Accepted Manuscript

Food web structure and trophodynamics of deep-sea plankton from the Bari
Canyon and adjacent slope (Southern Adriatic, central Mediterranean Sea)

I. Conese, E. Fanelli, S. Miserocchi, L. Langone

PII: S0079-6611(18)30096-X
DOI: https://doi.org/10.1016/j.pocean.2019.03.011
Reference: PROOCE 2095

To appear in: Progress in Oceanography

Received Date: 6 April 2018

Revised Date: 19 March 2019
Accepted Date: 27 March 2019

Please cite this article as: Conese, I., Fanelli, E., Miserocchi, S., Langone, L., Food web structure and trophodynamics
of deep-sea plankton from the Bari Canyon and adjacent slope (Southern Adriatic, central Mediterranean Sea),
Progress in Oceanography (2019), doi: https://doi.org/10.1016/j.pocean.2019.03.011

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 Food web structure and trophodynamics of deep-sea plankton from the Bari Canyon and
adjacent slope (Southern Adriatic, central Mediterranean Sea)

Conese I.1*, Fanelli E.2,3*a, Miserocchi S.1, Langone L.1

1
CNR-ISMAR, Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine, sede di Bologna, via
Gobetti 101, 40129 Bologna, Italy

2
Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce
Bianche, 60131 Ancona, Italy.
3
Stazione Zoologica Anton Dohrn di Napoli, Villa Comunale, 80121 Napoli, Italy
*These authors equally contributed to this work.
a
corresponding author: Emanuela Fanelli, e-mail: e.fanelli@univpm.it; Ph. +39 071 2204335

Submitted for consideration to:

Progress in Oceanography, Special Issue “Mediterranean canyons”

Key-words: food webs, deep-sea zooplankton, stable isotopes, sediment traps, Mediterranean
sea
Abstract

Zooplankton represent a key component of deep-sea ecosystems, linking Particulate Organic

Matter (POM) to higher trophic levels through both vertical migrations from the photic zone and
being prey of several megafaunal species, including demersal and benthopelagic organisms.
Nevertheless, this key group in deep-sea ecosystems is far to be well known, especially concerning
trophic aspects. In this study, we aimed to depict the trophic web structure of deep-sea zooplankton
collected in sediment traps from the Southern Adriatic, central Mediterranean Sea, and adjacent
slope from 600 to 1200 m, and its trophodynamics. To achieve these objectives, we considered a
long time-series set of samples, from March 2010 to October 2012 and we used stable isotope
analysis of nitrogen and carbon. The area is characterised by cascading events of dense shelf waters,
and the possible influence of this periodic phenomenon on seasonal changes in the isotopic
composition of deep-sea zooplanktonic species was also analysed. Our results evidenced a complex
structure for the deep-sea zooplankton food web with taxa organized in three trophic levels from
POM feeders to ultra-specialized carnivores. Temporal changes in the isotopic signatures of deep-
sea zooplankton species were observed, although the influence of cascading events was not so clear.
The high swimming capability of deep-sea zooplankton and their ability to perform vertical
migrations may be the cause of such unclear relationship, with species more relying on vertical
fluxes from surface primary production than on lateral advective transport of organic particles.
 1. Introduction

Although in the last decades the biology and ecology of deep-sea organisms received an
increasing attention, the information on some components of deep-sea fauna is still very scarce.
This is particularly true for deep pelagic fauna (i.e., meso- and bathypelagic organisms) and
especially deep-sea zooplankton (Burd et al., 2002; Koppelmann et al., 2003; Tamelander et al.,
2008), which are more difficult to collect compared to other components such as the deep-sea
benthos (O’ Dor et al. 2009). Deep-sea zooplankton represent a key component of deep-sea
ecosystems, linking Particulate Organic Matter (POM) to higher trophic levels through both vertical
migrations from the photic zone, i.e. the so-called swimmer flux (Miquel, 1994), and being prey of
several megafaunal species, including demersal and benthopelagic organisms (Fowler and Knauer,
1986). Recently, some studies in the Mediterranean Sea analysed the species composition of deep-
sea zooplankton (Cartes et al., 2010, 2013; Fanelli et al., 2009, Danovaro et al., 2017), but very few
investigations analysed its trophic structure or changes in their trophodynamics (Koppelmann et al.,
2003, 2009; Fanelli et al., 2009, 2011b). Depicting the food-web structure is fundamental to
understand the exchange of matter among organisms within an ecosystem, including the energy
flow from basal resources to top predators (Krumins et al., 2013). In this sense, classical studies
encompassed the use of stomach contents analysis (SCA), which is still a powerful tool to depict
food web structure or trophic relationships in aquatic organisms, but has the limitation of offering a
snapshot of the diet of a species in a specific time. It also requires strong taxonomic skills and is
heavily time-consuming (Fanelli and Cartes, 2010). Further, SCA is hard to perform for macrofauna
such as the deep-sea zooplankton, due to their small size. In the last decades, stable isotope analyses
(here after SIA) of nitrogen and carbon (δ13C and δ15N) have been widely used in food-web studies
to analyze community structure and ecosystem function (Post, 2002), to understand source links and
processes of marine and estuarine food webs (Cabana and Rasmussen, 1996), and to provide time-
integrated data about feeding relationships and energy flow (Peterson and Fry, 1987). SIA is based
on the predictable and quantifiable way that tissue nitrogen (15N) and carbon (13C) isotopes
change along the food web (Hobson and Clark, 1992). 15N increases about 3‰ per trophic level
(Post, 2002) and 13C changes according to the area prey items inhabit. More specifically, carbon
isotope ratios of organisms in a marine trophic system are influenced by the primary producers at
the base of the food web, which are affected in turn by different factors (i.e. phytoplankton size
growth rate, amount and types of primary productivity etc.). These contribute to geographic patterns
of 13C values in marine environments, including higher 13C values in benthic versus pelagic
habitats and in nearshore versus off-shore food webs (France, 1995). SIAs provide a continuous
measure of trophic position that integrates the assimilation of energy or mass flow through all the
different trophic pathways leading to an organism. In this way, complex interactions are
simultaneously captured, including trophic omnivory, and energy or mass flows are tracked through
ecological communities (Peterson and Fry, 1987; Cabana and Rasmussen, 1996). SIA is also very
effective for macrofaunal species: if the analyses can be performed at species level, the results
mirror the high trophic complexity of lower trophic level species that can include in the same family
carnivorous, deposit-feeders, filter feeders or herbivorous species.

