The mean trophic position (TP) of mesozooplankton largely determines how much mass and energy is available for higher trophic levels like fish.  Unfortunately, the ratio of herbivores to carnivores in mesozooplankton is difficult to identify in field samples.  Here we investigated changes in the mean TP of mesozooplankton in a highly dynamic environment encompassing four distinct habitats in the southern South China Sea: the Mekong River plume, coastal upwelling region, shelf waters, and offshore oceanic waters.  We used a set of parameters derived from bulk and amino acid nitrogen stable isotopes from particulate organic matter (POM) and four mesozooplankton size fractions to identify changes in the nitrogen source and structure of the planktonic food web across these habitats.  We found clear indications of a shift in N sources for biological production from nitrate in near-coastal waters towards an increase in diazotroph-N inputs in oceanic waters where diazotrophs shaped the phytoplankton community.  The shift in N source was accompanied by a lengthening of the food chain (increase in the TP), which may provide further support for the connection between diazotrophy and the indirect routing of N through the marine food web.  Our combined bulk and amino acid δ¹⁵N approach also allowed us to estimate the trophic enrichment (TE) of mesozooplankton across the entire regional ecosystem.  When put in the context of literature values, our high TE of 5.1‰ suggested a link between ecosystem heterogeneity and the less efficient transfer of mass and energy across trophic levels.

Samples of seawater and plankton were collected on two legs on R/V Falkor cruise FK160603 (3 June – 18 June, 2016) to the SCS off southern central Viet Nam.

Water samples were obtained using a Seabird CTD-rosette system outfitted with a sensor for chlorophyll a fluorescence (in the following Chl. a).

Samples of particulate organic matter (POM) for elemental and bulk isotope analyses were collected from the same depths as the nutrient samples.  A volume of 1–19 L of seawater was filtered through pre-combusted (450°C for 2 h) 47-mm GF/F filters (0.7 µm nominal pore size) using gentle pressure filtration (5-10 psi).  Filter samples were dried at 60°C onboard and stored over desiccant for analysis ashore.  At selected stations, additional samples of POM for CSIA were collected by gentle pressure filtration of 20-60 L of seawater from either the surface or the chlorophyll-maximum through a series of pre-weighted 47-mm polycarbonate filters (Whatman Nuclepore, 0.2 µm).  Samples were frozen at -20°C and dried at 60°C ashore.

Zooplankton for elemental, bulk isotope, and compound specific isotope analyses were collected in vertical tows around local noon and local midnight with a 0.5 m² ring net (200 µm mesh size) equipped with a USBL transponder.  We sampled through the upper 100 m of the water column or to ~10 m above bottom at shallower stations.  Animals were size-fractionated using a graded series of Nitex sieves with mesh sizes of 2000 µm, 1000 µm, 500 µm and 250 µm, and dried onboard at 60°C for 48 h.  Dry samples were ground to a fine powder, then stored in pre-combusted aluminum foil (450°C for 2 h) at -20°C for the duration of the cruise.

All elemental concentration and bulk stable isotope measurements of POM and size-fractionated zooplankton samples were made by elemental analysis with coupled isotope ratio mass spectrometry (EA-IRMS).  Analyses were carried out using a Thermo Scientific Delta V Advantage interfaced to a Flash 2000 elemental analyzer in Warnemünde, Germany or a Micromass Isoprime IRMS coupled to a Carlo Erba NA2500 elemental analyzer in Atlanta, GA, USA.  The overall precision of our bulk isotopic analysis is better than ±0.2‰ for δ¹³C and δ¹⁵N for both instruments, which were cross-calibrated by running splits of the same standard sets.

