Biogeochemistry DOI 10.1007/s10533-011-9630-y
Tracking seasonal changes in North Sea zooplankton trophic dynamics using stable isotopes Benjamin Ku¨rten • Suzanne J. Painting • Ulrich Struck • Nicholas V. C. Polunin • Jack J. Middelburg
Received: 27 July 2010 / Accepted: 24 July 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Trophodynamics of meso-zooplankton in the North Sea (NS) were assessed at a site in the southern NS, and at a shallow and a deep site in the central NS. Offshore and neritic species from different ecological niches, including Calanus spp., Temora spp. and Sagitta spp., were collected during seven cruises over 14 months from 2007 to 2008. Bulk stable isotope (SI) analysis, phospholipid-derived fatty acid (PLFA) compositions, and d13CPLFA data of mesozooplankton and particulate organic matter (POM) were used to describe changes in zooplankton relative trophic positions (RTPs) and trophodynamics. The aim of the study was to test the hypothesis that the RTPs of zooplankton in the North Sea vary spatially
Electronic supplementary material The online version of this article (doi:10.1007/s10533-011-9630-y) contains supplementary material, which is available to authorized users. B. Ku¨rten (&) Leibniz Institute of Marine Sciences (IFM-GEOMAR), Research Division Marine Ecology, Du¨sternbrooker Weg 20, 24105 Kiel, Germany e-mail:
[email protected] S. J. Painting Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Pakefield Road, Lowestoft NR33 0HT, UK U. Struck Museum fu¨r Naturkunde, Leibniz-Institut fu¨r Evolutionsund Biodiversita¨tsforschung an der Humboldt-Universita¨t zu Berlin, Invalidenstraße 43, 10115 Berlin, Germany
and seasonally, in response to hydrographic variability, with the microbial food web playing an important role at times. Zooplankton RTPs tended to be higher during winter and lower during the phytoplankton bloom in spring. RTPs were highest for predators such as Sagitta sp. and Calanus helgolandicus and lowest for small copepods such as Pseudocalanus elongatus and zoea larvae (Brachyura). d15NPOM-based RTPs were only moderate surrogates for animals’ ecological niches, because of the plasticity in source materials from the herbivorous and the microbial loop food web. Common (16:0) and essential (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) structural lipids showed relatively constant abundances. This could be explained by incorporation of PLFAs with d13C signatures which followed seasonal changes in bulk d13CPOM and PLFA d13CPOM signatures. This
N. V. C. Polunin School of Marine Science and Technology, Newcastle University, Claremont Road, Newcastle upon Tyne NE1 7RU, UK J. J. Middelburg Faculty of Geosciences, Utrecht University, PO Box 80021, 3508 TA Utrecht, The Netherlands J. J. Middelburg Centre for Estuarine and Marine Ecology, Netherlands Institute of Ecology (NIOO-KNAW), PO Box 140, 4400 AC Yerseke, The Netherlands
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Biogeochemistry
study highlighted the complementarity of three biogeochemical approaches for trophodynamic studies and substantiated conceptual views of size-based food web analysis, in which small individuals of large species may be functionally equivalent to large individuals of small species. Seasonal and spatial variability was also important in altering the relative importance of the herbivorous and microbial food webs. Keywords Calanus Compound-specific stable isotope analysis GC-c-IRMS North Sea Phospholipids Size-based food web Stable isotopes Zooplankton
Introduction The North Sea (NS) is a shelf sea of the North Atlantic Ocean where relatively shallow water and high nutrient inputs sustain a highly productive ecosystem. Copepods comprise up to 90% of mesozooplankton (referred to hereafter as zooplankton) biomass in both shallow-mixed and summer-stratified regions (Williams et al. 1994). The abundance of zooplankton in central and northern regions is influenced by the inflow of North Atlantic water, which re-introduces species such as Calanus finmarchicus, Calanus helgolandicus and Candacia armata each spring from overwintering stocks off the North Atlantic shelf (Fransz et al. 1991). Their distributions within the shallow NS are largely determined by currents and atmospheric features including seasonal forcing (Backhaus et al. 1994; Bonnet et al. 2005; Helaoue¨t and Beaugrand 2007). Prior to 1997, C. armata for example, was barely found in the NS, but its’ abundance has increased with increasing inflow of North Atlantic water into the northern NS (Edwards et al. 1999). In North Atlantic shelf seas calanoid copepods such as Calanus spp., Temora spp. and Centropages spp. play major roles in transferring energy mainly in the form of (essential) fatty acids (FAs) to higher trophic levels such as cod and herring (St. John and Lund 1996; Rothschild 1998; Beaugrand et al. 2003). Their primary food source, the primary producers, rapidly transform and incorporate dissolved inorganic elements into organic material, driving numerous biogeochemical fluxes. These include the depletion of
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dissolved inorganic nutrients (nitrogen [N], phosphate [P], silicon [Si]), shifts in the isotopic compositions of carbon (C) and nitrogen (N) (13C:12C, 15N:14N, henceforth d13C and d15N) of marine organisms, changes in the biochemical composition of suspended particulate organic material (POM) and lipid synthesis vital for the reproduction and growth of zooplankton (Cloern 1996; Sterner and Schulz 1998). The evaluation of d13C and d15N values of different animals in comparison to those of their food sources enables analysis of food web structures and fluxes of organic matter (Fry 2006). The transfer of C isotopes can be useful for tracing food sources, particularly where there are clear differences in d13C values between potential food sources. In addition, values of d15N potentially indicate trophic positions (Peterson and Fry 1987). High levels of 13C and 15N enrichment point towards a higher relative trophic position (RTP). However, isotopic fractionation varies at the molecular level depending on the metabolic pathway, so RTP calculations are subject to error (Hobson and Welch 1992; Hobson 1999; Michener and Kaufman 2007). A number of studies, of which many were conducted in polar regions, have used stable isotope (SI) analysis to describe zooplankton trophodynamics in marine environments. The notion emerged that seasonal changes in the composition and availability of primary producers cause trophic plasticity of zooplankton, which may be observed in changes in consumer trophic levels, lipid fingerprints and shifts in food preferences (Schmidt et al. 2003; Soreide et al. 2006, 2008; Tamelander et al. 2006a, 2008; Petursdottir et al. 2008; El-Sabaawi et al. 2009). Potential uncertainties in assessing lower trophic food webs using stable isotopes are often due to rapid d13C and d15N changes at the primary producer scale and heterogeneity in isotopic C and N fractionation associated with trophic transfer (Soreide et al. 2006; Tamelander et al. 2006b; Ku¨rten 2010). Bulk seston, considered to be representative of the end-member isotopic baselines (d13C and d15N) of the herbivorous food web on which zooplankton potentially prey, usually consist of complex mixtures of non-living detritus and living organisms. These include bacteria, protozoa and other microplanktonic heterotrophs with overlapping body sizes. It is, therefore, difficult to use isotope signatures of particulate organic matter (POM) as surrogates for those of obligate autotrophic phytoplankton especially when
Biogeochemistry
