DOI: 10.5061/dryad.bk3j9kdbv
title: Bulk and amino acid nitrogen specific isotope data from particulate organic matter and mesozooplankton (1000-2000 µm) from the Mekong River plume and southern South China Sea

description: 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.
description: 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
 <i>a</i> fluorescence (in the following Chl<i>.
 </i><i>a</i>). 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 m<sup>2</sup> 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 δ<sup>13</sup>C and
 δ<sup>15</sup>N for both instruments, which were
 cross-calibrated by running splits of the same standard sets.
 The δ<sup>15</sup>N 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<i>.</i><i> a</i> 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 <sup>15</sup>N.  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.
description: 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 δ<sup>15</sup>N 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 δ<sup>15</sup>N
 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
 δ<sup>15</sup>N 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.