Complex trophic interactions of calanoid copepods in the Benguela upwelling system

SEARES-01087; No of Pages 11 Journal of Sea Research xxx (2013) xxx–xxx

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Complex trophic interactions of calanoid copepods in the Benguela upwelling system Anna Schukat a,⁎, Holger Auel a, Lena Teuber a, Niko Lahajnar b, Wilhelm Hagen a a b

Marine Zoology, BreMarE — Bremen Marine Ecology, University of Bremen, P.O. Box 330440, 28334 Bremen, Germany Institute for Biogeochemistry and Marine Chemistry, University of Hamburg, Bundesstraße 55, 20146 Hamburg, Germany

a r t i c l e

i n f o

Article history: Received 10 July 2012 Received in revised form 22 April 2013 Accepted 26 April 2013 Available online xxxx Keywords: Zooplankton Fatty acids Stable isotopes Biomarkers Trophic level Food web

a b s t r a c t Life-cycle adaptations, dietary preferences and trophic levels of calanoid copepods from the northern Benguela Current off Namibia were determined via lipid classes, marker fatty acids and stable isotope analyses, respectively. Trophic levels of copepod species were compared to other zooplankton and top consumers. Lipid class analyses revealed that three of the dominant calanoid copepod species stored wax esters, four accumulated triacylglycerols and another three species were characterised by high phospholipid levels. The two biomarker approaches (via fatty acids and stable isotopes) revealed a complex pattern of trophic positions for the various copepod species, but also highlighted the dietary importance of diatoms and dinoflagellates. Calanoides carinatus and Nannocalanus minor occupied the lowest trophic level (predominantly herbivorous) corresponding to high amounts of fatty acid markers for diatoms (e.g. 16:1(n−7)) and dinoflagellates (e.g. 18:4(n−3)). These two copepod species represent the classical link between primary production and higher trophic levels. All other copepods belonged to secondary or even tertiary (some deep-sea copepods) consumers. The calanoid copepod species cover the entire range of δ15N ratios, as compared to δ15N ratios of all non-calanoid taxa investigated, from salps to adult fish. These data emphasise that the trophic roles of calanoid copepods are far more complex than just interlinking primary producers with pelagic fish, which should also be considered in the process of developing realistic food-web models of coastal upwelling systems. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Copepods were traditionally considered as being herbivorous, acting as a key link between primary producers and planktivorous fish (Anraku and Omori, 1963; Lowndes, 1935). This view of the trophic role of calanoid copepods has changed during the last decades. Trophic studies in different regions of the world ocean revealed that many copepod species are omnivorous with picoplankton, nanoplankton and microzooplankton as major components of their diet (e.g. Calbet et al., 2007; Escribano and Pérez, 2010; Kleppel et al., 1996). However, the natural diets of most planktonic copepod species are still not well characterised and it is difficult to clearly assign them to specific trophic levels. A classical method to determine dietary components is gut content analysis, which provides detailed taxonomic information (Hyslop, 1980). However, this method fails for soft-bodied and fragile organisms or in case of advanced digestion, and it can only provide a snap-shot impression. In contrast, trophic biomarkers (e.g. fatty acids and stable isotopes) integrate dietary signals over longer time spans of days to

⁎ Corresponding author. Tel.: +49 42121863032. E-mail address: [email protected] (A. Schukat).

weeks (Gentsch et al., 2009; Graeve et al., 1994). They may supplement conventional gut content analyses and represent a complementary approach to determine dietary preferences and trophic positions. The analysis of lipids, particularly fatty acid compositions, is used for the identification of trophic relationships in marine ecosystems. Some fatty acids are characteristic of specific groups of phyto- or zooplankton and are incorporated into consumers' body tissues largely unmodified, thus retaining a dietary signature (e.g. Dalsgaard et al., 2003). Stable isotope ratios of naturally occurring nitrogen (15N/14N) and carbon (13C/12C) are useful in aquatic food-web analyses for identifying trophic levels and different carbon sources, respectively (Hobson and Welch, 1992; Michener and Shell, 1994). Via isotopic fractionation, heavier isotopes accumulate in the animals' body tissues, while lighter isotopes are more rapidly metabolized and excreted (Bode and Alvarez-Ossorio, 2004; Vander Zanden and Rasmussen, 2001). Thus, the stable isotope composition of a consumer reflects the isotopic composition of its prey, but is further enriched in heavier isotopes. For marine food webs, the enrichment in 15N has been determined with 3–4‰ per trophic level (Cabana and Rasmussen, 1994; Minagawa and Wada, 1984; Peterson and Fry, 1987). Isotopic fractionation for carbon from prey to predator is less pronounced (~1‰) and this isotope ratio is therefore a useful indicator of origins and sources of primary production (Hecky and Hesslein, 1995; Hobson et al., 1995).

1385-1101/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seares.2013.04.018

Please cite this article as: Schukat, A., et al., Complex trophic interactions of calanoid copepods in the Benguela upwelling system, Journal of Sea Research (2013), http://dx.doi.org/10.1016/j.seares.2013.04.018

2

A. Schukat et al. / Journal of Sea Research xxx (2013) xxx–xxx

The Benguela Current off the coast of southwest Africa is characterised by a predominantly equatorward flow and high levels of Ekman-driven coastal upwelling (Boyer and Hampton, 2001; Shannon and O'Toole, 2003). Together with the Humboldt Current, it is the most productive ‘Large Marine Ecosystem’ of the world ocean (Heileman and O'Toole, 2008) with an annual primary productivity ranging from 400 to 900 g C m−2 yr−1 (Brown et al., 1991; Carr, 2002; Monteiro, 2010), which supports a high standing stock of zooplankton and fish. Copepods and euphausiids generally dominate the zooplankton community in upwelling regions and constitute the key link for channelling primary production into fish production (Cushing, 1990; Hidalgo et al., 2010). The most dominant calanoid copepod species of the northern Benguela Current with relatively high perennial abundances are Calanoides carinatus and Metridia lucens (Timonin et al., 1992; Verheye et al., 1991, 1992). Other frequently occurring copepods include Centropages brachiatus, Nannocalanus minor, Aetideopsis carinata, Pleuromamma spp., Eucalanus hyalinus and Rhincalanus nasutus (Hansen et al., 2005; Loick et al., 2005). Little is known about the feeding ecology and dietary spectra of these subtropical species, compared to their high-latitude congeners. Precise knowledge about the feeding preferences of dominant copepods is necessary to understand the trophodynamics and energy flux of the respective ecosystems. The aim of this study was to reveal strategies of calanoid copepods to cope with the variability of coastal upwelling systems and to identify their dietary relationships and trophic levels in this environment. Therefore, a large dataset of dominant calanoid copepods, as well as of other dominant taxa of zooplankton and upper trophic levels was assessed and trophic interactions were analysed combining the two complementary methodological approaches, marker fatty acids and stable isotopes.

Fig. 1. Study area in the northern Benguela upwelling system with stations sampled for zooplankton in March/April 2008 (open circles) and December 2009 (black dots) and for NO− 3 (2009; black crosses) and phytoplankton (2009; grey crosses) stations.