In this study we analysed, by means of SIA of carbon and nitrogen, deep-sea

macrozooplanktonic species collected by automatic sediment traps in the Bari Canyon and the
adjacent slope of the Southern Adriatic (central Mediterranean Sea). Submarine canyons play a
fundamental role in organic matter distribution in oligotrophic deep-sea environments and therefore
in the contribution of food sources to deep zooplankton. Mediterranean canyons are responsible for
inorganic/organic material accumulation (e.g., Alvarez et al., 1996, Granata et al., 1999) strongly
stimulating biological production. Further, shelf-slope water exchanges occurring in canyons have a
significant impact on the dispersion and recruitment processes of fish larvae (Sabates and Masó,
1992; Cowen et al., 1993), with greater abundances found on the canyon rims than in the shelf area
(i.e. the case of Palamós canyon: Alvarez et al., 1996) and high densities of migratory micronekton
observed over submarine canyons of the NW Mediterranean (Macquart-Moulin and Patriti, 1996).
The area of the Bari Canyon is characterized by North Adriatic Dense Water (NAdDW)
cascading events, which play a relevant role in biogeochemical cycles (Shapiro et al., 2003) as in
other areas of the Mediterranean, such as the Gulf of Lions (north-western Mediterranean) and the
Aegean Sea (eastern Mediterranean). The Mediterranean is an oligotrophic system, with increasing
oligotrophy eastwards, with low surface primary production and in turn low organic matter sinking
to deep waters. As in the Adriatic Sea Dense Shelf Water (DSW) cascading sustains transport of
particles and organic matter from shallow areas to the deep sea (Turchetto et al., 2007; Tesi et al.,
2008; Cantoni et al., 2016; Langone et al, 2016; Taviani et al., 2016). Thus new food inputs from
cascading events have a strong influence on deep-water communities by altering food availability
and especially the origin of food sources for deep-sea food webs.
Here, we used SIA of carbon and nitrogen to depict the food-web structure of deep-sea
macrozooplanktonic species collected by automatic sediment traps in the Bari Canyon and the
adjacent slope of the Southern Adriatic. Specifically, we aimed to: 1) depict the food web structure
of deep-sea macrozooplanktonic communities of south Adriatic and analyse seasonal changes in the
isotopic signatures, 2) identify which environmental variables best explain the observed patterns,
particularly if the NAdDW cascading has a role in such trends, and 3) define the main food sources
for deep-sea zooplankton species.

2. Materials and Methods

2.1. Study area

The Adriatic Sea is a shallow semi-enclosed basin (ca. 200 × 800 km) situated in the
northern central portion of the Mediterranean Sea (Cattaneo et al., 2003). The basin is commonly
divided into three sub-basins: the northern Adriatic, a broad shelf characterized by a low
longitudinal gradient (~0.02°) with depths shallower than 100 m (north of Ancona), the middle
Adriatic with a relatively steep shelf (~0.5°) where the water depth reaches 260 m in the Middle
Adriatic Pit, and the Southern Adriatic basin, with a depression (the South Adriatic Pit) of about
1200 m (Cattaneo et al., 2003). This study was performed in the western Southern Adriatic,
particularly the Bari Canyon and the adjacent open slope of the Southern Adriatic Sea (Fig. 1). The
Bari Canyon is a morphological structure (Trincardi et al., 2007a; Ridente et al., 2007) that dissects
with a West–East trend the western Adriatic shelf. It is a ~10 km-wide and elongated basinward for
~30 km, showing several heads at about 200 m water depth (Trincardi et al., 2007a).
Three main water masses characterize the study area: Levantine Intermediate Water (LIW,
T>13.5 °C; S>38.6 psu, Russo and Artegiani, 1996), Southern Adriatic Deep Water (also known as
Adriatic Deep Water – ADW, T=13.16 °C ± 0.3; S= 38.61 psu ± 0.09, Russo and Artegiani, 1996),
and NAdDW. This latter is the densest water (T ~ 11°C, S ~ 38.5, t ~ 29.5–29.6) of the whole
Mediterranean (Zore-Armanda, 1963; Vilibic and Supic, 2005). NAdDW is generated on the broad
and shallow (< 40 m deep) North Adriatic shelf through intense winter cooling and evaporation
during January and February and is largely affected by severe winter outbreaks of the cold and dry
northeast Bora wind (Belusic and Klaic, 2004; Grubisic, 2004), which shows large year to year
variability (Vilibic and Supic, 2005; Bignami et al., 2007). The process of generation of NAdDW is
principally temperature driven (Shapiro et al., 2003), although lateral advection of salinity from the
Southern Adriatic, as well as the freshwater discharge from rivers along the coast, of which the Po
River is the largest (average discharge of 1511 m3 /s, Boldrin et al, 2005), do have an impact in the
preconditioning phase in assisting or limiting the production. The NAdDW production is active
every year for a relatively short time interval and, depending on the meteorological conditions in
any year, may or may not reach a density that enables it to sink to the deepest part of the basin at
1200 m (Verdicchio and Trincardi, 2008). After it forms, the NAdDW spreads toward the south
moving along the Italian coast and generally reaches the Southern Adriatic shelf ca. 2 months later
(late March–early April; Zoccolotti and Salusti, 1987), although occasionally the transit time can be
exceptionally short (ca. 2 weeks at early stages of its formation, Benetazzo et al., 2014; Langone et
al., 2016).
When the NAdDW arrives at the western margin of the Southern Adriatic Margin (SAM), it
cascades to the bottom of the South Adriatic Pit (Bignami et al., 1990a; Mancua and Giorgetti,
1999; Trincardi et al., 2007b; Benetazzo et al., 2014), strongly interacting with the slope topography
by enhanced turbulent mixing (Bignami et al., 1990a; Bonaldo et al., 2018), also flushing the deeply
incised Bari Canyon (Trincardi et al., 2007a). This is the main canyon system along the Adriatic
margin and plays an important role in the NAdDW spreading (Bignami et al., 1990b; Vilibić and
Orlić, 2002; Turchetto et al., 2007). NAdDW cascading ventilates the intermediate and bathyal
water layers of the Southern Adriatic and sustains transport of particles and organic matter
(Turchetto et al., 2007; Tesi et al., 2008; Cantoni et al., 2016; Langone et al, 2016; Taviani et al.,
2016). In this way, the Bari Canyon represents an efficient conduit in delivering suspended
sediment from the continental shelf to the deep southern Adriatic basin (Turchetto et al., 2007). The
southern basin represents the ultimate repository, where sediment in transit, also derived from the
Po River (~600 km far away), are captured and transported.

