of fatty acids from ragworms collected from different habitats (F1,4 = 40.46, P = 0.002, R2 = 0.931). The concentrations of fatty acid 18:2n-6 and 18:3n-3 (ALA) was substantially higher in the FA pool of ragworms collected from areas colonised by halophytes (Table 2). Together, 18:2n-6 and 18:3n-3 represented nearly 20% of all PUFA (excluding HUFA) recorded in ragworms from areas colonised by halophytes, and only about 10% of total PUFA for ragworms occupying unvegetated sediments. Ragworms from unvegetated sediments displayed substantially higher levels of oleic acid (18:1n-9) (as well as lower cis-vaccenic/oleic acids ratio, 18:1n-7/18:ln-9). Mercury concentrations in the ragworms collected from Juncus and Bolboschoenus rhizosediment were similar (P > 0.05) (respectively, 1.23 ± 0.02 and 0.77 ± 0.01 ng Hg mg1, analytical STD). Mercury concentrations in ragworms collected from unvegetated sediment (1.88 ± 0.08 ng mg1 ± analytical STD) were similar (P > 0.05) to concentrations in ragworms collected from Juncus rhizosediment, but significantly different (P < 0.05) from concentrations in ragworms collected from Bolboschoenus rhizosediment. Mercury concentrations in halophyte rhizosediment (0–5 cm depth) for Juncus and Bolboschoenus were respectively 17.40 ± 1.17 ng Hg mg1 and 21.48 ± 6.33 ng Hg mg1. Mercury concentration in the

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Fig. 2. Barplots (error bars represent 1 standard deviation) representing the concentration of (a) SAT, (b) MUFA, (c) PUFA, (d) HUFA, (e) 18:3n-3 and (f) 20:3n-3 in ragworms, roots and sediment collected from area colonised by J. maritimus and B. maritimus and adjacent unvegetated sediment.

adjacent unvegetated sediment (0–5 cm depth) was 16.44 ± 0.09 ng Hg mg1. The concentration of mercury between halophyte rhizosediment and the adjacent unvegetated sediment did not differ significantly (P > 0.05). Differences between the sediment physicochemical parameters in areas colonised by Juncus or Bolboschoenus and the adjacent unvegetated sediment, are due to the ability of these halophytes to change the environmental biochemistry of the rhizosediment (Oliveira et al., 2010). Additionally, halophytes can act as sediment traps, allowing the accumulation of fine sediments and playing an important role in the settling of suspended matter (Chmura, in press). Despite their similar APP and BPP (Marques et al., 2011), these two halophytes differ in their annual dynamics: Juncus is an annual plant with a continuous and slow growth, in which leaf senescence progresses from the tip towards the base, and they become fully senescent after 230–250 days (Menéndez, 2008); the growth of Bolboschoenus stands can be divided into three phases: (i) a juvenile phase (winter/early spring); (ii) a mature phase, with higher shoot density and height (late spring/early summer) and; (iii) a senescent phase, with a dominance of belowground plant parts (late summer–winter) (Marques et al., 2011). In January, new shoots emerge but the total biomass is still dominated by the belowground biomass (Marques et al., 2011). The different aboveground biomass dynamics displayed by Juncus and Bolboschoenus at the time of sampling may explain the contrasting fatty acid signatures recorded in the present study for these two halophytes. The higher levels of SAT and MUFA displayed by Bolboschoenus probably reflect the translocation of nutri-

ents from senescent leaves to belowground biomass (Charpentier et al., 1998), while the higher PUFA content of Juncus is probably related to its continuous growth. The absence of 16:1n-7 and 18:1n-7 from sediment samples collected from mudflats may be related to a higher abundance of bacteria in halophyte rhizosediment (Oliveira et al., 2010; Cleary et al., 2012); their ability to biosynthesise large amounts of these FAs is well known (Volkman et al., 1998). While these FA’s are also commonly used as trophic markers for diatoms (Dalsgaard et al., 2003), their absence from sediment samples, along with the sampling season (winter), suggests that bacteria may have been responsible for the differences recorded. Signature FA’s, namely 18:3n-3, reveal a trophic contribution of below-ground halophyte biomass to ragworms, as ragworms in areas with no halophytes (i.e., unvegetated sediment) displayed less than half the content of this fatty acid. The absence of LCFA in the fatty acid profiles of ragworms collected in vegetated areas can be explained by the fact that these fatty acids accumulate in the waxy leaves of vascular plants and not in their below-ground biomass. Additionally, the above ground biomass of these two halophytes in the study area (Laranjo basin) is mostly exported (up to 90% in Juncus) (Marques et al., 2011), thus is less trophically available to ragworms. Budge and Parrish (1998) defined that if 18:2n-6 + 18:3n-3 represented more than 2.5% of the total pool of fatty acids of plankton samples it could be considered that terrestrial material was a significant source of organic matter. If the same threshold value is used for the present study, the combined percentage of 18:2n-6 and 18:3n-3