POM samples include terrestrial material or are dominated by members of the microbial food web, which are involved in rapid recycling of POM, nutrients and dissolved organic matter (DOM) in the water column (Azam et al. 1983; Cabana and Rasmussen 1996; Post 2002). There is now increasing evidence for multiple direct links from the herbivorous and microbial pathways to higher trophic levels through facultative opportunistic predation by zooplankton on autotrophic phytoplankton or microzooplankton of the microbial food web, with the relative dominance by different trophodynamic pathways being influenced by transient seasonal effects or changes in ontogenetic niche exploitation (Werner and Gilliam 1984; Legendre and Rassoulzadegan 1995; Schmidt et al. 2003; Soreide et al. 2008; Tamelander et al. 2008; de Laender et al. 2010). Compound-specific SI analysis (CSIA) of phospholipid-derived fatty acids (PLFAs) has been introduced for the characterisation of 13C isotope signatures at the base of the food web and has been used to estimate 13C fluxes attributable to bacteria and phytoplankton (Boschker and Middelburg 2002; Boschker et al. 2005). Due to their structural function, phospholipids are sensitive markers for unicellular organisms which do not generally store lipids, such as prokaryotic bacteria and some marine eukaryotic phytoplankton (Boschker and Middelburg 2002). Variability of phospholipid concentrations in cells tends to be lower than those of other lipid classes because phospholipids typically regulate the integrity of membrane fluidity (homeoviscosity), e.g. in response to temperature changes, rather than having a function as energy storage (Farkas 1979). Similarly, in zooplankton, phospholipids have the same function as structural membrane components, and storage of energy is a function of other lipid categories, such as neutral lipids or wax esters (see Lee et al. 1971). However, small copepods, such as C. typicus, P. elongatus and T. longicornis do not store significant amounts of lipids and rely on a steady food supply (Lee et al. 2006). Taxon-specific storage mechanisms, metabolism and lipid fingerprints of marine organisms have been widely documented, which has helped in assessing dietary relationships (Canuel et al. 1995; Hagen and Auel 2001; Dalsgaard et al. 2003; Berge´ and Barnathan 2005). In fact, the expression of some PLFAs parallels evolutionary traits. Bacteria usually contain
noteworthy concentrations of methyl-branched or odd-numbered FAs (e.g. i15:0, 10Me16:0; Canuel et al. 1995; Boschker and Middelburg 2002; Boschker et al. 2005). Similarities in PLFA fingerprints have been used to illustrate phylogenetic relationships of phytoplankton taxa such as Bacillariophyceae and Prymnesiophyceae, which both share Rhodophyceaetype plastids (Yoon et al. 2002; Falkowski et al. 2004). The ancestry of the Prasinophyceae and Chlorophyceae within the Chlorophyta are also shown in this way (Viso and Marty 1993). Among the large range of different lipid isomers, a number of fatty acid trophic markers (FATMs) have been identified that reveal likely trophic relationships between zooplankton species and their food sources including bacteria and phytoplankton. Two main prey items are the Bacillariophyceae (diatoms) which contain eicosapentaenoic acid [EPA; (20:5(n-3)] and the Dinophyceae (dinoflagellates) with docosahexaenoic acid [DHA; 22:6(n-3)] (Cushing 1989; Calbet 2001). Relative quantities of EPA and DHA often indicate seasonal changes in diet (Kattner et al. 1983; Kattner and Krause 1989; Peters et al. 2006). The suitability of polar-lipid FAs to track zooplankton diet was proven in studies at the Kattegat, the Mediterranean and the Antarctic (Virtue et al. 2000; Graeve et al. 2001). Since lipids are transported quite conservatively through marine food webs and because a range of essential FAs, such as EPA and DHA, cannot be synthesized de novo by animals, but only by phytoplankton, these FAs must be taken up directly or indirectly. d13C signatures of FATMs in zooplankton therefore provide valuable information on the changes of d13C signatures in the diet of zooplankton, which reflect changes in primary producers, including changes in relative abundances of Bacillariophyceae, Rhodophyceae and Dinophyceae and bacteria. The aim of this study was to test the hypothesis that the relative trophic positions of zooplankton in the North Sea are spatially and seasonally variable, in response to hydrographic variability, with the microbial food web playing an important role at times. Zooplankton were collected during seven cruises over 14 months in the southern and central NS. Bulk stable isotope (SI) analysis of d13C and d15N, phospholipid-derived fatty acid (PLFA) compositions and d13CPLFA data of zooplankton and particulate organic matter (POM) were used to describe changes in the relative trophic positions (RTPs) and trophic
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Biogeochemistry Fig. 1 Bathymetric chart of the North Sea and the three study sites situated in the Southern Bight (SB), at the Oyster Grounds (OG) and at North Dogger (ND)
dynamics. The biogeochemical processes that affected bulk d13C signatures of zooplankton and of their PLFA are discussed with respect to uncertainties regarding d13C offsets between bulk sample materials and CSIA of baseline proxies.
Materials and methods Study sites North Sea zooplankton communities were studied at three sites of contrasting hydrography (Fig. 1). One study site was located in the well-mixed Southern Bight (station SB; 53°9.8 N, 2°48.6E) where shallow water (maximum depth & 30 m) and tidal currents cause particulate material to remain in suspension all year round. This site is influenced, at times, by the East Anglia Plume, a zone of high turbidity which receives large amounts of nutrients from UK estuaries (Thames, Humber, Wash) and extends across the Southern Bight towards the German Bight (Nedwell et al. 2002; Weston et al. 2004; Spokes and Jickells
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2005). The second study site was located in the semidepositional Oyster Grounds (OG; 54°24.6 N, 4°2.6E; maximum depth & 48 m), which is a seasonally stratified area in the Dutch sector of the central NS, and which receives coastal zone-derived organic matter, including resuspended material transported with the East Anglia plume (Otto et al. 1990; van Raaphorst et al. 1998). The third study site (North Dogger, ND; 55°41.0 N, 2°17.2E; maximum depth & 80 m), 111 km north of the Dogger Bank, is representative of deeper seasonally-stratified areas of the central NS with little tidal resuspension and low concentrations of suspended matter. Water masses here are predominantly of North Atlantic origin, with limited coastal influence (Brown et al. 1999; Eisma and Kalf 1987; Harwood et al. 2008; Laevastu 1963). Sample collection The present investigation took place during the research project on ‘‘Marine Ecosystem Connections—Essential indicators of healthy, productive and biologically diverse European Shelf Seas’’ (The
Biogeochemistry