2.2. Sample collection

Deep-sea zooplankton was sampled at three mooring stations in areas where the passage of
dense shelf water is most likely to occur, based on morphological inferences by multibeam
acquisition (Trincardi et al., 2007a, b, 2014; Verdicchio and Trincardi, 2006; Foglini et al., 2016)
and model predictions (Bonaldo et al., 2016; Carniel et al., 2016). The first station was situated
within the Bari Canyon (site BB, 606 m water depth), the second one on the adjacent open slope
offshore the Gargano promontory (site FF, 733 m water depth) and the third one in the deepest part
of the Southern Adriatic basin (site EE, 1194 m water depth) (Fig. 1). Samples where obtained from
March 2012 to October 2012 from all the stations. Only for station BB a longer time-series dataset
was considered as the deployment started earlier, in March 2010 (March 2010-October 2012).
Each mooring line was equipped with a time-series Technicap sediment trap (12 receiving
cups, models PPS3/3 or PPS4/3, 0.125 m2 or 0.05 m2 collection area, respectively) fastened 35 m
above the sea bottom. To prevent organic degradation during deployment, trap sample cups were
filled with 5% formaldehyde solution buffered in filtered seawater (Knauer and Asper, 1989). The
interval of rotation of the sediment traps was variable between 7 and 36 days (on average, 15 days).
The shorter sampling periods correspond to the spring season in which a greater variability in
physical–chemical conditions was expected (for more details see Langone et al. 2016).
 2.3. Environmental variables collection

Near-bottom potential temperature and salinity were available from time-series acquired
during the periods of mooring deployment from March 2010 to October 2012. Each trap was
typically coupled with Sea-Bird Electronics CTD recorders (SBE16plus, SBE19plus or SBE37);
results of the accurate and complete analyses of physical parameters are shown in Chiggiato et al.,
2016. Values of surface chlorophyll-a concentration (mg/m3), obtained by the “Giovanni online
data system” available at https://giovanni.gsfc.nasa.gov/giovanni/, MODIS-Aqua 9 km, was used as
an indicator of the primary production in the area. Monthly average readings of concentrations at
the mooring positions were used. The daily water discharge of the Po River (in m3 s-1) was provided
by “Annali Idrologici” of Arpa Emilia-Romagna (http://www.arpa.emr.it) and monthly averaged.
Considering that the incorporation of 13C and 15 N in animal tissues is not immediate and also that
at bathyal depths the response of the community could be delayed with respect to the inputs
occurring at the surface, δ13C of zooplankton was compared with variables recorded previously to
the samples collection (i.e., one, two and three months before), as well as those recorded
simultaneously (Fanelli et al. 2011a). Thus, for the analyses of environmental variables, values of a
and Po river discharge were used considering different lag intervals before the sampling periods,
i.e., 1, 2 and 3 months before, and simultaneously to them (Chlasim, Chla1mo, Chla2mo, Chla3mo and
Rivsim, Riv1, Riv2 and Riv3, respectively for chlorophyll-a concentration and river discharge,
respectively).

2.4. Stable isotopes analyses (SIA)

After recovery during oceanographic cruises, sediment trap samples were stored at 4°C until
being processed in the laboratory. Sediment trap samples were processed according to Heussner et
al. (1990), slightly modified by Chiarini et al. (2014). Samples were first rinsed and wet sieved with
1 mm nylon net to retain the largest swimming organisms (the “swimmers”, mostly euphausiids,
fish larvae or hyperiids). Then the smaller ones (i.e. the mesozooplankton) were manually removed
under a dissecting microscope (Zeiss Stemi 2000 C, magnification x4-x10). Swimmers were
counted and determined at the lowest taxonomic level possible to obtain qualitative information on
the macrozooplankton composition. The food web structure was investigated using stable carbon
and nitrogen isotopes (δ13C and δ15N) on a total of 27 taxa: whole individuals were analyzed.
Samples were weighed (ca. 1 mg of dry weight) into tin cups. Total organic carbon (TOC) and total
nitrogen (TN) contents, and the δ15N and δ13C of zooplankton samples were determined by a
Finnigan DeltaPlus mass spectrometer directly coupled to a EA Flash2000 TermoFisher Scientific
via CONFLO III interface (EA-C-IRMS, CNR-ISMAR, Bologna, Italy). At the beginning of each
sequence three capsules of Atropine (IAEA standard), one of USGS-40 and one of IAEA-N2
(IAEA standards) were analyzed. Then, every 8-10 samples, one capsule of urea was analyzed, in
order to compensate for potential machine drift and as a quality control measure. The average
standard deviation of each measurement, determined by replicate analyses of the same sample, was
±0.07% for TOC and ±0.009% for TN. All isotopic compositions are presented in the conventional
δ notation and reported as parts per thousand (‰). Experimental precision (based on the standard
deviation of replicates of the internal standard) was < 0.2% for δ 15N and < 0.1% for δ13C. A
minimum of three replicates was analyzed when sufficient samples were available, and each
replicate included just one individual in order to reduce pseudo-replication (Hurlbert, 1984). When
the biomass of a specimen was not sufficient, several organisms were pooled together to obtain
sufficient mass for the isotope measurement (as in the case of small copepods).
C/N atomic ratios and δ13C values of particulate organic matter in sediment (SPOM)
collected by sediment traps were also measured (see Langone et al. 2016 for more details).