2. Material and methods 2.1. Sampling 2.1.1. Copepods Samples were collected in the northern Benguela upwelling system (Fig. 1, for details see Table A1, appendix) during low to moderate upwelling intensities (temperature range 15–19 °C, CTD data, Mohrholz, 2012). Copepods for lipid and stable isotope measurements were sampled from 6th to 13th December 2009 (FRS Africana). They were sampled by different net types: vertical and oblique Multinet hauls (Hydro-Bios Multinet Midi, mesh size 150–500 μm), towed 1 m2-MOCNESS (Multiple Opening and Closing Net with Environmental Sensing System, 333 μm), Current Underway Fish Egg Sampler (CUFES, 500 μm) and driftnet (200 μm). At stations on the shelf and upper continental rise, the whole water column was sampled by the vertical opening and closing nets. Further offshore the maximum sampling depth was 1000 m (2008) and 750 m (2009). Copepod samples for lipid and stable isotope analyses were taken from the same station, net and depth stratum, if possible. Immediately after each haul, samples were rapidly but gently sorted into species and stages and deep-frozen at − 80 °C for further analyses in the home lab. 2.1.2. Other zooplankton and higher trophic levels For comparison of trophic levels, also other taxa of the northern Benguela ecosystem were sampled on the same cruise and from 10th to 31st March 2008 (RV Maria S. Merian). Zooplankton (amphipods, euphausiids, decapods, chaetognaths and gelatinous zooplankton, e.g. salps) were collected from hauls of Multinet (150–200 μm), 1 m2-MOCNESS (333 μm) and Tucker trawl (1000 μm). Fish samples were also taken from the Tucker trawl and a demersal fish trawl. Feathers of seabirds (Hydrobatidae) were collected from specimens that accidentally landed on board. All samples were immediately deep-frozen at −80 °C.

2.1.3. Phytoplankton and water samples for isotopic baseline At some stations in December 2009, extensive blooms of large phytoplankton cells occurred and were sampled by Multinet hauls (200 μm) in the upper 30 m. Although microzooplankton (dinoflagellate Noctiluca) could not be excluded from these seston samples, the vast majority of seston during these extreme bloom events consisted of large centric diatoms (Coscinodiscus wailesii) and smaller chain-forming diatoms (e.g. Chaetoceros sp.). Water samples (5–18 l) were taken regularly of almost every CTD cast in 2009 from epi- to mesopelagic depth (0–800 m) and filtered through pre-combusted GF/F filters (Whatman). The filtrate was used to measure the isotopic composition (15N) of nitrate (NO− 3 ) to determine the ecosystem isotopic baseline for the calculation of δ15N of phytoplankton. 2.2. Lipid analysis Body dry mass of copepods was determined after lyophilisation of the deep-frozen samples for 48 h. Identical species and stages from the same station, net and depth stratum were pooled to obtain sufficient biomass. Lipids were extracted with dichloromethane:methanol (2:1 per volume) according to Folch et al. (1957) and total lipid content was measured gravimetrically after Hagen (2000). Lipid classes were determined by high-performance (HP) TLCscanning densitometry as described by Stübing et al. (2003). 2–3 μg lipid was applied in duplicate on HPTLC plates (silica gel 60, Merck), which were developed for 17 min in hexane:diethyl ether:acetic acid (80:20:2, v/v) for the separation of neutral lipids. The lipid bands were quantified at 550 nm wavelength and calibrated using commercial standards for each detected lipid class. Fatty acids were converted to their methyl ester derivatives (FAME) by transesterification in methanol containing 3% concentrated sulphuric acid (Kattner and Fricke, 1986; Peters et al., 2007) and

Please cite this article as: Schukat, A., et al., Complex trophic interactions of calanoid copepods in the Benguela upwelling system, Journal of Sea Research (2013), http://dx.doi.org/10.1016/j.seares.2013.04.018

A. Schukat et al. / Journal of Sea Research xxx (2013) xxx–xxx

analysed together with the fatty alcohols using a gas chromatograph (Agilent Technologies 7890A) equipped with a DB-FFAP column of 30 m length and 0.25 mm diameter and a programmable temperature vaporiser injector (Peters et al., 2007). Peaks were identified according to retention times in comparison to natural standards (marine fish oil and lipids of the copepod Calanus hyperboreus) of known composition. Fatty acid compositions are evaluated following the trophic biomarker concept (Dalsgaard et al., 2003). 16:1(n−7), as well as 16:4(n−1) and 18:1(n−7) are used as indicators of a diatom-dominated diet, while 18:4(n−3) is applied as dinoflagellate marker (Dalsgaard et al., 2003). 18:1(n−9) is commonly used as carnivory marker (Falk-Petersen et al. 1990). The ratio 18:1(n−9)/18:1(n−7) was thus calculated to estimate the degree of carnivory versus herbivory (Auel et al., 2002; Dalsgaard et al., 2003). In addition, we chose the fatty acid ratio of 18:1(n−9)/ [16:1(n−7) + 16:4(n−1) + 18:1(n−7) + 18:4(n−3)], in the following named 18:1(n−9)/∑herb. markers, as a new relative index of carnivory. 2.3. Stable isotope analysis Dried samples of usually 2 to 5 mg were transferred to tin capsules and stable isotope analyses were performed by Agroisolab GmbH in Jülich, Germany, using a mass spectrometer (EA NA1500 Series 2, Carlo Erba Instruments) and helium as carrier gas. Copepods were used as entire animals, whereas sub-samples (fraction of the whole grinded animal) were used for larger zooplankton taxa. For fish species, homogenized parts of their muscles were taken, which have a constant δ15N with size, time and temperature (Sweeting et al., 2007). Feathers (fraction of grinded tissue) were used for stable isotopes of seabirds (non-destructive sampling in live birds) (Bond and Jones, 2009). Samples of small species (e.g. epipelagic copepods) were pooled to obtain sufficient biomass (0.5–5.0 mg) for proper isotope analyses. Stable isotope ratios of carbon and nitrogen were determined using the standards IAEA-VPDB (IAEA-C1) and atmospheric air (IAEA-N1), respectively. Isotopic ratios are expressed as δ13C and δ15N in ‰, according to the equation given by Hobson et al. (2002). Lipids were not extracted prior to stable isotope analysis as suggested by some studies (e.g. Hobson et al., 2002; Mintenbeck et al., 2008), since biomass values of epipelagic copepods were generally low and any bias of δ15N by lipid extraction was to be avoided (Mintenbeck et al., 2008). However, lipid analysis revealed that copepods had a high variability in their lipid deposits (Table 1). Therefore, δ13C values of crustaceans were corrected on the basis of a molar C/N ratio, applying a lipid-normalisation model (applicable for C:N ratios ≥ 4.0) published by McConnaughey and McRoy (1979) and again tested for zooplankton by Smyntek et al. (2007). 2.3.1. Creation of a δ15N baseline and calculation of trophic levels An isotopic baseline of a food web is required to estimate distinct trophic levels. We applied the following three methodological approaches to obtain food-web baselines for the northern Benguela Current: i) Calculation of δ15N of phytoplankton via nitrate assimilation by phytoplankton. Here, the 15N/14N ratio of nitrate (NO− 3 ) from seawater samples (Table A2, appendix) was determined following the denitrifier method (Casciotti et al., 2002; Sigman et al., 2001) using the internationally recognised nitrate standard (IAEA-N3) for isotopic comparison with air N2. The isotope ratio is expressed as δ15N in ‰ according to the equation in Granger et al. (2004). δ15N of phytoplankton was then calculated via the following equation: 15