Centre for Environment, Fisheries, and Aquaculture Science, CEFAS). Seven cruises on RV Cefas Endeavour were spaced to capture seasonal changes in ecosystem structure and function: during winter (20–27 February 2007; 16–22 January 2008), spring (17–25 April 2007, 16–23 May 2007; 21–30 April 2008) and autumn (13–20 September 2007, 26–03 October/November 2007). For POM, Niskin bottles (10 and 30 l) attached to a CTD rosette sampler collected seawater at night from the surface mixed layer (*4–6 m depth). Once on deck, larger zooplankton and debris (plastics) were excluded by passing the water through a 60 lm NitexÒ mesh. The seawater was then filtered immediately using a manifold of 47 mm NalgeneÒ filter columns for lipid samples (Sartorius AG, FRG) and 25 mm glass frit columns for bulk POM. POM was filtered onto pre-combusted (5–8 h, 500°C), preweighed GF/F filters (nominal pore size 0.7 lm; Whatman, UK). Filtration volumes varied depending on seston concentrations, but water was filtered until the filters were clogged. Whenever possible, sample collection included a minimum of six filters for bulk POM (Vol.: 0.5–3 l) and six filters for pigment and lipid analysis (Vol.: 3–16 l). Filters were wrapped in hexane-wiped aluminium foil, stored in individual petri dishes, blast-frozen to -28°C and stored at -80°C in the laboratory. Zooplankton were collected with ring nets at night using vertical tows of ring nets (Ø 1 m, 200 lm NitexÒ mesh, non-filtering cod end, towed at *1 m s-1 from 2 to 4 m above the bottom to the surface) and double-oblique net tows through the surface mixed layer (Ø 2 m, 1000 lm NitexÒ mesh; at a bottom speed of 1–1.5 kn for 10–20 min). Animals were rinsed off the sampling nets with seawater, washed through 4 mm sieves to remove debris and large gelatinous forms, and collected in containers filled with fresh seawater to allow for gut evacuation (30–60 min, 15 l containers). Afterwards zooplankton were concentrated on 20 lm NitexÒ mesh, washed with GF/F filtered seawater into glass jars and immediately blast frozen to -28°C. Samples were later transferred to -80°C freezers ashore. Sample preparation After freeze-drying, POM filters were randomly assigned to three sets, one of which was kept as
back-up. One set was exposed to HCl fumes in a glass desiccating cabinet under light vacuum for 24 h to remove inorganic carbonates prior to bulk d13CPOM SIA. One set was not acidified and was used to derive d15NPOM values. Samples were then stored in desiccating cabinets until they were analyzed. Zooplankton were sorted to the lowest practical taxonomic level using stereomicroscope. Representing a range of feeding strategies, ten different zooplankton taxa were picked for bulk SI analysis including herbivorous, omnivorous and carnivorous Copepoda (order Calanoida, eight species), one raptorial predator (Sagitta elegans, Chaetognatha) and a complex of zoea larvae (Decapoda, Brachyura). Usually, adult females (Copepoda), adults regardless of gender (S. elegans), and zoeae were picked, cleaned from attached algae and detritus and sorted into petri dishes. The petri dishes were placed on freeze packs and filled with chilled GF/F filtered seawater. Depending on body mass and availability, 5–150 animals per sample were briefly washed in Ultrapure water (UPW) and placed in acetone-rinsed Sn capsules (5 9 8 mm, Elemental Microanalysis Ltd., UK). Samples were prepared in triplicates whenever possible, oven-dried (60°C, 24 h), folded and stored in desiccating cabinets prior to bulk SI analysis. Lipid extractions and acidifications prior to bulk SI analysis were avoided, precluding potential uncertainties in comparisons of results from this study with isotopic signatures of other studies (see Mintenbeck et al. 2008). For compound-specific analysis, specimens were pipetted onto pre-weighed GF/F filters, briefly rinsed with UPW, lyophilized and stored at -80°C pending lipid extraction. Bulk d13C and d15N stable isotope analysis Natural abundances of C and N stable isotopes were measured on a ThermoElectron Delta V isotope-ratio mass spectrometer (IRMS). All stable isotope ratios are expressed in conventional d notation as a measure of heavy to light isotope using the equation dX (%) = [(RSample:RStandard) - 1] 9 1000, where dX is 13C or 15N, and R is the 13C:12C or 15N:14N ratio. A peptone standard with known isotopic composition (N-content 11%; C-content 44%; d15N 7.6%; d13C -14.3%) was used after every 5th unknown sample as an internal standard and calibrated with IAEA-N1 and IAEA-N2 once a month. The stability of the
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Biogeochemistry
instrumentation, analytical precision, drift correction and linearity performance were calculated from the repetitive analysis of the peptone standard and corrected for if necessary. No blank correction was applied. The standard deviation for replicate samples, the analytical precision, was \0.2% for both isotopes. Lipid extraction and compound-specific stable isotope analysis of PLFAs Lipid extraction, separation of the polar lipid fraction, FA derivatization and isotope ratio mass spectrometry used the method described by Boschker et al. (1999, 2005). In brief, after lyophilization total lipids were extracted according to a slightly modified Bligh and Dyer (1959) method in a mixture (1:2:0.9/v:v:v) of chloroform, methanol and ultra-pure water from whole filters by shaking the extraction tubes (100 rpm, approx. 2 h). Total lipid extracts were separated into polarity classes on heat activated (120°C, 2 h) silicic acid gel columns (Silicic acid gel 60, 0.063– 0.200 mm) by sequential elution with 7 ml chloroform (neutral lipid fraction), 7 ml acetone (most pigments) and 15 ml methanol (polar lipid fraction). Polar lipid fractions were derivatized by mild alkaline transmethylation to yield fatty acid methyl esters (FAMEs). FAME compositions and d13C values were determined on a gas-chromatograph combustion-interface isotope-ratio mass spectrometer (GC-c-IRMS; Hewlett Packard G1530 GC; polar BPX-70 column 50 m, 0.32 mm diameter, film thickness 0.25 lm, SGE054607; Type-III combustion interface; Thermo Finnigan Delta-plus IRMS). Individual FAMEs were identified by comparisons of relative retention times to known amounts of internal reference standards (FAs 12:0, 19:0) and other alkane reference mixtures which were included to check the accuracy of isotopic determinations (mean ± 1SE; FA 12:0 = -27.96 ± 0.04%, N = 445; FA 19:0 = -30.76 ± 0.10%, N = 447). The values for d13C of individual PLFAs were calculated from FAME data by correcting for methyl C atoms added during derivatization using the equation d13CPLFA = ((n ? 1) 9 d13CFAME – 1 9 d13CMethanol)/n, where n = number of C atoms in PLFAs, d13CFAME = isotope ratio of measured methylated PLFAs and d13CMethanol = isotopic ratio of the methanol used (±46%, SuprapurÒ grade).
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Data evaluation Bulk d15NPOM data from Ku¨rten (2010) were used to evaluate spatial and temporal variability in zooplankton SI results, and to calculate relative trophic positions (RTPs) of zooplankton. d15NPOM-based RTP estimates (Eq. 1) assumed that RTPPOM values of 1 represented the isotopic baseline, and that a trophic fractionation factor of 3.4% represented one trophic transfer (Minagawa and Wada 1984): RTP ¼ d15 NZooplankton d15 NPOM =3:4 þ 1 ð1Þ P Bacterial lipids were calculated as PLFA Bacterial (i14:0, i15:0, a15:0, 15:1, i16:0, 16:1(n-7)t, 10Me16:0, 17:0, 17:1, br17:1, 18:1(n-7), cy17:0, cy19:0), frequently occurring in Gram-positive bacteria, Cytophaga/Flavobacteria and Gram-negative Eu-/Proteobacteria (Canuel et al. 1995; Boschker and Middelburg 2002; Boschker et al. 2005). For d13C PLFATotal and d13C PLFABacterial, numerical means were replaced by concentration-weighted d13C means. The isotopic offsets between bulk d13C (d13CPOM) and mean concentration-weighted d13C PLFATotal values were calculated as the difference between mean values for each set of sample replicates following Riebesell et al. (2000): Dd13COffset = d13Cbulk - d13CPLFA. For multivariate analysis of PLFA fingerprints (Field et al. 1982), Primer 6 was used (Clarke and Gorley 2006). PLFA abundances were expressed as the percentage (%) of PLFATotal before analyzing the data. PLFA abundance data were then square-root transformed to produce a Bray-Curtis index of similarity matrix for subsequent analysis of the similarity by multidimensional scaling, including a stress level as a measure of the ‘goodness-of-fit’. Other statistical analyses used SPSS 19.0 (SPSS Inc. USA).