2.5. Data analysis

2.5.1. Food web structure of deep-sea zooplankton

Isotope data were normally distributed, thus they were not transformed for univariate and
multivariate analyses. Because of sampling constraints, suspended POM was not sampled in this
study, thus we used as baseline of the food web, for the estimate of trophic levels, the δ 15N value of
Salpa sp. (i.e., a primary consumer, Alldredge and Madin, 1982) collected by a 500 micron mesh-
size plankton net at 150 m water depth, above the sediment trap at site EE, during an oceanographic
cruise in March 2014. δ15N values were converted to trophic level based on the assumption of a
2.75‰ fractionation per trophic level (as proposed by Caut et al., 2009), and that the base material
(i.e., the filter feeder Salpa sp.) had a trophic level of 2.
The calculation of the trophic levels is based on the following formula:

TLi = (δ15Ni - δ15NPC) + 2/TEF

where TLi is the trophic level of species i, δ 15Ni is the mean δ15N of species i, and δ15NPC is the
mean δ15N value of a primary consumer (in this case Salpa sp.= 1.81‰), TEF (Trophic Enrichment
Factor) is 2.75‰.
In order to give an overall picture of the trophic web structure of deep-sea
macrozooplankton community, a hierarchical cluster analysis (average grouping method) was
carried out on the resemblance matrix (Euclidean distance) on δ 13C and δ15N mean values per
species or taxon (Davenport and Bax, 2002; Fanelli et al. 2009, 2011a, b). A further graphical
representation of trophic groups was obtained with a non-metric Multidimentional Scaling (nMDS)
(untransformed data, Euclidean distance). The groups obtained were then compared with postulated
trophic groups (TG) based on data acquired from literature, according to TG reported in Table 1.
These are filter feeders (Ff), carnivores (C), omnivores (OMN) and species with unknown feeding
guild (UNK).
A PERMANOVA test (Permutation Analysis of Variance based on Euclidean distance,
Anderson, 2001) was then run on a one-factor design (trophic group, fixed with four levels as
described above) in order to detect significant differences among trophic groups; then a pair-wise
comparison was done between groups. Significance was set at p=0.05 and p-values were obtained
using 9999 permutations of residuals under unrestricted permutation of raw data, which is
recommended when there is only a single factor (Anderson, 2001).

2.5.2. Spatial and temporal variability in isotopic composition of deep-sea zooplankton

First, in order to analyse patterns in both spatial and temporal variability and to test if
temporal variability can be attributed to dense shelf water cascading events, a PERMANOVA
(Anderson, 2001) design was created based on two factors (cascading, fixed with two levels – Y vs.
N, indicating period of cascading occurrence vs. absence, and site, fixed with three levels, BB, FF
and EE) crossed within each other; then pair-wise comparisons were run for significant
PERMANOVA factors. The two isotopic markers were then analysed separately through two-way
univariate PERMANOVA test for the effect of cascading and site.
Finally, a δ13C-δ15N biplot was constructed in order to highlight eventual differences
between periods of cascading occurrence, usually occurring between March and June in the area
(Langone et al., 2016), and period without cascading. The r values of the δ13C-δ15N correlations
were then calculated.

2.5.3. Correlation with environmental variables

To identify which environmental factors best explain the observed patterns, in particular if
cascading events have a role in such trends, δ 15N and δ13C data of each species/taxon, per each
station and sampling date (month), were coupled to environmental variables. The DistLM
(distance‐based linear modeling) analysis was run on the whole dataset based on Aikake’s
Information Criterion (AIC) and Best as selection procedure. The procedure was run with 9999
permutations. Before the analysis, a draftsman plot (i.e. scatter plots of all pair-wise combinations
of variables: Clarke and Warwick, 2001) was applied to environmental variables to identify whether
any of them were strongly correlated, thus providing redundant information. Redundant variables (r
> 0.70) could be discarded, simplifying the matrix.
In order to determine the environmental drivers of carbon sources sustaining the deep-sea
zooplanktonic food web, δ13C values of zooplanktonic taxa were compared with independent
explanatory variables by means of Generalized Linear Models (GLM) after normalizing the
dependent variables by log(x+1) transformation (Sokal and Rohlf, 1995). The distribution family
used was Gaussian with identity link. The model selection was computed by adding single terms
based on minimizing Akaike’s Information Criterion (AIC) and only including variables that were
significant (P < 0.05).
The starting environmental variables included in both models (multi- and univariate) were:
1) C/N and δ13C values of SPOM analysed from the same sediment traps by Langone et al. (2016);
2) near-bottom temperature (°C); 3) near-bottom salinity; 4) chlorophyll-a concentration (Chlasim
and Chla1mo-Chla3mo); 5) Po river discharge (Rivsim and Riv1-Riv3) and 6) presence or absence of
event of cascading (yes/no).

2.5.3. Mixing models

To estimate the potential food sources for zooplanktonic species in the different areas, i.e.
BB, EE and FF, during the period of cascading (i.e. from March to June-July) we applied a
Bayesian model in SIAR 4.1.1 (Stable Isotope Analysis in R 2.12.2). This model runs under the free
software R (R Development Core Team 2009). The model allows the inclusion of sources of
uncertainty. In particular, the variability in the isotope signatures (mean and standard deviation) of
prey species can be incorporated into the model (Parnell et al. 2010). SIAR uses Markov-chain
Monte Carlo modelling, takes data on animal stable isotopes and fits a Bayesian model of the diet
habits based on a Gaussian likelihood function with a Dirichlet prior mixture distribution for the
mean. The model also assumes that each target value (i.e. the stable isotope data for each
individual) comes from a Gaussian distribution with an unknown mean and standard deviation. The
structure of the mean is a weighted combination of the food sources’ isotopic values. The standard
deviation depends on the uncertainty in the fractionation corrections and the natural variability
among target individuals within a defined group. We used the trophic enrichment factor (TEF) of
1.70 ± 0.10‰ for C and 2.75 ± 0.10‰ for N as the average of enrichment factors estimated for
zooplankton species provided in Fanelli et al. (2009, 2011a). As potential sources for zooplankton
we considered the averaged isotopic signatures of marine phytoplankton (Harmelin-Vivien et al.
2008), the values of POM from the North Adriatic (Faganeli et al., 2009; Berto et al. 2013) and
values of soil-derived OM and aged OM as in Langone et al. (2016) (Table 2).
 All analyses were performed using PRIMER 6 and PERMANOVA+ (Clarke and Warwick,
2001, Anderson et al., 2008) and R 2.14.2 (www.r-project.org).