15

δ Nphytoplankton ¼ δ Nnitrate =εphytoplankton

3

where ε is the isotope effect (Granger et al., 2004) for nitrate assimilation by phytoplankton. The 15N/14N of nitrate in the ocean typically suggests an isotope effect of 5–10‰ for nitrate assimilation (Altabet, 2001; Sigman et al., 1999; Wu et al., 1997). We calculated a mean ε value (6.6 ± 3.6) from all published data available for marine diatoms (45 data points, seven species) (Granger et al., 2004; Montoya and McCarthy, 1995; Needoba et al., 2003; Waser et al., 1998). Regional values of δ15N of nitrate and thus calculated δ15Nphytoplankton did not differ between the transects at 17°S vs. 23°S (Mann–Whitney U-test, p = 0.27). Depth-related differences in δ15N of nitrate (0–100 m vs. >100–800 m) were not significant either (Mann–Whitney U-test, p = 0.78). Therefore, we used the mean value of all data (1.0 ± 0.1‰, Table A2, appendix) to calculate trophic levels. ii) Direct measurement of δ15N of mixed phytoplankton subsamples. iii) Use of primary consumers as baseline by defining the copepod with the lowest δ15N (N. minor, 4.8‰) as trophic level 2.0. Trophic levels were then calculated according to the equation:   15 15 Trophic level ¼ λ þ δ Nconsumer −δ Nbase =Δn where λ is the trophic level of the organism used to estimate δ15Nbase and Δn is the enrichment in δ15N per trophic level. We applied the common average enrichment of 3.4‰ per trophic level (Hobson and Welch, 1992; Peterson and Fry, 1987). 2.4. Statistical analyses Prior to statistical analysis, data distribution was tested for normality. Species-specific differences in biochemical composition were analysed using one-way ANOVA and a proximate post-hoc test (Tukey) within the Prism software package (5.0). A two-tailed unpaired t-test (confidence interval 95%) was applied to test for intraspecific differences between sex and stage using the same software package. For data with no Gaussian curve (e.g. δ15N of nitrate, temperature), differences were tested with the Mann Whitney U-test (two-tailed). 3. Results 3.1. Dry mass and lipid composition C. brachiatus, M. lucens and N. minor belonged to the smallest copepods (0.05–0.07 mg dry mass (DM), Table 1) in this study. These were followed by C. carinatus, A. carinata and Euchaeta marina (0.11–0.20 mg DM). Pleuromamma robusta, E. hyalinus, R. nasutus and Euchirella similis were the largest and heaviest copepods with 0.30–1.05 mg DM (Table 1). Total lipid contents (TL) ranged from 7% to 50% DM with lowest lipid levels in females of E. similis (6.7 ± 4.0% DM), followed by females of C. carinatus (11.3 ± 2.5% DM), A. carinata (2008: 12.2 ± 4.7%), M. lucens (12.5 ± 2.3% DM) and males of E. hyalinus (12.8 ± 1.6% DM) (Table 1, Fig. 2). Females of E. hyalinus showed a moderate lipid amount of 16.8 ± 2.2% DM, similar to E. marina (14.7% and 18.9% DM) and N. minor (17.5% and 19.8% DM). Higher lipid levels occurred in females of A. carinata from 2009 (23.9 ± 3.2% DM) and in copepodite stages C5 of C. brachiatus (27.9 ± 17.0% DM). Females of R. nasutus and copepodids C5 of C. carinatus (both years) had significantly higher lipid levels (46–50% DM) than the other copepods (ANOVA, Tukey, p ≤ 0.01, F = 27.1, df = 57). Copepod species also differed in lipid class compositions (Table 1, Fig. 2). High wax ester (WE) levels (51–87% TL) were found in females of E. marina, R. nasutus and copepodids C5 of C. carinatus. Females of C. carinatus had lower but highly variable WE contents (26.7 ± 17.4% TL) and a significantly higher phospholipid (PL) level compared to the copepodids C5 (ANOVA, Tukey, p ≤ 0.001, F = 364.6, df = 27). Triacylglycerols (TAG) were the main neutral lipid class in E. similis,

Please cite this article as: Schukat, A., et al., Complex trophic interactions of calanoid copepods in the Benguela upwelling system, Journal of Sea Research (2013), http://dx.doi.org/10.1016/j.seares.2013.04.018

4

Calanidae

Eucalanidae

C. carinatus

N. minor

E. hyalinus

Aetideidae R. nasutus

A. carinata

Euchaetidae

Metridinidae

E. similis

E. marina

M. lucens

P. robusta

Centropagidae C. brachiatus

C5

C5

♀

C5

♂

♀

♀

♀

♀

♀

♀

♀

♀

♂

Month/year n (ind) Dry mass (mg ind−1) Total lipid (% DM)

March/2008 6 (200) 0.15 ± 0.02 50.1 ± 6.7

Dec/2009 5 (160) 0.15 ± 0.01 50.2 ± 4.2

Dec/2009 3 (90) 0.11 ± 0.02 11.3 ± 2.5

Dec/2009 2 (100) 0.07/0.07 17.5/19.8

Dec/2009 3 (30) 0.33 ± 0.01 12.8 ± 1.6

Dec/2009 4 (40) 0.77 ± 0.05 16.8 ± 2.2

Dec/2009 5 (32) 0.54 ± 0.08 46.2 ± 7.5

March/2008 4 (88) 0.19 ± 0.02 12.2 ± 4.7

Dec/2009 3 (90) 0.18 ± 0.01 23.9 ± 3.2

March/2008 7 (37) 1.05 ± 0.1 6.7 ± 4.0

March/2008 2 (16) 0.20/0.16 14.7/18.9

Dec/2009 4 (200) 0.06 ± 0.01 12.5 ± 2.3

Dec/2009 3 (48) 0.30 ± 0.80 13.9 ± 1.7

Dec/2009 4 (220) 0.04 ± 0.02 28.3 ± 20.0

Lipid classes (% TL) WE TAG CHOL FFA PL

84.4 ± 2.7 7.0 ± 2.3 1.0 ± 0.2 0.7 ± 0.5 6.9 ± 0.9

87.4 ± 2.2 6.2 ± 1.2 0.2 ± 0.4 1.1 ± 0.2 5.0 ± 0.8

26.9 ± 17.4 10.5 ± 6.9 7.1 ± 3.0 5.3 ± 1.6 50.2 ± 8.4

– 9.3/14.3 11.9/11.3 6.8/1.9 71.9/72.4

– 38.9 ± 6.7 9.7 ± 1.8 2.2 ± 0.7 49.1 ± 5.0

– 59.3 ± 4.7 6.0 ± 0.7 2.8 ± 0.5 31.8 ± 4.8

83.3 ± 1.6 1.2 ± 1.0 2.0 ± 0.7 1.7 ± 1.0 11.8 ± 1.4

17.8 ± 7.3 50.6 ± 5.4 4.1 ± 0.7 2.3 ± 0.6 25.2 ± 2.1

12.1 ± 4.1 59.7 ± 3.2 3.3 ± 0.8 2.8 ± 0.5 22.1 ± 2.1

3.2 ± 3.4 36.5 ± 10.8 8.2 ± 3.6 4.3 ± 3.2 47.8 ± 5.1

51.3/82.4 4.5/– 5.4/2.2 2.1/1.2 36.7/14.2

3.3 ± 0.6 5.0 ± 1.4 18.1 ± 2.3 6.7 ± 2.4 66.9 ± 4.7

– 44.0 ± 0.8 14.2 ± 1.4 3.5 ± 0.7 38.3 ± 0.9

– – 14.5 ± 3.4 7.0 ± 2.1 78.6 ± 3.4

Fatty acids (% TFA) 14:0 16:0 18:0 16:1(n−7) 16:2(n−4) 16:4(n−1) 18:1(n−7) 18:1(n−9) 18:2(n−6) 18:4(n−3) 20:1(n−9) 20:2(n−6) 20:5(n−3) 22:1(n−11) 22:6(n−3) 24:1(n−11) 18:1(n−9)/18:1(n−7) 18:1(n−9)/∑herb. markers

5.9 ± 1.5 6.8 ± 0.6 1.4 ± 0.1 10.4 ± 0.6 1.6 ± 0.2 2.2 ± 1.1 0.6 ± 0.1 4.2 ± 0.4 1.3 ± 0.1 3.8 ± 0.3 13.5 ± 0.6 – 14.3 ± 2.5 16.7 ± 1.2 7.4 ± 1.0 0.5 ± 0.1 6.6 ± 0.8 0.2 ± 0.0