Results Relative trophic positions Seasonal heterogeneity of isotopic baselines of North Sea food webs from bulk and compound-specific stable isotope analysis of POM are presented by Ku¨rten (2010). Spatial and seasonal variability in d15NPOM-based RTPs of the entire zooplankton community (Fig. 2) shows that zooplankton RTP estimates
Biogeochemistry Fig. 2 Relative trophic positions (RTPs) of zooplankton, based on d15NPOM, from February 2007 to April 2008 at North Dogger, at the Oyster Grounds and in the Southern Bight. Values below RTP = 1 indicate d15NZooplankton \ d15NPOM. POM particulate organic matter
largely followed a unimodal pattern, being high in winter and low during the phytoplankton blooms in spring and early summer. Zooplankton at the ND site showed a small decrease in RTP after the autumn bloom, whereas RTP values increased until January 2008 at the two other sites. The selected taxa occupied a range of trophic positions, with Sagitta elegans and Metridia longa showing highest RTP (2.7 and 2.9 respectively, Table 1; mean value of all sites), and zoea larvae, Paracalanus elongatus and Candacea armata showing the lowest mean RTPs (B1, Table 1). At the OG, Anomalocera patersoni, considered an omnivorous species, occupied a RTP similar to Centropages typicus and zoeae larvae (Table 1). At the ND site, RTPs were similar for Calanus finmarchicus and Calanus helgolandicus (1.8 and 1.7, respectively), while C. helgolandicus exhibited higher RTPs (C2) at the SB and OG sites. Very low RTP values of zoeae (ND: 1.1; SB: 0.9) indicated that Brachyura larvae preferred food from a lower trophic level than represented by bulk POM samples of the present study. Trophodynamics differed for Candacia armata between the northern (ND: RTP 2.2) and the southern NS (SB: RTP 0.9). Stable isotope analysis of POM At the ND site, there was a maximal bulk POM 13C depletion in May 2007 (-26.7%), whereas the overall seasonal d13C amplitude was relatively small (4.8%; Fig. 3). At the OG site, 13C depletion occurred during spring 2008, but from April 2007 until January 2008, d13C remained at -22.8 ± 0.7%. In the SB, POM samples were generally 13C enriched and showed larger seasonal d13CPOM amplitudes, with greatest
d13CPOM enrichment in winter (Fig. 3) during times of elevated land run-off. POM d13C PLFATotal differed from bulk d13CPOM according to season and site, with d13C offsets being mostly negative in spring and positive in winter (Fig. 3). Across all sites PLFATotal d13C ratios were on average 1.2% depleted compared to bulk d13CPOM values, but variable (range: -3.1 to ?7.2%, n = 23). One-way ANOVAs on PLFA 13C data showed that PLFATotal d13C values were indistinguishable among sites (F(2, 55) = 1.428, P = 0.239). PLFA data at the ND site showed maximal 13 C enrichment in April and September, followed by a pronounced depletion in May and moderate depletion until April 2008 (-26.0 ± 0.5%; Fig. 3). Similar trends were observed at the OG and SB, but maximal 13 C enrichment was attained in May and October 2007 (Fig. 3). Zooplankton bulk stable isotope analysis The overall mean values for NS zooplankton stable isotopes were -21.0 ± 1.7% for d13C (±1SD) and 8.6 ± 2.4% for d15N (n = 253 for both). Tests for the effect of site on isotope signatures indicated a difference among sites for both d13C (one-way ANOVA, F(2, 248) = 42.216, P \ 0.001) and d15N (F(2, 248) = 11.653, P \ 0.001). Tukey HSD tests revealed that zooplankton were on average 13C enriched in the southern NS and most depleted at ND; the SB and the OG did not differ in d15N values (P = 0.994, N = 83). For the copepods C. finmarchicus, C. helgolandicus, C. typicus, T. longicornis and the chaetognath S. elegans, more detailed spatial and temporal comparisons of bulk SI data were possible (Fig. 4).
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Biogeochemistry Table 1 Average (mean ± SE) carbon (C) to nitrogen (N) ratios, d13C and d15N (%) and d15NPOM-based relative trophic positions (RTPs) of North Sea zooplankton collected at Site
ND
OG
SB
Species
the North Dogger site (ND), the Oyster Grounds (OG) and the Southern Bight (SB), from February 2007 to April 2008
d13C
C:N n
Mean
SE
Candacia armata
4
6
0.1
Calanus finmarchicus
10
9.4
0.9
Calanus helgolandicus
12
7
0.4
Centropages typicus
4
6.5
Metridia longa
n.d.
Pseudocalanus elongatus
2
Sagitta elegans
n
d15N Mean
SE
n
8
-23.5
0.2
8
16
-22.3
0.3
16
22
-22.1
0.3
0.4
7
-23.4
n.d.
n.d.
3
7.9
0.8
2
12
6.9
0.7
Temora longicornis
2
5.6
Zoea (Brachyura)
2
7.1
Anomalocera patersoni
3
C. finmarchicus C. helgolandicus
RTP Mean
SE
7.9
0.3
7
0.5
22
6.9
0.3
0.3
7
5.9
-20.9
0.2
3
-22
0.8
2
19
-20.6
0.3
0.6
2
-19.6
0.1
5
-22.4
6.1
0.3
4
7
10
1.3
10
7
0.4
C. typicus
5
5.6
M. longa Paracalanus elongatus
3 1
6.1 8.6
S. elegans
8
T. longicornis
3
Zoea (Brachyura)
n
Mean
SE
Min.
Max.
Range
8
2.2
0.1
16
1.8
0.2
1.4
2.4
1
0.6
2.9
22
1.7
2.3
0.2
0.3
3.1
0.4
7
2.8
1.4
0.2
0.8
2.7
1.9
11
0.4
7
0.2
3
2.9
0.1
2.8
3.1
0.3
2
1.1
0.1
1
1.2
0.2
19
9.7
0.5
19
2.7
0.2
0.8
4.3
3.5
0.6
2
0.3
5
7.9
1.2
2
2.0
0.3
1.7
2.2
0.5
5.3
0.8
5
1.1
0.0
1
1.2
-20.5
0.9
0.2
4
9.8
0.9
4
1.6
0.2
1.2
1.9
0.7
10
-22.1
17
-21.7
0.4
10
8.1
0.8
10
1.9
0.1
1.3
2.4
1.1
0.3
17
8.9
0.6
17
2.2
0.2
1
3.9
2.9
0.1
9
0 n.d.
3 1
-20.6
0.5
9
9.3
0.3
9
1.7
0.2
1.1
2.8
1.7
-21.1 -21.9
0.1 n.d.
3 1
4.4 6.6
0.1 n.d.
3 1
2.0 1.0
0.0 n.d.
2 1
2.1 1
0.1 n.d.
6.5
0.2
6.8
1
16
-20.5
0.3
16
11.6
0.5
16
2.6
0.2
1.6
4.2
2.6
14
-21
0.4
14
8.4
0.6
14
1.7
0.2
1.1
3.1
11
7.5
2
0.4
15
-20.3
0.2
15
9
0.3
6
1.5
0.1
1.1
1.8
A. patersoni
2
0.7
5.7
0.1
3
-18.8
0.1
3
10.1
0.2
3
1.3
0.1
1.2
1.4
0.2
C. armata C. finmarchicus
2
5.6
0.1
2
-23
0.2
2
8.9
0.3
2
0.9
0.1
0.8
1
0.2
4
8.2
1.2
8
-21.7
0.3
8
7.8
0.5
8
1.4
0.2
0.7
2.2
1.5
C. helgolandicus
10
6.4
0.4
16
-20.6
0.2
16
9.7
0.4
16
2.0
0.2
0.8
3.4
2.6
C. typicus
7
4.9
0.2
8
-19.2
0.2
8
8.1
0.4
8
1.3
0.4
0.2
3.3
3.1
P. elongatus
1
7.1
n.d.