3. Results

Overall, 27 taxa were identified and analyzed (Table 1): copepods, hyperiids and
euphausiids were the most represented groups. The δ15N mean values of deep-sea zooplanktonic
taxa ranged between 1.32‰ (Clio sp.) and 9.25‰ (Clione sp.), measured at stations EE and BB,
respectively. The range of δ13C mean values was between -22.00‰ (Ostracoda sp.) and -14.76‰
(Clio sp.) both at EE.

3.1. Food web structure of deep-sea zooplankton

The calculation of trophic levels identified three main levels: the lowest level (TL 2),
characterized by filter feeders and low-TL omnivores; the intermediate level (TL 3) composed by
carnivores, omnivores (i.e., species feeding on phytodetritus, exuviae of small crustaceans and also
small zooplankton) and scavengers (N. borealis), but also of species with unknown feeding guild
(Ostracoda sp., Euphausiacea and Brachyura larvae); the highest level (TL 4) characterized by
carnivores (Table 1).
The trophic groups identified by cluster analysis were in general agreement with the
postulated feeding habits of the group members. The cluster analysis based on δ 15N and δ13C values
identified three main groups, designated as group I, II and III, and two subgroups within group III
(subgroups IIIA and IIIB: Fig. 2a). Group I included filters feeders: the hyperiid Vibilia sp. and the
calanoid copepod Acartia sp. The pteropod Clio sp., an omnivore species of low trophic level
feeding on mucus net of salps, was also included in this group. Within group II, the cluster
algorithm placed three species belonging to the same trophic level (TL=4): the calanoid Calanus
helgolandicus, reported as an omnivore species in the literature, Decapoda larvae of type II, which
encompass larvae belonging to different trophic guilds, essentially omnivore decapod larvae, and
the carnivore Clione sp. Group III comprised the remaining species. Within subgroup IIIA mostly
carnivores and scavengers (i.e., the isopod Natatolana borealis) were included, together with
species of unknown feeding habits (Decapoda larvae of type I -probably carnivore larvae-
Brachyura and Euphausiacea larvae). Finally, within the subgroup IIIB we mostly found low-TL
omnivores. The clear separation of organisms of Group II from other organisms of low TL (filter
13
feeders and omnivores of group III), is due to the particularly C-enriched values of these latter
species.
 Carnivores and omnivores were separated in two subgroups (IIIA and IIIB). Consistently,
the nMDS plot with overlaid results of the cluster analysis showed a separation of species into three
main groups, according to their position on the food web and the resource use, with filter feeders
(on the right part of the graph) clearly separated from carnivores and omnivores (Fig 2b). The
separation at 1.7 distance highlighted further differences within each group, i.e. within group II the
two carnivores C. helgolandicus and Clione sp. were separated from Decapoda larvae type II,
having this latter higher 13C values. Similarly, within group I the two strictly filter feeders Vibilia
sp and Acartia sp. were segregated from Clio sp. because of the greater 13C of the latter.
Average stable isotopic ratios differed significantly among trophic groups identified by
cluster analysis (PERMANOVA pseudo-F4,26 = 4.74, p < 0.01). The pair-wise comparisons showed
significant differences between the isotopic signatures of carnivores and both filter feeders and
omnivores, the latter two being statistically similar (Table 3). Species with unknown feeding habits
differed significantly from filter feeders but not from carnivores and omnivores.

3.2. Spatial and temporal changes in the isotopic composition of deep-sea zooplankton

The two-way PERMANOVA test showed significant differences for factor site and
cascading but not for the interaction term (Table 4). The pairwise comparisons based on factor
“site” evidenced significant differences between EE site and the other two, but not between FF and
BB (Table 4).
The analysis of the individual isotopic markers δ 15N and δ13C through a two-way univariate
PERMANOVA, showed that factor cascading was always significant, the factor site was only
significant for 15N, and the interaction term was never significant (Table 5). The pairwise
comparisons for the factor site for 15N showed again significant differences only at site EE.

δ13C-δ15N biplot showed slight differences between the isotopic signatures of species
collected in period of cascading vs. no cascading (Figure 3), with a larger 13C range observed
during cascading vs. no cascading periods. The value of the correlation was rather higher for species
analysed when no cascading occurred (r=0.42, p<0.01) than for those during the cascading period
(r=0.32, p<0.05).
3.3. Trends in environmental variables

During the period March-October 2012, the mean value of near-bottom temperatures and
salinities at the three sites ranged between 12.91°C, at site EE, and 13.33°C, at site BB (Fig. 4a),
and from 38.70 psu at EE, to 38.71 psu at the other sites. During the first half of 2012, the three
mooring stations exhibited high temporal variations but the pattern of variability was different from
station to station. At station FF, situated on the open slope, in the northern part of the study area,
short-term high-amplitude multiple events followed by time intervals with modest temperature
changes occurred; the frequency of these fluctuations were weekly and bi-weekly. Mooring EE, in
the deepest part of the basin, showed a weak variability with small oscillations. At station BB, in
the northern branch of the canyon, temperature and salinity abruptly dropped during the second
week of February 2012. Here, temperature showed strong high-frequency oscillations until June
because of the intrusion of colder water. From July to October 2012, during the period of low
sedimentation, all three stations showed an increasing trend of bottom temperature and salinity,
without oscillations.
Throughout the whole sampling period, at the station BB, the mean value of near-bottom
temperature and salinity measured were 13.47 °C (Fig. 4b) and 38.71 psu, respectively. Because of
the limited salinity range, density variability was more strictly related to temperature oscillations
(Langone et al., 2016). Throughout this period, time-series bottom temperature showed a small but
permanent increasing trend (0.083 °C year -1).
The chlorophyll-a concentration pattern (Chl a), as a proxy of primary production, exhibited
a seasonal trend at all sites (Fig. 5), with a main peak in winter: in December-January throughout
the observation period. A generally lower but prolonged Chl a peak was observed in 2012 with high
Chl a values until April. During the whole sampling period, the Po river discharge reached the
greatest value in May 2009 (> 7000 m3 s-1) and the lowest in August 2012 (~355 m3 s-1) (Fig. 6).
Overall, the river discharge was high in spring (March-May) and late autumn (November).