4.4 ± 0.8 6.6 ± 1.0 1.4 ± 0.2 7.9 ± 0.4 1.7 ± 0.1 4.4 ± 0.6 0.5 ± 0.1 3.6 ± 0.3 1.1 ± 0.0 3.3 ± 0.4 12.6 ± 1.1 – 20.8 ± 1.7 15.2 ± 0.3 5.6 ± 0.2 0.5 ± 0.1 7.0 ± 0.8 0.2 ± 0.0

3.2 ± 1.2 22.3 ± 9.3 5.3 ± 2.4 8.0 ± 2.5 0.8 ± 0.7 0.7 ± 0.6 1.3 ± 0.2 2.9 ± 0.3 1.6 ± 1.3 1.4 ± 0.7 1.2 ± 1.5 0.1 ± 0.1 16.0 ± 3.0 1.8 ± 2.9 20.0 ± 3.3 2.6 ± 1.5 2.3 ± 0.4 0.3 ± 0.1

1.3/2.7 12.5/18.2 4.5/5.0 1.4/2.9 –/0.2 – 0.7/1.0 2.8/6.4 8.0/1.4 9.2/0.7 –/0.5 7.5/– 12.0/15.7 – 28.7/35.1 1.6/3.7 3.9/6.6 0.2/1.4

3.5 ± 1.1 34.7 ± 6.1 7.9 ± 1.8 6.7 ± 2.2 0.9 ± 0.4 0.5 ± 0.3 2.4 ± 0.1 9.0 ± 0.7 1.2 ± 0.3 0.6 ± 0.3 0.3 ± 0.0 0.3 ± 0.3 12.1 ± 1.6 – 10.7 ± 2.0 2.8 ± 1.0 3.7 ± 0.1 0.9 ± 0.2

4.0 ± 0.3 24.1 ± 1.9 3.8 ± 0.5 8.6 ± 0.3 1.8 ± 0.1 2.8 ± 0.3 3.3 ± 0.1 6.0 ± 0.6 0.8 ± 0.1 1.4 ± 0.1 0.4 ± 0.0 – 26.4 ± 2.0 – 5.8 ± 0.1 0.9 ± 0.1 1.9 ± 0.2 0.4 ± 0.0

1.0 ± 0.4 3.8 ± 0.8 1.1 ± 0.2 17.9 ± 1.5 2.4 ± 0.3 3.9 ± 0.6 1.4 ± 0.4 33.8 ± 3.4 1.2 ± 0.2 1.6 ± 1.5 0.6 ± 0.1 0.2 ± 0.2 20.0 ± 4.2 – 3.8 ± 1.3 0.8 ± 0.1 24.2 ± 4.1 1.4 ± 0.2

4.7 ± 1.2 20.5 ± 2.0 2.6 ± 0.2 6.6 ± 0.7 0.8 ± 0.4 0.5 ± 0.2 4.2 ± 0.2 9.5 ± 2.4 1.0 ± 0.1 0.9 ± 0.2 4.0 ± 1.3 0.2 ± 0.1 14.8 ± 1.5 7.4 ± 2.5 11.5 ± 1.0 2.0 ± 0.5 2.3 ± 0.6 0.8 ± 0.2

4.2 ± 0.8 24.4 ± 0.2 4.4 ± 0.4 5.5 ± 0.3 0.9 ± 0.0 1.1 ± 0.1 3.8 ± 0.0 10.0 ± 0.6 1.1 ± 0.1 1.4 ± 0.1 1.3 ± 0.3 – 21.2 ± 0.5 2.1 ± 0.6 8.4 ± 0.4 1.3 ± 0.1 2.6 ± 0.2 0.8 ± 0.1

2.0 ± 0.7 18.0 ± 2.9 3.7 ± 0.8 3.2 ± 1.5 0.3 ± 0.2 – 2.6 ± 0.3 14.8 ± 5.2 1.2 ± 0.2 0.5 ± 0.4 0.7 ± 0.4 0.5 ± 0.3 10.3 ± 0.9 0.4 ± 0.2 29.7 ± 6.8 5.8 ± 2.1 5.8 ± 1.7 2.3 ± 0.4

1.3/0.3 11.7/1.6 2.4/0.4 4.5/9.5 0.9/1.3 0.4/0.8 1.6/0.7 20.4/44.0 1.5/1.3 1.7/1.8 0.8/– 0.2/0.2 14.6/12.1 0.7/– 28.7/18.9 2.4/0.3 12.9/66.3 2.5/3.5

1.0 ± 0.7 14.7 ± 1.6 3.7 ± 0.2 2.0 ± 0.5 0.1 ± 0.1 – 1.6 ± 0.0 2.5 ± 0.1 1.3 ± 0.1 2.0 ± 0.1 0.8 ± 0.1 0.4 ± 0.4 20.5 ± 1.7 – 35.9 ± 2.6 5.5 ± 0.6 1.5 ± 0.0 0.4 ± 0.0

3.4 ± 1.5 21.4 ± 1.1 3.1 ± 0.5 5.0 ± 2.4 0.8 ± 0.4 0.3 ± 0.4 3.0 ± 0.4 8.2 ± 3.4 1.5 ± 0.6 1.1 ± 0.8 1.4 ± 2.0 0.1 ± 0.2 13.4 ± 7.6 2.5 ± 2.6 16.6 ± 2.4 5.7 ± 4.0 2.7 ± 0.8 1.0 ± 0.4

2.3 ± 0.7 18.2 ± 1.1 4.4 ± 0.3 1.5 ± 1.3 0.5 ± 0.3 0.1 ± 0.2 2.5 ± 0.5 2.1 ± 0.7 0.7 ± 0.2 0.5 ± 0.2 0.3 ± 0.1 – 16.1 ± 1.1 0.1 ± 0.2 40.3 ± 3.8 1.6 ± 0.3 0.8 ± 0.2 0.5 ± 0.1

Fatty alcohols (% TFAlc) 14:0 16:0 18:0 20:1 22:1

11.7 ± 0.8 16.2 ± 1.1 1.7 ± 0.2 19.1 ± 1.0 50.7 ± 1.9

12.3 ± 1.0 16.8 ± 1.2 2.1 ± 0.1 19.9 ± 0.6 48.8 ± 1.7

10.1 ± 18.7 17.8 ± 19.5 1.9 ± 8.8 16.1 ± 9.3 54.1 ± 31.2

– – – – –

– – – – –

– – – – –

36.0 ± 3.6 58.0 ± 3.0 6.1 ± 0.6 – –

16.3 20.6 – 13.8 47.2

17.1 40.2 – 13.5 29.2

38.1 ± 6.9 61.9 ± 6.9 – – –

4.0/31.6 89.5/63.7 3.2/2.3 – 1.6/0.9

52.3 ± 7.1 47.8 ± 7.1 – – –

– – – – –

– – – – –

± 3.3 ± 4.6 ± 2.1 ± 4.0

± 1.8 ± 15.1 ± 4.5 ± 10.1

A. Schukat et al. / Journal of Sea Research xxx (2013) xxx–xxx

Please cite this article as: Schukat, A., et al., Complex trophic interactions of calanoid copepods in the Benguela upwelling system, Journal of Sea Research (2013), http://dx.doi.org/10.1016/j.seares.2013.04.018

Table 1 Dry mass (DM), total lipid content (TL) and lipid class compositions as well as fatty acid and fatty alcohol compositions and carnivory ratios of dominant calanoid copepods from the northern Benguela upwelling system. WE: wax esters; TAG: triacylglycerols; CHOL: cholesterol; FFA: free fatty acids; PL: phospholipids; TFA: total fatty acids; TFAlc: total fatty alcohols; ∑herb. markers: sum of 16:1(n−7), 16:4(n−1), 18:1(n−7), 18:4(n−3); n (ind): number of samples (total number of individuals).