2
-22
1.2
2
9.2
2.2
2
2.8
1.3
1.5
4.1
2.6
S. elegans
8
6
0.2
15
-19.6
0.2
15
11.7
0.4
15
2.7
0.3
1.1
4.1
3
T. longicornis
10
5.4
0.2
15
-19.7
0.3
15
7.7
0.5
15
1.2
0.2
-0.2
3
3.2
Zoea (Brachyura)
7
7
0.2
9
-17.4
0.3
9
7.5
0.4
4
0.9
0.4
0.1
1.6
1.5
RTP values given as mean, minimum, maximum, and range n number of samples, n.d. no data, POM particulate organic matter
In the SB, zooplankton had higher d13C values (mean ± SE enrichment of 1.6 ± 0.2%, Fig. 4) than the average d13CZooplankton at the ND site (-21.8%; one-way ANOVA; F(2,190) = 23.655, P \ 0.001; Tukey HSD, P \ 0.001, N = 61). In May and September, zoeae attained -15.9% (data not shown), while T. longicornis d13C reached -18.2% in October 2007 at the SB site. Higher mean d13C values were also measured for S. elegans (-
123
19.6 ± 0.2%) and C. typicus (-19.2 ± 0.2%). At all sites, C. finmarchicus were most 13C depleted with low d13C values in October (-25.3%), similar to C. armata in September 2007 at ND (-24.4%). However, in spring 2008 all zooplankton species had almost identical bulk d13C values (approx. -22%) (Fig. 4). Highest d15N values were measured for S. elegans (*14.5%) and C. helgolandicus (*13.8%) in February 2007 at the OG and SB and lowest d15N
Biogeochemistry
Fig. 3 Bulk d13C (%) and concentration-weighted PLFATotal d13C values (mean ± SE) of POM and zooplankton collected from February 2007 to April 2008 at North Dogger, the Oyster
Grounds and in the Southern Bight. PLFA phospholipidderived fatty acids, POM particulate organic matter
values (1.8%) occurred in T. longicornis in February 2007 at the OG (data not shown).
Bulk d13CZooplankton and zooplankton d13C PLFATotal (mean ± SD = 24.8 ± 2.0%) were significantly correlated with each other (Spearman, coefficient = 0.535, P \ 0.001, n = 65). Comparisons of these values (Fig. 4) showed that d13C offsets were similar at all sites but variable over an annual cycle (3.7 ± 0.2%, n = 65, mean ± 1SE; one-way ANOVA, F(2,62) = 0.352, P = 0.704). Offsets ranged from -1.3% 13C enrichment of PLFAs compared to bulk tissue (C. finmarchicus, October 2007, ND) to a maximal depletion of 8.1% (T. longicornis, January 2008, SB). Although not significant (F(6,58) = 2.247, P = 0.51), PLFAs tended to be more 13C depleted in winter and during bloom conditions (September 2007; January, April 2008) and less depleted after blooms (May, October 2007). Overall mean values for PLFATotal d13C signatures were ca. -24.5% for C. finmarchicus and C. helgolandicus, ca. -24.9% for C. typicus and S. elegans and ca. -23.9% for T. longicornis. The most 13C enriched and depleted FAs were found in T. longicornis at ND in October (18:1(n-7): -18.9%) and at the OG in April 2008 (EPA), respectively. The largest range in d13C values
Zooplankton PLFA analysis and CSIA The polar lipid fractions of zooplankton contained a total of 69 PLFAs, some of which were present in trace amounts only. Data evaluation was carried out on 16 FAs accounting for 89.4 ± 12.3% (mean ± 1SD) of PLFATotal. Generally, DHA was the largest and EPA the second largest essential PLFA component in the five main zooplankton species. The relative contribution of the major structural membrane FAs (16:0, EPA, DHA) to the PLFATotal remained fairly constant with little seasonal changes (Fig. 5). Similar and parallel changes in bulk and PLFATotal d13C values of zooplankton and POM suggest that zooplankton attained homeoviscosity of cell membranes by incorporating PLFAs with variable d13C signatures (Fig. 6). Like most of the 16 more abundant zooplankton PLFAs, PLFAtotal d13C signatures (Fig. 3) and those of 16:0, EPA, DHA (Fig. 6) largely followed the isotopic signatures of POM.
123
Biogeochemistry Fig. 4 Bulk d13C (%) and concentration-weighted PLFATotal d13C values (mean ± SE) of five selected zooplankton taxa collected from February 2007 to April 2008 at North Dogger, the Oyster Grounds and in the Southern Bight (species: Cfi Calanus finmarchicus, Che Calanus helgolandicus, Cty Centropages typicus, Sel Sagitta elegans, Tlo Temora longicornis). PLFA phospholipid-derived fatty acids
was for DHA in C. typicus at the OG (range 9.2%; Fig. 6). The d13C changes of FA 18:1(n-7), which occurs mainly in bacteria and only in small concentrations in other organisms, also followed d13C changes of EPA and DHA (Fig. 6). PLFA fingerprinting revealed temporal and taxonP specific changes in concentrations of PLFABacteria in zooplankton. Bacterial PLFA concentrations generally showed seasonal succession, with lower PLFABacteria abundances in May (OG) and October (SB) 2007 and in April 2008 (all sites; Fig. 7), and higher bacterial PLFAs in zooplankton in September 2007 (SB) and January 2008 (OG, SB). Moreover, P across all zooplankton taxa PLFABacteria tended to be higher in the southern NS than at the ND site (oneway ANOVA; F(2,79) = 2.397, P = 0.098; Tukey
123
HSD; P = 0.101, n = 25; ND: 3.1 ± 1.6%, SB: 4.3 ± 2.3% [mean ± 1SE]). Several clusters comprising mainly S. elegans and zoeae were separated from other zooplankton taxa in an MDS plot by the presence of bacterial markers such as i15:0, i17:0, a17:0 and cy19:0 (Cluster I; Fig. 8). During phytoplankton blooms in September 2007 and April 2008, FA 20:3(n-3) and bacterial markers such as t16:1(n-9) grouped the small copepods C. typicus, T. longicornis and P. elongatus together (Cluster II). C. armata, C. finmarchicus and C. helgolandicus collected in late autumn/winter (October 2007, January 2009; Cluster III) contained no i17:0 and relatively few other bacterial markers (Fig. 8). In April 2007, Calanus spp. and T. longicornis contained several bacterial markers, particularly t18:2(n-6), but also larger
Biogeochemistry
Fig. 5 Relative contribution (%) of major PLFAs [16:0; 18:1(n-7); 20:5(n-3), EPA; 22:6(n-3), DHA] to PLFATotal (mean ± SE) in five selected North Sea zooplankton species collected from February 2007 to April 2008 (for species
acronyms see caption of Fig. 4). 18:1(n-7) = often present in bacteria. EPA dominant in Bacillariophyceae, DHA dominant in Dinophyceae (after Boschker and Middelburg 2002)
quantities of 20:0 (Clusters IV), whilst these PLFAs were absent from these species in May 2007 (Cluster V). Summaries of zooplankton PLFA compositions and CSIA data are presented in Supplementary Material 1 (% of PLFATotal) and Supplementary Material 2 (d13CPLFA).