The δ¹⁵N values of triflouroacetyl/isopropyl ester (TFA) AA derivatives (Hofmann et al., 2003; Veuger et al., 2005) were measured on a Thermo GC Isolink CN with Trace 1310 (GC), coupled via a ConFlo IV combustion interface (C) to a Mat 253 (IRMS, all Thermo Fisher Scientific GmbH, Dreieich, Germany) in Warnemünde.  The precision of our GC-C-IRMS measurements varied among individual AAs and sample type but the standard deviation of 3-5 analyses typically was ≤1.0‰.  Due to the resource intensive nature of this approach, CSIA analyses were performed on only a subset of POM and zooplankton samples, which included POM from the surface and Chl. a maxima and mesozooplankton from the 1000-2000 µm size fraction.  This size fraction was chosen because it provided good coverage across the various habitats (zooplankton in the larger size fractions were not always present at adequate abundances) and because samples from this size fraction had minimal phytoplankton contamination (explained in more detail below).  In total, 9 POM samples from 6 stations (Supporting Table S1) and 21 zooplankton samples from 15 stations (Supporting Table S2, Fig. 1) were analyzed for CSIA.  Individual nitrogen stable isotopes of 12 AAs included the so-called “source” AAs glycine (Gly), lysine (Lys), phenylalanine (Phe) and tyrosine (Tyr); the “trophic” AAs alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), isoleucine (Ile), leucine (Leu), proline (Pro), and valine (Val); and the “metabolic” AA threonine (Thr), categorized by Germain et al. (2013), McClelland and Montoya (2002), and Chikaraishi et al. (2009) according to the sensitivity of each AA to trophic enrichment in ¹⁵N.  Asp and Glu also include the amide forms asparagine and glutamine, respectively, with the N isotopic signature coming only from the α-amino-N from both compounds as the amide N is lost during hydrolysis.  Details of the EA-IRMS and GC-C-IRMS analyses are provided in the Supporting Information S1 of Weber et al.

The readme file contains an explanation of each of the variables in the dataset, its measurement units, and -if it concerns a derived variable like TP or ∑V- including the literature reference as found in the associated manuscript. #NA =  habitat type not available. #NV = isotope values not measured. Information on how the measurements were done can be found in the associated manuscript referenced above.

All outlier values due to contamination of mesozooplankton with phytoplankton or fish or transient isotope fractionation events (Stn. 6) have been included but marked by colour in the xls sheet. The outlier identification has been done as follows and as descibed in detail in the associated manuscript:

We used a two-step process based on the samples´ C:N ratios and bulk δ¹⁵N values in relation to the mixed layer depth to identify POM and zooplankton samples that should be excluded from further analysis due to either contamination with fish or large phytoplankton or influence from transient events of isotopic fractionation related to nitrate uptake.  Contamination was determined both with visual inspection and on the basis of elemental C:N ratios.  The C:N ratio of tropical mesozooplankton biomass is expected to vary between 4.0 and 5.4 (Steinberg & Saba, 2008), and departure from this range is indicative of sample contamination by fish (comparatively lower C:N values) or large phytoplankton (comparatively higher C:N values) (Supporting Fig. S3).  Identifying the influence of transient isotope fractionation events on the δ¹⁵N value of plankton samples is more difficult, particularly in complex systems where there is also significant mixing of isotopically unique N sources (Fry, 2006; Glibert et al., 2019).  Since the sources of and processes acting on the DIN pool are highly system specific (Montoya, 2007), there exists no defined method for distinguishing the influences of major sources from transient processes.  For our study area, we have used the highly significant relationship between the bulk δ¹⁵N values of all five plankton compartments and MLD to identify outliers influenced by transient processes.  The MLD is known to play an integral role in regulating the supply of DIN to surface waters and consequently the relative contributions of subthermocline nitrate and diazotroph nitrogen (when DIN is strongly limiting, Hutchins & Fu, 2017) to POM – the two major sources of isotopically unique N in POM of the SCS.  This regulatory role of MLD has previously been shown in the SCS (Voss et al., 2006) and in the central Baltic Sea (Loick-Wilde et al., 2019), making it ideal for our purpose here.