A. Schukat et al. / Journal of Sea Research xxx (2013) xxx–xxx

5

amounts of the two shorter-chain saturated fatty alcohols 14:0 and 16:0, while 16:0 dominated with 90% in E. marina.

Fig. 2. Total lipid content (% dry mass, DM) and percentage contribution of major lipid classes to total lipid of dominant calanoid copepods. WE: wax esters, TAG: triacylglycerols, PL: phospholipids. Staged columns with error bars represent two sampling years (2008, 2009), no error bars: only two replicates were analysed. See Table 1 for the number of samples.

P. robusta, E. hyalinus and A. carinata with 37–60% TL. Males of E. hyalinus had significantly lower TAG levels compared to females (t-test, p ≤ 0.001). PL was also a dominant lipid class in these four copepod species. Maximum PL contents (63–79% TL) were determined in M. lucens, N. minor and C. brachiatus, where TAG and WE occurred only in small amounts or were completely absent (i.e. C. brachiatus). Cholesterol (CHOL) and free fatty acids were minor lipid classes (combined b 10% TL), except for M. lucens, P. robusta and C. brachiatus, which exhibited higher portions of CHOL (14–18% TL).

3.2.1. Fatty acid biomarker ratios The ratios 18:1(n−9)/18:1(n−7) and 18:1(n−9)/∑herb. markers are used as indicators for the degree of carnivory (Table 1, Fig. 3). E. marina showed the highest degree of carnivory with significantly higher fatty acid ratios (ANOVA, Tukey, p ≤ 0.05, F = 11.0 and 39.4, df = 54), as compared to all other copepods (except for E. similis for 18:1(n−9)/∑herb. markers, p = 0.13). R. nasutus and E. similis also exhibited higher values for both ratios. The two samples of N. minor varied between 3.9 and 6.6 for 18:1(n−9)/18:1(n−7) and 0.2 and 1.4 for 18:1(n−9)/∑herb. markers. Intermediate ratios were found in females of A. carinata, P. robusta and males of E. hyalinus (18:1(n−9)/ 18:1(n−7): 2.3–3.7, 18:1(n−9)/∑herb. markers: 0.8–1.0). Females of E. hyalinus exhibited low ratios, similar to C. brachiatus and M. lucens (18:1(n−9)/18:1(n−7): 0.8–1.5, 18:1(n−9)/∑herb. markers: 0.4– 0.5). Deviating results with regard to the two marker ratios occurred in C. carinatus, particularly in the copepodids C5, with higher values (2.3–7.0) for 18:1(n−9)/18:1(n−7) and lowest values (0.2–0.3) for 18:1(n−9)/∑herb. markers. 3.3. Stable isotope ratios and trophic levels Stable isotope ratios (δ15N, δ13C) of dominant calanoid copepods are shown together with isotopic ratios of phytoplankton in Table 2. Other taxa of the northern Benguela food web are also listed for comparison of stable isotope ratios and calculated trophic levels. Mean δ15N values

3.2. Fatty acid and alcohol compositions Fatty acid compositions of most copepod species were characterised by high amounts of typical membrane fatty acids, e.g. 16:0, 20:5(n−3) and 22:6(n−3) (Table 1). Principal fatty acids in copepodids C5 of C. carinatus (both years) were the marker fatty acid for diatoms 16:1(n−7) (7.9–10.4% total fatty acids (TFA)) and the long-chain mono-unsaturated fatty acids typical of calanid copepods 20:1(n−9) (12.6–13.5% TFA) and 22:1(n−11) (15.2–16.7% TFA). Females of C. carinatus also showed elevated values of 16:1(n−7), whereas 20:1(n−9) and 22:1(n−11) occurred in low amounts b 2% TFA. One of the two samples of N. minor had a high amount (9.2% TFA) of the dinoflagellate marker 18:4(n−3). E. hyalinus (both sexes), A. carinata (both years), E. similis and P. robusta had, in addition to the membrane fatty acids, higher amounts of the carnivory marker 18:1(n− 9). The diatom marker 16:1(n− 7) also showed enriched values in these species (except in E. similis). A. carinata from 2008 exhibited higher amounts of 20:1(n− 9) (4.0 ± 1.3% TFA) and 22:1(n− 11) (7.4 ± 2.5% TFA). E. marina and R. nasutus had significantly higher amounts of 18:1(n− 9) with up to 44% TFA, as compared to all other copepods (ANOVA, Tukey, p ≤ 0.001, F = 29.0, df = 54). Lipids of these two species also typically contained 16:1(n− 7). R. nasutus was the only species, which contained phytanic acid (~ 7% TFA, not listed in Table 1). M. lucens and C. brachiatus were clearly dominated by the three membrane fatty acids (Table 1). Six of the calanoid copepod species stored wax esters in variable amounts and thus also contained fatty alcohols (Table 1). The fatty alcohol compositions of C. carinatus and A. carinata were characterised by the alcohol moieties 14:0, 16:0, 20:1 and 22:1, with the long-chain mono-unsaturated fatty alcohols as dominant components (together > 60% and > 40%, resp.). R. nasutus, E. similis and M. lucens had high

Fig. 3. Fatty acid ratios A) 18:1(n−9)/18:1(n−7) and B) 18:1(n−9)/∑herb. markers. Copepod species are arranged according to their level of carnivory versus herbivory. See Table 1 for the number of samples.

Please cite this article as: Schukat, A., et al., Complex trophic interactions of calanoid copepods in the Benguela upwelling system, Journal of Sea Research (2013), http://dx.doi.org/10.1016/j.seares.2013.04.018

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A. Schukat et al. / Journal of Sea Research xxx (2013) xxx–xxx

Table 2 Stable isotope compositions (δ15N and δ13C) of several components of the northern Benguela food web. Trophic levels are calculated via i) δ15N of phytoplankton (calculated by NO− 3 ), ii) δ15N of phytoplankton samples and iii) δ15N of primary consumer (Nannocalanus minor) using a δ15N enrichment of 3.4‰. δ13C′ = lipid corrected, values in brackets signify species with a C:N ratio b4.0, n = number of samples (total number of individuals). Species

Stage

Nitrate Phytoplankton (calculated by NO− 3 ) Mixed phytoplankton Copepods (b700 m depth) Nannocalanus minor Aetideopsis carinata Calanoides carinatus Calanoides carinatus Eucalanus hyalinus Euchaeta marina Rhincalanus nasutus Centropages brachiatus Metridia lucens Pleuromamma robusta Copepods (>700 m depth) Paraeuchaeta gracilis Paraeuchaeta hanseni Gaetanus pileatus Megacalanus princeps Pseudochirella sp. Lucicutia sp. Gaussia princeps Other crustaceans Decapods Oplophorus novaezeelandiae Acanthephyra pelagica Sergestes orientalis Gennadas brevirostris Sergestes armatus Sergia robusta Euphausiids (Nematoscelis sp.) Amphipods Hyperia galba Themisto gaudichaudi Other zooplankton Pteropods (Cymbulia sp.) Chaetognaths (Sagitta sp.) Jellyfish Chrysaora hysoscella Beroe sp. Aequorea aequorea Salps Salpa fusiformis Pyrosoma sp. Fish Anchovy (Engraulis capensis) Cyclothone sp. Cape hake (Merluccius capensis) Snoek (Thyrsites atun) Storm petrel (Hydrobatidae)

δ13C (‰)