14 months was considered to be sufficient to capture seasonal signals in zooplankton. We assumed that changes in the isotopic composition of primary producers would be manifested in zooplankton isotopic composition during the subsequent sampling event. This assumption was based on evidence that the selected taxa generally have relatively short generation times (e.g. 30 days for C. helgolandicus, 17 days for C. typicus [Halsband-Lenk et al. 2002; Bonnet et al. 2005]), and that changes in diet can be reflected in isotope compositions within a few weeks. For C. finmarchicus, for example, a switch in diet (from Bacillariophyceae to Dinophyceae) changes the PLFA fingerprints within 2 weeks, and even non-growing C. finmarchicus in the Arctic rapidly accumulate 13C of 13C-labelled algae in their structural PLFAs (Graeve et al. 2005).
Discussion The present study revealed seasonal trophodynamic changes among adult Copepoda, S. elegans and Brachyura larvae (zoeae) as well as differences among central and southern NS sampling sites that were reflected in phospholipid and isotopic composition. Zooplankton sampling during seven cruises over a period of
123
Biogeochemistry
Fig. 6 d13C values of major PLFAs [16:0; 18:1(n-7); 20:5(n3), EPA; 22:6(n-3), DHA] in five selected North Sea zooplankton species collected from February 2007 to April 2008 (for species acronyms see caption of Fig. 4). 18:1(n-
7) = often present in bacteria. EPA dominant in Bacillariophyceae, DHA dominant in Dinophyceae (after Boschker and Middelburg 2002)
Comparison of relative trophic positions and SI data
However, the wide range of RTP values we observed in C. typicus and T. longicornis suggest a wide range of food sources and concur with recent reports of their omnivory (Calbet et al. 2007; Carlotti et al. 2007; Gentsch et al. 2009). Comparisons with estimates based on dietary information (Yang 1982) suggest that RTP values from this study were on average 0.7 trophic levels lower than those of Yang (1982, Table 2). For C. finmarchicus, for example, Yang (1982) calculated a value for RTP of 2.3, whereas we estimated a mean value of 1.8, and a maximum of 2.9. These are similar to RTPs reported from Arctic regions (1.6–3.1), where higher RTP were also recorded in winter (Soreide et al. 2006, 2008; cf. Fig. 2). A range of isotopic data and RTPs have been presented for NS macrofauna (Das et al. 2003) also
The relative trophic positions of zooplankton during this study were generally lowest during the spring bloom, relatively high at the SB and OG sites in late summer/early autumn, but variable at ND during this period, and highest in late autumn and winter. In late summer/early autumn, variable RTP values at ND were influenced by the late breakdown of thermal stratification (Greenwood et al. 2009). There are few North Sea studies to which the results of this study can be compared. Bulk isotope values for Temora longicornis at ‘Helgoland Roads’ during early spring (d13C: -20.9 to -16.7%; d15N: 7.7–16.1%, Gentsch et al. 2009) fell within the range of values reported here.
123
Biogeochemistry
Fig. 7 Spatial and P seasonal variability (%; mean ± 1SE) of bacterial lipids ( PLFABacterial of PLFATotal) present in five selected North Sea zooplankton species collected from
February 2007 to April 2008 at North Dogger, the Oyster Grounds and in the Southern Bight. PLFA phospholipidderived fatty acids
Fig. 8 Two-dimensional MDS ordination plot based on PLFA compositions of North Sea zooplankton. The abundance of the fatty acid (FA) i17:0, commonly expressed by some bacteria, is superimposed (bubble size = % of PLFATotal). Group clusters indicated by overlaid boundaries show similarity at the 75% (solid line) and 82% (dashed line) resemblance level (species: Apa Anomalocera patersoni, Car Candacia armata, Cfi Calanus finmarchicus, Che C. helgolandicus, Cty Centropages typicus, Pel Pseudocalanus elongatus, Tlo Temora longicornis, Sel Sagitta elegans, Zoe Zoea). PLFA phospholipid-derived fatty acids
assuming RTPPOM = 1, and using a fixed d15NPOM value of 9% (Middelburg and Nieuwenhuize 1998). Das et al. (2003) showed that the mean d15N of zooplanktivorous fish in the southern NS was 14.7%, while d15N values of herring (Clupea harengus) were somewhat lower (13.0 ± 1.1%) than for sprat (Sprattus sprattus, 16.6 ± 0.5%). Assuming an average trophic enrichment of 3.4% (Minagawa and Wada 1984), zooplankton d15N values can be inferred from
d15N values of zooplankton consumers, as e.g. herring typically selects adult Calanus sp. females (Dalpadado et al. 2000). On the basis of the herring values, a d15N of 9.6% can be inferred for NS zooplankton. The observed annual mean (±1SE) d15N of C. helgolandicus in the southern NS (this study) was 9.7 ± 0.4% and that of the similar-sized A. patersoni 10.1 ± 0.2%. The lower d15N, reported for C. harengus from the northern NS (12.1 ± 0.53%; Jennings et al. 2001),
123
Biogeochemistry Table 2 North Sea zooplankton d15NPOM-based relative trophic positions (RTPs) and estimates from dietary studies by Yang (1982) Species
A. patersoni
n
RTP Mean
1SE
Min.
Max.
Range
Yang (1982)
7
1.5
0.1
1.2
1.9
0.7
n.d.
C. armata
10
1.9
0.2
0.8
2.4
1.6
n.d.
C. finmarchicus
34
1.8
0.1
0.6
2.9
2.3
2.3
C. helgolandicus
55
2.0
0.1
0.3
3.9
3.6
n.d.
C. typicus
24
1.5
0.2
0.2
3.3
3.1
2.4
M. longa
6
2.5
0.2
2.0
3.1
1.1
n.d.
P. elongatus
5
1.8
0.6
1.0
4.1
3.1
2.1
S. elegans
50
2.7
0.1
0.8
4.3
3.5
3.5
T. longicornis
31
1.5
0.1
-0.2
3.1
3.3
2.4
Zoea (Brachyura)
29
1.3
0.1
0.1
3.1
3.0
2.5
may be related to the somewhat lower d15N values of zooplankton at the ND compared to the SB (Table 1). Noteworthy were high RTPs and d15N values for S. elegans and C. helgolandicus (Table 1), with the latter exceeding those of NS top predators such as harbour porpoise (Phocoena phocoena), harbour seal (Phoca vitulina) and seabirds (great skua, Stercocarius skua, common guillemots, Uria aalge; Das et al. 2003; Ka¨kela et al. 2006). These results departs from linear associations found in some cases between body size and d15N (Jennings et al. 2001, 2002), and routine bulk d15NPOM data may inadequately represent the real variety of food sources available to zooplankton. Most studies that exploit linear, direct associations of d15N with trophic level do not take account of the microbial-loop and the utilization of various source materials with variable 15 N. As a result, common d15NPOM-based RTP estimates require cautious interpretation, particularly if some NS zooplankton species are selective feeders, for which there is sufficient evidence (Lebour 1922; Peters et al. 2006; Gentsch et al. 2009). Difficulties are mainly attributable to overlapping size ranges of bacterio-, phyto- and zooplankton, trophodynamic complexity (due to non-conforming mixotrophs) and changes of the baseline signatures equivalent to more than two trophic levels due to effects of the microbial loop (Azam et al. 1983; Jennings et al. 2002). Rapid biogeochemical processes that alter the isotopic signatures at the base of the food web may not necessarily propagate up to top predators, due to lag effects associated with assimilation, tissue turnover and longevity (Jennings et al. 2002).