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Fig. 3. Principal Coordinates Analysis (PCO), Axis 1 and 2 are shown of fatty acid profiles sampled from (a) sediment, roots and ragworms combined, (b) sediment only, (c) roots only and (d) ragworms only. Samples from isolation sources and habitats are indicated by distinct symbol-shade combinations (Ju, Juncus; Bo, Bolboschoenus; M, unvegetated sediment; Rh, belowground biomass (rhizomes and roots), Sd, Sediment; Ra, ragworms). Codes in the figures represent fatty acids.

recorded for ragworms sampled in vegetated areas clearly exceeds this value (3.3% and 3.5% for ragworms in Juncus and Bolboschoenus rhizosediment, respectively). While our study confirms the validity of using C18 PUFAs as useful markers of vascular plants, it must be highlighted that these FA’s are not unique to halophytes and other potential sources (macroalgae and seagrasses) may also be involved in the trophic dynamics of FA’s (Hall et al., 2006). In addition, as already highlighted by Kelly and Scheibling (2012), certain invertebrate taxa display the ability to modify dietary FA’s, thus masking potential trophic interactions through their FA profiles. Caution must also be taken when comparing FA profiles recorded in other studies for the same target organism (Luis and Passos, 1995), as methodological aspects related to the collection of specimens (buying from commercial fishing bait wholesalers vs. sampling in situ) and the processing of ragworms prior to biochemical analysis (processing of non depurated ragworms vs. ragworms depurated for gut cleansing) may cause significant bias to FA profiles. As revised by Dalsgaard et al. (2003), lower values of the cis-vaccenic/ oleic acids ratio (18:1n-7/18:ln-9) can be used as a proxy to infer the predominance of carnivory vs. herbivory in marine animals (as cis-vaccenic acid (18:1n-7) is present in very low levels in

animal tissues). In our study, the lowest 18:1n-7/18:ln-9 ratio was displayed by ragworms collected in unvegetated areas, thus reinforcing the lack of contribution of halophyte below-ground biomass detritus to their FA pool. Ragworms collected in unvegetated areas also presented significantly higher concentrations of Hg compared to ragworms collected from Bolboschoenus rhizosediment. This suggests that, despite the similar concentration of Hg in the sediment, carnivory may lead to a higher transference of Hg from prey to predator. At vegetated areas, it is possible that halophytes auto-remediate the systems by reducing Hg availability, thus representing an ecosystem service provided through Hg phytostabilisation and/or phytoaccumulation (Marques et al., 2011). In conclusion, this study shows that FA profiles of ragworms can provide important information on their habitat and feeding behaviour, including Hg accumulation. Acknowledgements Portuguese Foundation for Science and Technology supported this study through the project (PTDC/MAR/67752/2006) FCOMP01-0124-FEDER-007378 (also supported by FEDER funding through COMPETE-Programa Operacional Factores de Competiti-

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vidade). FCT funding through CESAM (Centre for Environmental and Marine Studies) is also acknowledged. References Almeida, C.M.R., Mucha, A.P., Vasconcelos, M.T.S.D., 2006. Comparison of the role of the sea club-rush Scirpus maritimus and the sea rush Juncus maritimus in terms of concentration, speciation and bioaccumulation of metals in the estuarine sediment. Environ. Pollut. 142, 151–159. Barbier, E.B., Hacker, S.D., Kennedy, C., Koch, E.W., Stier, A.C., Silliman, B.R., 2011. The value of estuarine and coastal ecosystem services. Ecol. Monogr. 81, 169– 193. Budge, S.M., Parrish, C.C., 1998. Lipid biogeochemistry of plankton, settling matter and sediments in Trinity Bay Newfoundland. II. Fatty acids.. Org. Geochem. 29, 1547–1559. Castro, R., Pereira, S., Lima, A., Corticeiro, S., Válega, M., Pereira, E., Duarte, A., Figueira, E., 2006. Accumulation, distribution and cellular partitioning of mercury in several halophytes of a contaminated salt marsh. Chemosphere 76, 1348–1355. Charpentier, A., Mesléard, F., Thompson, J.D., 1998. The effects of rhizome severing on the clonal growth and clonal architecture of Scirpus maritimus. Oikos 83, 107–116. Chmura, G.L., in press. What do we need to assess the sustainability of the tidal salt marsh carbon sink? Ocean Coast. Manage. http://dx.doi.org/10.1016/ j.ocecoaman.2011.09.006. Cleary, D.F.R., Oliveira, V., Gomes, N.C.M., Pereira, A., Henriques, I., Marques, B., Almeida, A., Cunha, A., Correia, A., Lillebø, A.I., 2012. Impact of plant species on local environmental conditions, microbiological parameters and microbial composition in a historically Hg-contaminated salt marsh. Mar. Pollut. Bull. 64, 263–271. Coelho, J.P., Nunes, M., Dolbeth, M., Pereira, M.E., Duarte, A.C., Pardal, M.A., 2008. The role of two sediment-dwelling invertebrates on the mercury transfer from sediments to the estuarine trophic web. Est. Coast. Shelf Sci. 78, 505–512. Cohen, Z., Vonshak, A., Richmond, A., 1988. Effect of environmental conditions on fatty acid composition of the red algae Porphyridium cruentum: correlation to growth rate. J. Phycol. 24, 328–332. Dalsgaard, J., St John, M., Kattner, G., Muller-Navarra, D., Hagen, W., 2003. Fatty acid trophic markers in the pelagic marine environment. Adv. Mar. Biol. 46, 225– 340. Ghosh, M., Singh, S.P., 2005. A review on phytoremediation of heavy metals and utilization of its byproducts. Appl. Ecol. Environ. Res. 3, 1–18. Graeve, M., Kattner, G., Wiencke, C., Karsten, U., 2002. Fatty acid composition of Arctic and Antarctic macroalgae: indicator of phylogenetic and trophic relationships. Mar. Ecol. Prog. Ser. 231, 67–74. Gurr, M.I., Harwood, J.L., Frayn, K.N., 2002. Lipid Biochemistry. Blackwell Science, Oxford. Hall, D., Lee, S.Y., Meziane, T., 2006. Fatty acids as trophic tracers in an experimental estuarine food chain: tracer transfer. J. Exp. Mar. Biol. Ecol. 336, 42–53.