δ13C′ (‰)

Year

n

2009 2009 2009

36 25 3

−16.2 ± 0.5

f f C5 f f f f adult f f

2009 2009 2009 2009 2009 2008 2009 2009 2009 2009

3 (35) 4 (60) 4 (58) 3 (56) 7 (16) 2 (6) 5 (11) 1 (25) 2 (100) 1 (2)

−22.7 ± 0.8 −17.3 ± 0.1 −19.9 ± 0.2 −17.8 ± 0.1 −19.4 ± 0.4 −20.4/−20.8 −20.0 ± 0.9 −21.5 −19.5/−19.8 −18.5

(−23.2 ± 0.8) −16.3 ± 0.3 −17.1 ± 0.2 −17.5 ± 0.1 −19.5 ± 0.3 −20.3/−21.0 −17.8 ± 1.2 (−21.7) (−19.8/−20.0) −18.6

f f f C5 f f m,f

2008 2008 2008 2008 2008 2008 2008

5 (12) 1 2 1 1 1 3

−19.2 ± 0.7 −19.1 −18.1/−18.3 −18.6 −20.1 −21.9 −19.8 ± 0.6

Adult Adult Adult Adult Adult Adult Adult

2009 2009 2009 2009 2009 2009 2008

2 2 1 3 3 1 3

Adult Adult

2009 2009

(NO− 3 )

δ15N (‰)

C:N

Trophic level

4.8 ± 0.4 6.0 ± 0.3 6.1 ± 0.3 8.1 ± 0.1 6.9 ± 0.5 8.5/8.2 8.7 ± 2.1 8.9 10.6/10.1 10.9

3.6 5.1 10.4 4.3 4.0 4.0 7.9 3.8 3.8 4.0

i 2.1 2.5 2.5 3.1 2.7 3.2 3.3 3.3 3.7 3.9

ii 2.1 2.4 2.4 3.0 2.7 3.1 3.2 3.3 3.7 3.9

iii 2.0 2.4 2.4 3.0 2.6 3.0 3.2 3.2 3.6 3.8

−17.7 ± 0.4 −17.2 −17.8/−18.4 −18.5 −20.2 −19.3 19.0 ± 0.1

8.9 ± 0.6 9.3 9.4/9.1 10.8 11.0 11.9 12.8 ± 0.6

6.2 6.8 4.1 4.1 4.0 9.6 4.9

3.3 3.4 3.4 3.9 3.9 4.2 4.5

3.3 3.4 3.4 3.8 3.9 4.1 4.4

3.2 3.3 3.3 3.8 3.8 4.1 4.4

−16.3/−16.9 −15.1/−15.5 −16.7 −15.4 ± 0.3 −17.6 ± 0.6 −15.5 −18.2 ± 0.2

−15.9/−16.2 −14.2/−14.3 (−17.1) −15.0 ± 0.5 −17.5 ± 0.7 −14.8 (−18.3 ± 0.2)

5.7/7.3 6.6/7.1 6.9 8.0 ± 1.0 8.5 ± 0.8 8.8 9.6 ± 0.7

4.6 5.1 3.6 4.3 4.1 4.7 3.8

2.6 2.7 2.7 3.1 3.2 3.3 3.5

2.6 2.7 2.7 3.0 3.1 3.2 3.5

2.5 2.6 2.6 2.9 3.1 3.2 3.4

1 3

−18.1 −17.8 ± 0.5

−18.0 −17.7 ± 0.4

9.2 9.9 ± 0.6

4.1 4.1

3.4 3.6

3.3 3.6

3.3 3.5

2008 2008

4 4 (5)

−18.9 ± 0.5 −18.2 ± 0.5

7.3 ± 0.8 8.8 ± 0.9

3.4 4.8

2.8 3.3

2.8 3.2

2.7 3.2

2009 2009 2008

1 1 5

−17.8 −19.4 −17.5 ± 0.6

9.3 10.3 12.1 ± 1.6

3.9 4.0 3.3

3.4 3.7 4.3

3.4 3.7 4.2

3.3 3.6 4.2

2009 2008

8 2

−21.6 ± 0.9 −20.8/−20.4

6.4 ± 0.4 7.8/7.9

4.8 3.4

2.6 3.0

2.5 2.9

2.5 2.9

2008 2008 2009 2009 2008

2 2 1 2 4

−16.4/−16.0 −19.7/−18.1 −17.1 −21.4/−15.7 −17.3 ± 0.4

7.6/7.0 13.4/10.4 14.1 14.5/14.0 13.5 ± 0.7

5.9 5.4 3.9 3.8 2.9

2.8 4.2 4.9 4.9 4.7

2.8 4.1 4.8 4.8 4.6

2.7 4.1 4.7 4.8 4.6

varied greatly among taxa (1.0–14.5‰). As expected, the most depleted δ15N values were determined for phytoplankton and the most enriched values were found in fish and storm petrels. The lowest δ15N values of copepods (6–7‰) occurred in females of A. carinata, E. hyalinus and copepodids C5 of C. carinatus. Higher δ15N values (8–9‰) were found in females of C. carinatus, R. nasutus and C. brachiatus. Both species of the family Metridinidae, M. lucens and Pleuromamma sp., exhibited the highest δ15N values (10–11‰). δ13C values were lipid-corrected (δ13C′) for species with a C:N ratio ≥ 4.0. The majority of copepods clustered around δ13C′ values of −18‰ to −20‰. Higher δ13C′ isotope ratios were measured for females of A. carinata and C5s of C. carinatus with −16.3 ± 0.3‰ and −17.1 ± 0.2‰, respectively. The application of all three approaches for the baseline determination produced very similar results. The highest difference between the calculated trophic level of a species was 0.2 (Table 2). Calanoid copepod species from depths shallower than 700 m exhibited trophic levels of 2.0 to 3.9. N. minor, A. carinata, as well as copepodids C5 of C. carinatus showed low trophic levels of around

6.3 ± 0.8 1.0 ± 0.1 1.2 ± 0.8

2, whereas females of C. carinatus occupied a higher trophic level (3.0–3.1). The highest trophic level (3.8–3.9) among epi- to mesopelagic copepods was determined for Pleuromamma sp. Copepod species from deeper water layers (>700 m) had on average higher trophic levels (3.2–4.5) than those of shallower waters. Salps, pteropods, decapods and chaetognaths exhibited trophic levels from 2.5 to 3.3, whereas euphausiids, amphipods and jellyfish had higher trophic levels of 3.3 to 4.3. The trophic level for fish ranged from 2.7–2.8 for anchovy (Engraulis capensis) to 4.8–4.9 for snoek (Thyrsites atun). The storm petrel (Hydrobatidae) also occupied a high trophic level of 4.6–4.7. 4. Discussion 4.1. Life-cycle adaptations of calanoid copepods to upwelling Lipids may reveal information about life-cycle strategies (Hagen and Auel, 2001). Due to their specific biochemical and metabolic properties,

Please cite this article as: Schukat, A., et al., Complex trophic interactions of calanoid copepods in the Benguela upwelling system, Journal of Sea Research (2013), http://dx.doi.org/10.1016/j.seares.2013.04.018