3.4. Environmental drivers of temporal variability in food web structure

All correlations values obtained by Draftsman plots were lower than 0.7, thus all variables
were kept for the following analyses.
The DistLM performed for all the stations, showed that the best match between isotopic
values and environmental variables was obtained with three variables: chlorophyll-a recorded
simultaneously to sampling (Chl a sim, Pseudo-F=22.39, p=0.001), and Po river discharge recorded
2 and 3 months before sampling (Riv2, Pseudo-F=12.77, p=0.001; Riv3, Pseudo-F=10.84,
p=0.001). The best solution, obtained with AIC=272.66, explained 16% of the total variance.
The GLM models carried out on δ13C data of zooplankton showed that in the whole area
Riv2, Riv3 and salinity best explained the pattern of changes in δ 13C values (Table 6). The
explained deviance for the overall model was 21.9%.

3.5. Mixing models

 Mixing models (Fig. 6) showed that the main source of organic matter for deep-sea zooplankton
communities changed at each site with a greater contribution of POM from the North Adriatic at BB
site, where phytoplankton-derived OM was also important, contributing by ca. 40%. A similar
contribution of both POM from the North Adriatic and marine phytoplankton (as a proxy of vertical
inputs) was obtained at FF site, as was a strong dependence of this latter by the zooplanktonic
community at the EE site (Fig. 7). The contribution of both soil-derived OM and aged OM was
negligible at all sites.

4. Discussion

This study elucidates the food web structure of deep-sea zooplankton species collected in
sediment traps in southern Adriatic Sea, and presents, to our knowledge, the first isotopic data on
some species of the deep-sea swimming fauna of the Mediterranean, i.e. Heterorabdus sp.,
Haplostylus sp., Clio sp. and Clione sp. among others. The study included 27 taxonomic groups,
encompassing copepods, hyperiids, euphausiids, decapods, pelagic mollusks, chaetognats and
mesopelagic fishes, describing their feeding guilds.

4.1. Methodological approach

Samples in sediment traps were fixed in formaldehyde, which is found to affect the isotopic
signatures of marine species in different ways, the effects being species-specific (Fanelli et al.,
2011). Most of the studies analyzing the effects of fixative on stable isotope values of marine
species, especially deep-sea species, showed a minimal or no influence of formalin fixation on 15N
(Fanelli and Cartes, 2010; Fanelli et al., 2011), while formalin fixation affected δ 13C with 13
C-
depletion (Fanelli et al. 2016). While the increase in δ13C per trophic level is only ca. 1‰ on
average (DeNiro and Epstein, 1978), the mean shifts in carbon isotopes through formalin treatment
did not exceed -2.0‰ in any experiment. Since, in many ecosystems, carbon sources differ by more
than 2‰ (i.e. C3 vs. C4 plants or marine phytoplankton vs. terrigenous inputs), a -2.0‰ shift in
preserved specimens should not confound results (Edwards et al., 2002). Thus, considering a shift in
zooplankton lower than 2‰ (1.1‰ for carbon and 1.5‰ for nitrogen isotopic signatures:
Feuchtmayr and Grey, 2003), it should be possible in this case to use preserved samples to elucidate
food web structure. Additionally, all the samples were fixed, so the eventual bias should be similar
throughout the food web, with no consequence for the overall analyses here presented. However, a
bias in the C/N ratios cannot be excluded.

4.2. Food web structure of swimming fauna in the deep southern Adriatic Sea
 Our analysis using stable isotopes suggests a relatively complex food web in the deep
southern Adriatic Sea in which different food sources, i.e. from marine phytoplankton to terrestrial
organic matter, were used by deep zooplankton. The isotopic values of the macrozooplanktonic
species studied agreed with those reported in literature (Fanelli et al., 2009; 2011a). The stable
isotope ratios of this fauna displayed a continuum of values over the δ15N range of 8‰, confirming
a wide spectrum of feeding strategies, from filter feeders to the ultraspecialized carnivore pelagic
mollusk, the pteropod Clione sp., feeding on other pteropods like Limacina (Lalli and Gilmer, 1989;
Hermans and Satterlie, 1992).
Based on δ15N values, setting as baseline the POM feeder Salpa sp. and assuming a trophic
enrichment of 2.75 ‰ (Caut et al., 2009), our results showed that the deep macrozooplanktonic
community of the deep southern Adriatic Sea is characterized by taxa belonging to three trophic
levels (from filter feeders to carnivores), as already observed in other Mediterranean areas (i.e.,
Northwestern Mediterranean: Fanelli et al., 2009, 2011b; Eastern Mediterranean: Koppelmann
2003, 2009). Filter feeders (primary consumers) were located at the base of the trophic web, and
specialized carnivores placed at the highest trophic level (see also figure 8). Organisms feeding on
phytodetritus, exuviae and small zooplankton, as well as the scavenger isopod Natatolana borealis
were located at intermediate trophic levels. The deep-sea zooplanktonic food web in the Bari
Canyon and adjacent slope seemed to be represented by some strict carnivores with high TL and a
large number of omnivores that probably take advantage of particles that can be of low (particulate
phytodetritus) or high (remains of organisms) TL origin. The large numbers of carnivores and
omnivores here found is coherent with the oligotrophic conditions of the area. Indeed, in extremely
food-poor environments, such as the deep sea, macrophagy seems to be a better strategy than
microphagous suspension-feeding (Monniot and Monniot, 1991; Vacelet and Boury-Esnault, 1995).
Carnivores prey on organisms from several trophic levels, as well as omnivores, which
alternatively act as either particle feeders or carnivores, thus creating a complex food web in which
an organism may be prey and/or predator (Fanelli et al. 2011b). This is the case of omnivores and,
for example, of Calanus helgolandicus, which is described as an herbivore in laboratory conditions
or a feeder in surficial water (Koppelmann et al. 2003), but, in the deep sea, it seems to switch to a
carnivore diet, probably made up of small copepods or exuviae (Fanelli et al. 2011b) due to the
absence of phytoplankton at these depths. The species tends to be omnivorous to avoid competition
for food (Gage and Tyler, 1991; Jarre-Teichman et al., 1997), feeding not only on living organisms,
but also on dead particles (dead diatoms and faecal material; Paffenhöfer and Strickland, 1970).
 Based on comparisons among trophic groups, most of the species with unknown feeding
habits seemed to be omnivores with preference to carnivory, especially all the species in group IIIA.
More specifically, Ostracoda grouped with the carnivore mesopelagic fish Cyclothone sp. In this
case, it was not possible to identify to species level the ostracods collected in sediment traps, but the
isotopic signature of nitrogen was coherent with the values of the carnivore species Conchoecia sp.,
described in Lochhead (1968), that feeds on dead crustaceans and detritus. Omnivores included
organisms with isotopic nitrogen signature similar to carnivores: as mentioned before, C.
helgolandicus can be alternatively omnivore or strictly carnivore. The pteropod Clio sp. is a
phytophagous/omnivore taxon which grouped here with filter feeders, accordingly to its
consumption on tintiniids, radiolarians, and centric diatoms, reported in literature (Gilmer, 1990).
Carnivores included also the pteropod Clione sp., a specialist predator feeding on other
pteropods, especially Limacina spp. (Lalli and Gilmer, 1989) thus showing a high trophic level
(TL=4), and the amphipod Hyperia galba, a parasite species. Finally, the carnivore patterns found
in euphausiids’, brachyurans’ and decapod type I larvae have not been previously reported in
literature, as the few studies existing on the feeding ecology of these taxa larvae are focused on
shallow-water species. However, experimental studies on the larval development of brachyuran
species showed high growth rate with carnivore diet, i.e. Artemia nauplii or rotifers (Harms and
Seeger, 1989; Baylon, 2009), likely suggesting a preference of brachyuran larvae to carnivory.
The wide ranges of δ13C values for all the species suggest that meso-macrozooplankton
along the southern Adriatic margin probably exploits different food sources. δ13C varied from lower
values (i.e., −22‰), indicative of a pelagic or fresher food source, to greater ones (i.e., −15‰),
suggestive of a more benthic or recycled/refractory food source.