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Causes and consequences of changing d13C baseline signatures Phytoplankton d13CPOM signatures usually reflect the d13C values of the dissolved inorganic C source (DI13C) and subsequent modification by physiological processes that vary with taxon and responses to environmental variables including dissolved CO2 concentration, light and nutrient limitation, temperature and pH (Falkowski 1991; Fry and Wainwright 1991; Laws et al. 1995; Rau et al. 1996; Burkhardt et al. 1999a, b). Fractionation of C (d13C) by Bacillariophyceae, for instance, tends to be inversely correlated with nutrient availability, but bulk POM samples may include also Dinophyceae and other types of phytoplankton of which many are mixotrophic and potentially feed on a variety of isotopically different sources and seasonal effects of nutrient limitation remain masked (Pancost et al. 1999; Stoecker 1999). Although lipids are typically somewhat 13C depleted compared to the bulk source material, despite a wide range of fractionation values between substrate and PLFAs ranging from -11.4 to ?0.5% (Abraham et al. 1998; Cifuentes and Salata 2001), CSIA of PLFAs offers an alternative to bulk SI analysis to determine end-member d13C values from routine POM samples. In the present study, we observed a seasonal effect, with negative fractionation between bulk d13CPOM and d13CPLFA during spring, and positive offsets during late autumn and winter. The average 1.2% depletion of d13CPLFA against bulk d13CPOM matched previous reports (bacteria: 1.2%, average:
Biogeochemistry
3%) of offsets from the Scheldt estuary (Boschker et al. 2005). However, within one sample, individual d13CPLFA values varied by more than 10%. Deciphering the differences between species, between life stages and strains of marine phytoplankton is complicated due to differences in growth rates, which further depend on environmental factors such as temperature, light intensity and nutrient supply, factors that may be similar to CO2 concentration in importance to affect the bulk POC and lipid isotopic composition (Schouten et al. 1998; Riebesell et al. 2000). Among the polyunsaturated FAs, EPA has frequently been used as a marker for Bacillariophyceae. In this study, EPA was usually more 13C depleted than DHA, a FA more abundant in Dinophyceae. Bacterial PLFAs tended to be even more 13C enriched, and large differences in d13C values of different FAs within one sample indicated that bacterial PLFA synthesis may depend on C derived from various origins. The determination of POM d13CPLFA values provided detailed information of isotopic signatures from different groups of primary producers, masked in standard bulk POM SI analysis. The results from the present study suggested that rapid intrinsic isotopic fluctuations occur in PLFAs between consecutive blooms events, with 13C enrichment during bloom formation and 13C depletion at later bloom stages when senescent algae sink out of the surface water and new communities are established. The divergent fate of individual compounds with distinct d13C signatures and intrinsic d13C changes has methodological implications for food web reconstructions and palaeo-oceanographic studies, since some essential FAs may be incorporated directly into the consumers’ tissue, whereas dietary uptake of nonessential FAs that are also produced de novo by heterotrophs may be used in other metabolic pathways instead. Variability among d13CPLFAs within one sample may cause d13C isotopic baseline proxies derived from bulk POM or even from mean d13C of PLFATotal to become biased, particularly when individual FAs selectively accumulate in consumers’ tissues or exhibit large variability of unknown magnitude. Moreover, since isotopic end-members in the Southern Bight include terrestrial, riverine and estuarine sources from the Thames, the Humber and the Wash which contribute to the East Anglia plume, other end-members potentially include enriched 13 CPOM from these sources. The majority of turbid
European estuaries have d13C values of -24 to 26%, suggesting uniformity in the mixture of riverine, estuarine and marine sources with a dominance of terrestrial organic matter (TOM). Marine endmembers of the Thames estuary can be 13C enriched in winter to -21% (Middelburg and Herman 2007). Fichez et al. (1993) showed the marine end-member POM d13C value in the center of the Wash to be 19%. Typical 13C enrichment of marsh plants and macroalgae such as Carex, Spartina, Zostera, Fucus, Ulva and Enteromorpha, which occur naturally in the catchment of the Southern Bight, ranges from -10 to -13.1% (Smith and Epstein 1971), and can also not be excluded as potential isotopic end-members. Hence, regardless of the nature and interactions of the processes that alter the isotopic composition of d13CPOM, they act in parallel and cause isotopic signatures of d13C and d15N of bulk POM and those of d13CPLFA to vary in a complex fashion. Consequences for consumer d13C The variability of d13C values among PLFAs suggested that seasonal baseline d13C variations propagate to zooplankton even at the level of structural lipids. In this study, zooplankton PLFATotal d13C values were on average lower by 3.7 ± 0.2% (range: -1.3% to ?8.1%) compared to bulk isotopes. Ranges and patterns in zooplankton 13CPLFA depletion compared to bulk tissue suggested seasonal influences and dietary effects on d13CPLFA signatures. The PLFAs 16:0, EPA and DHA were present in relatively constant proportions throughout the sampling period in both adult Copepoda and S. elegans (Fig. 5). The magnitude of isotopic offsets between bulk tissue and lipids has been linked to the physiological condition of the organisms and tissue type. Extrapolating existent regularities of bulk tissue-lipid d13C offsets is usually confounded either methodologically, through removal of lipids prior to SI analysis, or subject to bias after arithmetic lipid corrections when using constants to estimate tissue protein content from C:N ratios, as well as due to variable amounts of total lipids (Mintenbeck et al. 2008). However, offsets that relate to the selective accumulation of individual FAs, with potentially different d13C signatures within consumers, have not been considered. Comparisons of bulk and concentration-weighted mean d13CPLFA values of zooplankton and POM suggest that
123
Biogeochemistry
homeoviscosity was achieved by incorporating PLFAs with variable d13C signatures, because PLFA13 Total d C signatures of zooplankton largely follow the isotopic signatures of POM, and POM shows marked changes in d13CPLFA signatures and composition during bloom events in the stratified central NS. At time scales studied here, phospholipids appeared to be suitable to study changes in zooplankton C sources. This study also revealed that even structurally similar phospholipids exhibit different isotopic signatures and still provide evidence of distinct feeding histories or food preferences (see also Evershed et al. 2007; Koch 2007). This result is not surprising, as essential FAs such as EPA and DHA are vital for growth and reproduction of Copepoda (Brett and Mu¨ller-Navarra 1997; Sterner and Schulz 1998; Klein Breteler et al. 2005). Zooplankton trophodynamics Bacteria are the most numerous organisms in marine ecosystems and through them flows a large fraction of annual primary production (Fenchel 1984, 1988). Several trophic pathways for fluxes of bacterialderived C towards higher trophic levels, such as fish, coexist in aquatic ecosystems. The abundance of bacterial FAs in zooplankton of the SB and seasonally stratified OG sites coincided with increased bacterial biomass in POM during the phytoplankton bloom (data not shown). The abundance of bacterial FAs was also higher at the SB during phases of increased river run-off in winter. In contrast, there was less seasonality at the ND site, where the influence of coastal water is small (Brown et al. 1999). Lipid analysis indicated that small copepods, S. elegans and zoea larvae relied to a larger degree on the microbial food web than the evaluation of their bulk d15N signature alone would indicate. The abundance of bacterial markers during the spring and autumn blooms (Fig. 7) suggested greater importance of the microbial food web as a food source for small copepods such as C. typicus, T. longicornis and P. elongatus (Fig. 8) during this time. Bacterial PLFAs were almost absent from C. armata, C. finmarchicus and C. helgolandicus in late autumn/ winter, but were markedly present in April 2007, suggesting a higher degree of omnivory by these species during spring. Certainly, both phytoplankton