A. Schukat et al. / Journal of Sea Research xxx (2013) xxx–xxx

different major lipid classes, e.g. wax esters, triacylglycerols and phospholipids, serve different functions in zooplankton (Albers et al., 1996; Hagen et al., 1993; Lee et al., 2006). Wax esters serve as long-term metabolic reserves, especially in copepods, whereas triacylglycerols are utilised for short-term demands (Båmstedt et al., 1990; Conover, 1988; Mayzaud et al., 1998). In contrast, phospholipids are typical components of biomembranes, only some Antarctic euphausiid species are known to accumulate phosphatidylcholine as energy reserve (Hagen et al., 1996). Studies of lipid compositions of zooplankton mainly focussed on high-latitude regions, since lipid accumulation and storage are much more pronounced in these seasonally-pulsed environments than in subtropical or tropical regions (Kattner and Hagen, 2009). Many of the polar calanoid copepods accumulate extensive lipid deposits as wax esters (Kattner and Hagen, 1995). In contrast, in the tropical regions usually lipid-poor copepods with low wax ester levels occur in epipelagic waters (Lee and Hirota, 1973). Data on lipid compositions of copepods from upwelling regions are scarce, except for C. carinatus from the Benguela Current (Verheye et al., 2005). Upwelling areas are highly variable ecosystems and – although at different time scales – the periodicity of active vs. inactive upwelling is comparable to the seasonal food supply at high latitudes and may lead to similar strategies of lipid accumulation in zooplankton to buffer the highly variable primary production. Among the ten dominant copepod species in the Benguela upwelling system covered in this study, three species stored wax esters (C. carinatus, R. nasutus, E. marina), four deposited triacylglycerols (E. hyalinus, A. carinata, E. similis, P. robusta) and another three were characterised by high phospholipid levels (N. minor, M. lucens, C. brachiatus). Wax esters are usually accumulated by primarily herbivorous copepod species that undergo diapause and cease feeding for extended periods of time (Båmstedt et al., 1990; Hagen and Schnack-Schiel, 1996). Accordingly, C. carinatus is known to (Arashkevich and Drits, 1997; Verheye et al., 2005) and R. nasutus is suspected to undergo diapause as part of their life-cycle in the Benguela Current (Schukat et al., 2013). The energy deposition via wax esters represents an adaptative strategy to buffer the variability of upwelling systems and to cope with food-limited periods (Lee et al., 2006). In the Benguela system, upwelling intensity fluctuates throughout the year in seven to ten-day cycles between active upwelling and relaxation. Lowest upwelling intensity occurs in austral summer and autumn over a period of four to six months (Branch et al., 1987). Lipid storage patterns of C. carinatus and R. nasutus closely resemble those of their Antarctic congeners Calanoides acutus and Rhincalanus gigas, respectively, and both species exhibit essentially similar life-cycle strategies in the Benguela upwelling system as those species in polar regions (Hagen, 1999). The wax ester deposits of E. marina in the present study are comparable to wax ester levels measured in the tropics (Lee and Hirota, 1973) and in high latitudes for Paraeuchaeta (Hagen et al., 1995; Laakmann et al., 2009a,b). In this species, wax esters are suggested to function primarily as buoyancy aids rather than as energy stores to ensure their ‘drift and wait’ feeding strategy (Auel and Hagen, 2005; Laakmann et al., 2009a). Triacylglycerols are generally used by copepod species that feed year-round and usually do not rely on extensive lipid reserves (Hagen, 1999; Hagen et al., 1993). In this study E. hyalinus, A. carinata, E. similis, and P. robusta contained low to moderate levels of triacylglycerol as main lipid class, which is in line with findings for species of the same genera in tropical and temperate regions of the ocean (Kattner and Hagen, 2009; Kotani, 2006). The smallest species in this study, N. minor, M. lucens and C. brachiatus, all contained high phospholipid levels (>70% TL) and very low neutral lipid reserves. Studies from other regions found high levels of wax esters in Metridia and triacylglycerols in Centropages (Albers et al., 1996; Lee and Hirota, 1973). Generally, phospholipids do not serve as storage lipids in copepods. Apparently, Nannocalanus, Metridia and Centropages do not

7

rely on lipid deposits and additional studies are needed to elucidate their life-cycle strategies in the northern Benguela upwelling system. Overall, the Benguela upwelling system is characterised by a mixture of calanoid copepod species with different energetic strategies. Some species exhibit strategies typical of congeners from coldtemperate to polar regions affected by a seasonally variable food supply, while others show traits of tropical species. Thus, the inventory of upwelling systems hosts species with at least two opposing strategies, in line with the highly variable environmental factors during active vs. inactive upwelling. 4.2. Comparison of methodological approaches (fatty acids vs. stable isotopes) Although our samples were collected during two cruises in March 2008 and December 2009, interannual variability between these years was not very pronounced. Most intense upwelling (sea surface temperature (SST) ≤13 °C, IBU-index: Intense Benguela Upwelling) (Hagen et al., 1981) in the northern Benguela is recorded for austral winter and spring from June to November (Branch et al., 1987; E. Hagen et al., 2001). Sampling in the present study was conducted during low to moderate upwelling periods in March and December. Comparison of SST (50–0 m, CTD data; Mohrholz, 2012) and chlorophyll a concentrations (MODIS-Aqua 4 km) of both sampling times indicated similar upwelling intensities. In both years SST varied between 15 and 19 °C (shelf and offshore, resp.). Chlorophyll a concentrations in shelf and offshore regions were also similar between years (2008: b 1–8 mg m−3, 2009: b1–10 mg m−3), indicating that sampling years were comparable. SST differences between transects (17°S vs. 23°S) were not very pronounced (0–2 °C). Only chlorophyll a concentrations indicated different food availability between transects (17°S: 1–3 mg m−3, 23°S: 2–10 mg m−3). Both methods (marker fatty acids and stable isotopes) resulted in rather similar trophic classifications of copepods. Most copepod species had calculated trophic levels between 2.5 and 3.9, based on δ15N ratios supporting a predominately omnivorous to carnivorous feeding mode. Correspondingly, relatively high indices of carnivory as derived from fatty acid trophic biomarkers suggest omnivorous to carnivorous feeding for many species. However, for some species (M. lucens, Pleuromamma sp., N. minor) both approaches differed in their indication of the carnivory degree and trophic level. M. lucens and Pleuromamma sp. showed surprisingly high δ15N values, but had low or intermediate “carnivory” fatty acid ratios. Likewise, N. minor with the lowest δ15N value had carnivory indices above the median, based on both fatty acid ratios. The differences for N. minor and Pleuromamma may be explained by the fact that samples for fatty acids and stable isotopes came from different stations with deviating food availability. The two methodological approaches may differ in their sensitivities to changes in dietary composition, particularly with regard to component-specific turnover times and, thus, the time span over which dietary signals are integrated by different trophic biomarkers (Gentsch et al., 2009; Graeve et al., 1994). Deviating results of the two indices of carnivory based on fatty acid compositions are mainly caused by the diatom marker 16:1(n−7), which is included in the 18:1(n−9)/∑herb. markers ratio, but not considered in the 18:1(n−9)/18:1(n−7) ratio. Particularly copepodids C5 of C. carinatus contained rather high amounts of 16:1(n−7), but only traces of 18:1(n−7). Therefore, they had the lowest degree of carnivory according to the 18:1(n−9)/∑herb. markers ratio, but intermediate values based on the 18:1(n−9)/18:1(n−7) ratio. We suggest that the new ratio with most relevant herbivory markers allows for a more robust interpretation than the previously used 18:1(n−9)/ 18:1(n−7) ratio, because this ratio shows a linear increase with augmenting lipid content (Stübing and Hagen, 2003) and 18:1(n−7) is often only a minor component of total fatty acids. Moreover, both 18:1 isomers can be derived from de novo synthesis; 18:1(n−9) via

Please cite this article as: Schukat, A., et al., Complex trophic interactions of calanoid copepods in the Benguela upwelling system, Journal of Sea Research (2013), http://dx.doi.org/10.1016/j.seares.2013.04.018