4.3. Spatio-temporal variability in isotopic composition of deep-sea zooplankton in

relation to environmental variables

Isotopic signatures of zooplankton are similar at BB and FF, and both differ from EE. Thus
differences in the food webs across sites seems to be mostly attributable to the different depth of the
stations within the southern Adriatic basin, with site FF and BB being positioned on the slope at
730 m and 600 m outside and inside the canyon, respectively, and site EE located in the deepest part
of the basin, at 1200 m (see Fig. 1).
At temporal scale, the lack of significant differences in the interaction term “cascading*site”
suggests that the effects of cascading can be considered parallel at all sites, although food webs in
the different sites are different (delivered from statistical significant differences between sites).
Such temporal variability is clearly highlighted by changes in the strength of the 13C-15N
correlation between periods of cascading vs. no cascading, which pointed out to the use of a larger
number of food sources by the community in periods of cascading originated by both advective and
vertical fluxes (see below). Indeed, the intermediate and bathyal layers of the SW Adriatic margin,
affected by NAdDW cascading events, are clearly influenced by lateral transport of particles and
organic matter (Turchetto et al., 2007; Tesi et al., 2008; Cantoni et al., 2016; Langone et al, 2016;
Taviani et al., 2016).
In the whole area, the best explanatory variables of changes in the isotopic composition of species
were chlorophyll-a recorded simultaneously to sampling period (as a proxy of primary production
and thus of vertical flux) and Po river discharge recorded 2 and 3 months before sampling period.
Although a riverine input should lower the 13C value of a species (Peterson & Fry, 1987), we
found a positive relationship between 13C and Po river discharge recorded 2 and 3 months before
sampling, in the GLM model. This unusual result may be due to the effect of nutrients supplied by
river discharge that stimulated the primary production in the studied area with a time lag due to the
transfer time of waters travelling from Northern to Southern Adriatic Sea, as demonstrated by the
significant correlation between these two variables, especially between river discharge recorded two
months before the sampling and chlorophyll-a recorded simultaneously to sampling (r=0.38,
p<0.05). Moreover, river input enhances the advective flux that can be composed of both
13
continental (i.e. C-depleted) and marine (13C-enriched) material transported from the shelf with a
consequent potential potential positive effect on the 13C of zooplanktonic species. This signal is
decoupled from the NAdDW formation, which is denser when the Po river discharge is lower, but
this is attributable to the lag time described above. Salinity is the other explanatory variable in the
GLM model, that further support periods with reduced supply of NAdDW and higher proportion of
ambient water, namely the Levantine waters (LIW). Both cascading and river discharge are less
saline than LIW, which are waters saltier and warmer (Millot, 2013). The three water masses of
different origin may bring different food sources. From our results, it is plausible that deep-sea
zooplankton lives and feeds most of the time in LIW waters: the greater the salinity, the higher the
13C values of deep-sea zooplankton, as observed in other marine species, including fishes (Elsdon
and Gillanders, 2002).

4.4. Potential food sources for deep-sea zooplankton in the South Adriatic

The deep-sea zooplankton collected in the sediment traps considered in this study (deployed
along the southern Adriatic margin and in the Bari Canyon) were affected both by a vertical flux
toward the deep ocean from the uppermost layers of the water column (i.e. the euphotic zone) and
by a lateral flux driven by physical processes related to sediment transfer through DSW cascading
(Turchetto et al., 2007; Tesi et al., 2008; Langone et al., 2016). The vertical flux is composed by
particulate organic matter (faecal pellets, detritus, etc.), phytodetritus, zooplankton exuviae and also
by the “swimmer flux” (i.e., fauna which undergoes diel vertical migration and transport organic
matter from the photic zone to the deep-sea, see Miquel 1994). The advective flux is composed by a
mix of fresh and old marine material from the shelf (Lalande et al., 2016) and it is evident as a 13C-
enriched signature in some deep-sea species.
Despite particle fluxes along the western margin of the southern Adriatic Sea are mainly
driven by lateral advection (Tesi et al., 2008; Langone et al., 2016), deep-sea zooplankton
communities seemed to rely mostly on the vertical marine fraction of these fluxes, as found also in
other studies (Fanelli et al., 2009, 2011a). Deep-sea zooplankton is characterized by organisms with
swimming activities that can move from some to hundred meters from the bottom upward, some of
them reaching the euphotic zone.
We observed a decreasing influence of the POM originated in the North Adriatic on the
species collected inside the canyon (BB site), on the adjacent slope (FF site) and on the farthest and
deepest station (EE site), where this influence is negligible and the community is essentially relied
on marine phytoplankton. Thus, the difference in food sources at each site here found by mixing
model results, demonstrated a certain influence of cascading events on species collected inside the
Bari Canyon. However, such relationship was not as clear as the temporal window explored in this
study could have not fully mirrored the effect of cascading events on organisms, and in turn to the
delay from the incorporation of organic matter and the assimilation in animal tissue. In general, in
deep-sea food webs where the response of the community is delayed with respect to the food inputs
from the surface (Fanelli et al., 2013), defining the actual contribution of vertical flux vs. the
influence of cascading is a challenging task that deserves further studies. Indeed, from an isotopic
point of view, phytoplankton bloom, as a proxy of marine contribution, and dense shelf water
cascading generate the same effects, i.e. less negative 13C coupled to lower 15N (Langone et al.,
2016). A similar seasonal trend was observed also in the particles collected in the South Adriatic Pit
(Turchetto et al., 2012) and was attributed to the enhanced primary production during the spring.