123
and grazers release DOM during blooms, fuelling the production of the microbial food web (Azam et al. 1983; Lancelot and Billen 1985; Fenchel 1988; Baines and Pace 1991; Elifantz et al. 2005; Suratman et al. 2009). DOM release and phytoplankton production of transparent exopolymeric particles under nutrient depleted growth conditions (Engel et al. 2002; Van den Meersche et al. 2004), typically create favorable growth conditions for bacteria and bacterivorous micro-zooplankton, which are preyed upon by meso-zooplankton. The bacteria themselves are likely to be too small to be selectively grazed by zooplankton sampled during this study, but cells may have been ingested passively, due to bacterial colonization of aggregates of senescent phytoplankton (e.g. Bacillariophyceae and Phaeocystis spp., Prymnesiophyceae), on which zooplankton prey (Riebesell 1991; Bequevort et al. 1998). Passive uptake of bacteria has been shown in feeding experiments with Calanus pacificus by Lawrence et al. (1993), who found high label retention after gut evacuation and thus demonstrated that passive uptake of bacteria provides a direct trophic link between the microbial food web and the herbivorous food chain. In addition, the abundance of bacterial FAs in zooplankton in this study may be partly due to feeding on bacterivorous Protozoa including Ciliata and Tunicata including Appendicularia and other members of the microbial food web, which has been reported from freshwater (Lake Michigan) and coastal and upwelling areas (Painting et al. 1993; Fessenden and Cowles 1994; Vargas and Gonza´lez 2004; Bundy et al. 2005; Vargas et al. 2007). It is unlikely that from a nutritional point of view bacteria contribute substantially to the diet of zooplankton throughout their lifecycle, but the abundances of PLFABacteria in zooplankton indicate strong linkages between the microbial food web and some mesozooplankton species, suggesting omnivorous rather than herbivorous feeding. Ecological implications This study has highlighted the complementarity of three biogeochemical approaches, viz. bulk SI analysis (d13C and d15N), PLFA fingerprinting and CSIA of d13CPLFA, and confirmed that body size and d15N signatures alone are weak surrogates of zooplankton trophic level (Jennings et al. 2001). Relatively low
Biogeochemistry
d15NPOM-based RTP values of *1.3, for example, suggest herbivory for zoeae larvae, but the presence of bacterial FAs clearly links zoeae to the microbial food web. Similarly, typically high RTPs values for the copepod predator S. elegans (e.g. Soreide et al. 2006), and abundances of bacterial PLFA markers indicate a trophic relationship with the microbial food web. This corroborates the concept of size-based food web analysis, in which small individuals (zoeae) of large species may be functionally equivalent to large individuals (S. elegans) of small species, see Jennings (2005). This study supports recent criticisms that linear trophic relationships in plankton food chains are too simple and poorly applicable to the classical views of trophic complexity (Landry 2002). Evaluation of all three methods used here showed more complexity than simplifying linear phytoplankton-zooplanktonfish food chains assume, although the use of d15N as a trophic level estimator is based on linearity and the inherent assumption of relatively constant trophic fractionation (Azam et al. 1983; Pomeroy 2001; Landry and Calbet 2004). It has been shown that linear food chains, underlying the use of d15N as trophic level indicator, may be inappropriate for routine assessments of natural abundances of C and N isotopes in food webs that include small-sized animals, in which a ‘‘secondary network of interactions involves individual developmental stages of each animal represented’’ (Landry 2002). Doubtless, trophic fractionation leads to moderately predictable 15 N enrichment from one trophic level to the next in higher metazoans. Legendre and Rassoulzadegan (1995) proposed that the herbivorous and microbial loop food webs are extreme cases of a trophodynamic continuum in the pelagic domain. Thus, isotopic variability at the base of the food web, attributable to co-existing auto-, mixo- and heterotrophic plankton organisms’ use of DOM released by phytoplankton, and of remineralized macronutrients from a variety of isotopically depleted or enriched sources, challenges conceptual linearity (Tamelander et al. 2009, this study) and highlights the complexity of multivorous food webs. This was shown most clearly by high RTPs and d15N of some zooplankton taxa, whose d15N exceeded those of NS top predators such as fish and seabirds. These discrepancies are likely to be due to differences in tissue turnover between zooplankton and higher animals (Rolff 2000), which reduce the
speed of isotopic baseline propagation up to higher trophic levels with increasing size, body mass and age. Thus, over the whole size-range of organisms within the NS food web, several food chains coexist that separately lead to increased RTPs, confounding correct assignments of RTPs to omnivorous species, as well as to small organisms or species that consume (Jennings et al. 2001; Sweeting et al. 2005).
Conclusions This study provides the first detailed descriptions of NS zooplankton RTPs and trophodynamics using bulk SI analysis, PLFA compositions and CSIA of PLFAs. Bulk SI analysis demonstrated that NS zooplankton occupy a range of RTPs, and PLFA fingerprints revealed temporal and species-specific changes in diet including the abundance of bacterial FAs in zooplankton. Besides physiological aspects, terrestrial run-off may be responsible for the increased availability and abundance of bacterial FAs in zooplankton collected in the Southern Bight, factors that were less important at North Dogger. CSIA revealed that 13C incorporation from essential FAs influenced by changes in d13C signatures of potential food sources, alters isotopic signatures of zooplankton phospholipids. The use of d15N signatures for source delineation may be compromised by the inherent complexity of isotopic end-members included in routine POM samples and their trophic position. The use of bulk d13CPOM and d13CPLFA, as proxies for environmental conditions and resource exploitation of microbial communities, merits ongoing research. The diet of NS zooplankton was shown to include components of the microbial food web, either through passive feeding on bacteria which colonize phytoplankton aggregates or through feeding on bacterivorous protozoa and ciliates, suggesting omnivorous rather than strictly herbivorous feeding for NS zooplankton species. This, in turn, has implications for numerical marine ecosystem models, which may be used to deduce fisheries yield from primary production, as bottom-up processes play important roles (Mackinson et al. 2009). Nutrient-phytoplankton-zooplankton (NPZ) models used for the study of macronutrient fluxes sometimes fail to account for intra-annual variation in zooplankton growth or prey preferences and typically assume a limited number of
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Biogeochemistry
trophic interactions (Edwards and Richardson 2004; Irigoien et al. 2005). Given that zooplankton have been shown to have seasonal plasticity in food preferences (Soreide et al. 2006, 2008; Tamelander et al. 2006a, 2008), seasonal changes in diet should be accounted for. Ecosystem models therefore need to take account of the trophodynamic plasticity among zooplankton and their prey (primary consumers) and changes in the relative dependencies of zooplankton on the herbivore or microbial food webs. Ideally, such models should also account of smaller juvenile stages, growth, development and reproduction cycles of zooplankton as well as the feedback loops between the lower and higher trophic levels of the food web (Fennel and Neumann 2001; Zhao et al. 2008). Acknowledgments This study was supported by the EURopean network of excellence for OCean Ecosystems ANalysiS (EUR-OCEANS) funded by the European Commission [project WP6-SYSNS-1098]. The field work was partially funded by the Centre for Environment, Fisheries and Aquaculture Science (CEFAS) and the Centre for Estuarine and Marine Ecology, Netherlands Institute of Ecology (NIOO-KNAW). We thank C. Vignot for preparatory assistance and M. Houtekamer, P. van Rijswijk, P. van Breugel and A. Knuijt for analytical support. We thank two anonymous reviewers for their valuable comments.
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