8

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desaturation of 18:0 and 18:1(n−7) via chain elongation of 16:1(n−7). Although most marine animals also have the potential to synthesize 16:1(n−7) de novo, several studies including experimental evidence confirmed this fatty acid as suitable trophic marker due to the strong uptake of dietary 16:1(n−7) in association with diatoms (Graeve et al., 1994; Stübing et al., 2003; W. Hagen et al., 2001). The combination of fatty acid and stable isotope measurements to provide complementary insights into trophic levels proved to be very useful, albeit certain inconsistencies still exist. 4.3. Trophic role of calanoid copepods in coastal upwelling systems Zooplankton taxa, especially copepods, play a central role in the energy flux of marine ecosystems interlinking primary producers with pelagic fish and higher trophic levels (Loick et al., 2005; Verheye et al., 2005). According to our data for the northern Benguela system, only the two calanid species C. carinatus and N. minor fit into the classical scheme of herbivorous copepods connecting primary production with secondary consumers. Both species occupy low trophic levels as primary consumers and contain high amounts of ‘herbivory’ fatty acid biomarkers. C. carinatus thrives in nutrient-rich, recently upwelled waters close to the coast, which are generally dominated by diatoms. Indeed, females and diapausing copepodids C5 of C. carinatus accumulated high amounts of the characteristic diatom marker 16:1(n−7), emphasising the importance of diatoms for the nutrition of copepods within upwelling plumes (Ceballos et al., 2006; Smith, 2001). However, the 2‰ higher δ15N ratio of C. carinatus females compared to C5s indicates that surface-dwelling females also rely on other resources than diatoms. Protozoa could serve as an additional food source (Ceballos et al., 2006). In contrast to C. carinatus, the epipelagic N. minor occurs further offshore in generally warmer and nutrient-depleted waters. The species contained higher levels of the dinoflagellate marker 18:4(n− 3). These data together with very low δ15N values suggest that autotrophic dinoflagellates are preferred by N. minor, consistent with a role as primary consumer. The differences in the fatty acid patterns of C. carinatus and N. minor are in line with the general zonation pattern of phytoplankton across coastal upwelling systems with diatomdominated assemblages in recently upwelled, nutrient-rich waters nearshore and dinoflagellate communities prevailing further offshore, where silicate has already been depleted (Gibbons and Hutchings, 1996; Probyn et al., 2000). Both species represent important food items for higher trophic levels such as euphausiids, chaetognaths and mesopelagic fish (Kinzer and Schulz, 1985; Liang and Vega-Peréz, 1995). C. carinatus occurs perennially in relatively high densities and in terms of biomass it belongs to the dominant zooplankton species in the northern Benguela system (Verheye et al., 1991, 1992). N. minor is less important in terms of abundance and biomass than C. carinatus, however, in warmer tropical water regions like the Angola Benguela Front, it may contribute significantly to zooplankton biomass (Timonin et al., 1992). Thus, these two species represent important ‘classical’ links between primary production and secondary consumers in the Benguela upwelling system. The third copepod species in our dataset with a low trophic level of 2.4 was A. carinata. This species differs in distribution pattern and life cycle from the previous two epipelagic herbivores. A. carinata occurred almost exclusively at mesopelagic depths above the continental rise, where dense blooms of senescent diatoms (mainly Coscinodiscus) are found that sank out of the euphotic zone. Under these conditions, females of A. carinata were very active and showed high egg production rates (pers. obs.). Hence, they seem to feed specifically on sinking phytodetritus after extreme bloom events to fuel their reproductive processes. With this feeding behaviour, A. carinata may have a strong impact on the sinking particle field and may regulate the export of organic material from the upwelling shelf to greater depths and the adjacent open ocean. At times of low phytodetritus

availability, A. carinata may switch to a carnivorous diet including younger stages of C. carinatus. Females of A. carinata collected in 2008 contained higher amounts of the fatty acids 20:1(n− 9) and 22:1(n− 11), typical components synthesized de novo by late copepodids of C. carinatus. Several studies from different regions of the world ocean have shown that supposedly herbivorous zooplankton taxa may rather have an omnivorous diet depending on local conditions (e.g. Gifford, 1993; Vargas et al., 2006). The present study supports the notion that pure herbivory is rarely found in copepods, whereas omnivory is very common also among calanoid copepods of the northern Benguela upwelling system. In our investigation, seven of ten epi- to mesopelagic and all deep-sea copepod species had calculated trophic levels ≥ 2.5, indicating a rather omnivorous or even carnivorous feeding behaviour. Copepods often show high clearance rates or even a preference for microheterotrophs such as ciliates as compared to phytoplankton (Fessenden and Cowles, 1994; Zeldis et al., 2002). Motile prey may be easier to detect and/or more attractive to ingest than immobile and armoured algal cells such as diatoms. Omnivory by copepods may play an important role, establishing a link between the microbial loop and the ‘classical food’ chain, with copepods ingesting large amounts of ciliates and flagellates (Calbet and Saiz, 2005). Moreover, many copepods, like Euchaeta spp., are true predators, which feed on comparably large prey items such as other mesozooplankton organisms. This has been demonstrated by feeding experiments, gut content analysis, trophic biomarkers, and functional morphology of their mouth parts (Hagen and Auel, 2001; Hopkins, 1987; Michels and Schnack-Schiel, 2005). In this study the predatory behaviour of euchaetid species is supported by the high level of carnivory, as indicated by fatty acid trophic markers and also the trophic level (3.0–3.2) for E. marina. Omnivorous/carnivorous copepod species together can occur in rather similar abundances (Hansen et al., 2005; Loick et al., 2005; Schukat et al., 2013) and biomasses (Timonin et al., 1992) in the Benguela Current as compared to the two herbivorous copepod species and thus also represent an important intermediate trophic link in the ecosystem. The fact that most copepod species occupy rather high trophic positions, leads to the unusual situation that the taxonomic group of calanoid copepods in total, from the small epipelagic N. minor to large deep-sea species such as Gaussia princeps, covers nearly the whole range of δ15N ratios, similar to all other higher taxa from salps to adult fish (Fig. 4). C. carinatus and N. minor share trophic positions with the herbivorous filter-feeding salp Salpa fusiformis at the base of the food web, whereas the majority of the copepod species have trophic positions similar to predatory zooplankton such as amphipods, the chaetognath Sagitta sp. and jellyfish, for which copepods usually serve as prey items (Liang and Vega-Peréz, 1995). This study revealed that most calanoid copepods of the northern Benguela upwelling system exhibit trophic roles that are far more complex than just interlinking primary producers with pelagic fish. Although it may be difficult to implement, this should be considered for the development of realistic food-web models. Such models often include just one box for zooplankton or a parameterisation of zooplankton by size class. Here we show that zooplankton taxa, particularly copepods, represent many different trophic guilds. Food-web models should therefore integrate dominant and ecologically important key species with their specific trophic positions to ensure an authentic description of trophic interactions in coastal upwelling ecosystems. Acknowledgments We thank the captains and crews of RV Maria S. Merian and FRS Africana for their skilful support during cruises. Special thanks go to Anja Hansen and Norbert Wasmund for helpful information on the

Please cite this article as: Schukat, A., et al., Complex trophic interactions of calanoid copepods in the Benguela upwelling system, Journal of Sea Research (2013), http://dx.doi.org/10.1016/j.seares.2013.04.018

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Fig. 4. Ranges of δ15N and their associated trophic levels for 39 plankton taxa and higher trophic levels, e.g. fish and birds, from the northern Benguela Current. Trophic levels were calculated by applying a trophic enrichment value of 3.4‰.

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Please cite this article as: Schukat, A., et al., Complex trophic interactions of calanoid copepods in the Benguela upwelling system, Journal of Sea Research (2013), http://dx.doi.org/10.1016/j.seares.2013.04.018