Kelly, J.R., Scheibling, R.E., 2012. Fatty acids as dietary tracers in benthic food webs. Mar. Ecol. Prog. Ser. 446, 1–22. Lefeuvre, J.-C., Laffaille, P., Feunteun, E., Bouchard, V., Radureau, A., 2003. Biodiversity in salt marshes: from patrimonial value to ecosystem functioning. The case study of the Mont-Saint-Michel bay. C. R. Biol. 326, 125–131. Lepage, G., Roy, C.C., 1986. Direct transesterification of all classes of lipids in onestep reaction. J. Lipid Res. 27, 114–120. Marques, B., Lillebø, A.I., Pereira, E., Duarte, A.C., 2011. Mercury cycling and sequestration in salt marshes sediments: an ecosystem service provided by Juncus maritimus and Scirpus maritimus. Environ. Pollut. 159, 1869–1876. Mattheus, C.R., Rodriguez, A.B., McKee, B.A., Currin, C.A., 2010. Impact of land-use change and hard structures on the evolution of fringing marsh shorelines. Est. Coast. Shelf Sci. 88, 365–376. Menéndez, M., 2008. Leaf growth, senescence and decomposition of Juncus maritimus Lam in a coastal Mediterranean marsh. Aquat. Bot. 89, 365– 371. Oksanen, J., Kindt, R., Legendre, P., O’Hara, R.B., Simpson, G.L., Solymos, P., Stevens, M.H.H., Wagner, H., 2008. Vegan: community ecology package version 1.15-1, http://www.cran.r-project.org/, http://www.vegan.r-forge.r-project.org/. Oliveira, V., Santos, A.L., Coelho, F., Gomes, N.C.M., Silva, H., Almeida, A., Cunha, A., 2010. Effects of monospecific banks of salt marsh vegetation on sediment bacterial communities. Microb. Ecol. 60, 167–179. Peláez, F., Collado, J., Arenal, F., Basilio, A., Cabello, A., Díez Matas, M.T., García, J.B., González del Vale, A., González, V., Gorrochategui, J., Hernández, P., Martin, I., Platas, G., Vicente, F., 1998. Endophytic fungi from plants living on gypsum soils as a source of secondary metabolites with antimicrobial activity. Mycol. Res. 102, 755–761. Pereira, M.E., Lillebø, A.I., Pato, P., Válega, M., Coelho, J.P., Lopes, C., Rodrigues, S., Cachada, A., Otero, M., Pardal, M.A., Duarte, A.C., 2009. Mercury pollution in Ria de Aveiro (Portugal): a review of the system assessment. Environ. Monit. Assess. 155, 39–49. Sousa, A.I., Lillebø, A.I., Pardal, M.A., Caçador, I., 2011. Influence of multiple stressors on the auto-remediation processes occurring in salt marshes. Mar. Pollut. Bull. 62, 1584–1587. Válega, M., Lillebø, A.I., Pereira, M.E., Duarte, A.C., Pardal, M.A., 2008. The longterm effects of mercury in a salt marsh: hysteresis in the distribution of vegetation following recovery from contamination. Chemosphere 71, 765–772. Valiela, I., Cole, M.L., Mcclelland, J., Hauxwell, J., Cebrian, J., Joye, S.B., 2000. Role of salt marshes as part of coastal landscapes. In: Weinstein, M.P., Kreeger, D.A. (Eds.), Concepts and Controversies in Tidal Marsh Ecology. Kluwer Academic Publishing, Dordrecht, The Netherlands, pp. 23–38. Volkman, J.K., Barrett, S.M., Blackburn, S.I., Mansour, M.P., Sikes, E.L., Gelin, F., 1998. Microalgal biomarkers: a review of recent research developments. Org. Geochem. 29, 1163–1179. Würzberg, L., Peters, J., Schüller, M., Brandt, A., 2011. Diet insights of deep-sea polychaetes derived from fatty acid analyses. Deep Sea Res. Part II 58, 153–162.