5. Conclusions

Meso- and macrozooplankton organisms play a key role in biological processes in all marine
ecosystems, linking phytoplankton/micro-zooplankton to the higher trophic levels. Through their
vertical migrations, zooplankton organisms may transfer the organic matter from the euphotic,
productive layer to the dark deep ocean (Siokou-Frangou et al., 2010; Williamson et al., 2011). In
this context, the present study represents an important contribution to the knowledge of the ecology
of deep-sea zooplankton. This important component of deep-sea macrofauna is far to be structurally
simple and the high subdivision in trophic levels here found has important implication for
ecosystem modeling but also for the correct evaluation of the good environmental status of a food
web within the context of national/international marine monitoring program, such as the EC Marine
Strategy Framework Directive (Directive 2008/56/EC). Food-web structure is a fundamental
descriptor within an ecosystem-based monitoring approach and a correct definition of its elements,
with deep-sea zooplankton being not a homogeneous compartment, but characterized by different
TL species/taxa, is crucial to provide reliable models.

Further, we highlighted the importance of both vertical and advective fluxes in driving
changes in the food web structure of deep-sea zooplankton. We identified the main drivers of the
food web dynamics as delayed records of river discharge (i.e. the Po River in the North Adriatic,
recorded two or three months before the sampling) which highlight a relationship with the NAdDW,
and salinity, and thus a potential link with the LIW. In this sense, changes in the Mediterranean
water masses, under a climate change scenario, with alteration (i.e., increase) of the salinity and
temperature regimes could have severe effects on the trophodynamics of deep-sea zooplankton.

Acknowledgements

This study was carried out within the framework of the projects HERMIONE (Grant
agreement No. 226354) and COCONET (Grant agreement No. 287844) of the European
Commission, and the Flagship project RITMARE SP5_WP3_AZ1 (the Italian Research for the
Sea). EF was also supported by IDEM (Implementation of the MSFD to the Deep Mediterranean
Sea; contract EU No 11.0661/2017/750680/SUB/EN V.C2.). We wish to acknowledge the cruise
participants who helped us with the mooring servicing, in particular the Captains and the crew
members of the R/Vs Urania, G. Dallaporta and Minerva Uno. Special acknowledgment to
Alessandra De Olazabal and Valentina Tirelli (OGS) for helping with sample sorting and
identification, and, in particular, to Fabio Savelli (CNR-ISMAR) for organic matter analyses.
Analyses of chlorophyll-a concentration used in this paper were produced with the Giovanni online
data system, developed and maintained by the NASA GES DISC. Finally, we wish to acknowledge
the valuable comments and suggestions provided by two anonymous reviewers that greatly
improved the original version of the manuscript.

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Tables

Table 1. Mean δ15N, δ13C and C/N values (±SD) of taxa analyzed. Trophic levels are based on the
equation reported in the text. Trophic groups (TG) and literature source (Source) are indicated. Ff =
Filter feeders; OMN = Omnivores; C = Carnivores; UNK = Taxon with unknown feeding habits.

Phylum/Class/Order Taxon δ15N δ13C C/N TL Feeding guild

YN
Hyperiidea Vibilia sp. 1.84 (0.63) -18.45 (2.41) 7.73 (0.03) 2 Ff
Pteropoda Clio sp.Y 2.32 (0.89) -17.62 (2.47) 4.77 (0.87) 2 OMN
Copepoda Acartia sp.Y 2.52 -19.54 6.00 2 Ff
Copepoda Haloptilus sp.YN 3.15 (0.05) -20.77 (0.91) 5.22 (1.12) 2 OMN
Copepoda Euchirella sp.N 4.02 -20.19 4.88 2 OMN
Copepoda Paracalanus sp.YN 3.98 (1.20) -20.68 (0.93) 4.96 (1.72) 2 OMN
Copepoda Pleuromamma sp.YN 3.61 (1.13) -19.70 (0.92) 6.60 (1.55) 2 OMN
Copepoda Euchaeta sp.YN 4.21 (0.78) -20.77 (0.51) 5.00 (0.68) 2 C
Siphonophora Lensia sp.YN 4.58 (1.29) -18.22 (1.08) 4.31 (0.49) 3 C
YN
Copepoda C. typicus 5.10 (0.40) -19.39 (0.99) 5.06 (0.36) 3 OMN
YN
Osteichthyes Cyclothone sp. 5.31 (0.83) -19.68 (0.65) 4.87 (0.76) 3 C
YN
Ostracoda Ostracoda 4.81 (1.43) -19.35 (1.62) 6.36 (0.98) 3 UNK
Euphausiacea Euphausia sp.YN 5.74 (1.09) -19.85 (0.61) 4.91 (1.35) 3 OMN
Siphonophora C. appendiculataY 5.86 -19.30 3.97 3 C
Chaethognatha SagittoideaYN 6.14 (0.92) -19.46 (1.01) 5.38 (1.22) 3 C
Decapoda Euphausiacea larvaeY 6.15 -18.29 6.14 3 UNK
Euphausiacea N. megalopsN 6.23 (0.54) -19.92 (0.36) 3.99 (0.06) 3 C
Cnidaria SiphonophoraYN 6.44 (0.21) -18.88 4.05 3 C
Hyperiidea H. galbaYN 6.82 (1.24) -19.77 (0.18) 4.77 (0.40) 3 C
Decapoda Brachyura larvaeY 6.78 -18.72 6.88 3 UNK
Copepoda Heterorhabdus sp.N 6.67 (1.44) -20.13 (0.80) 5.78 (0.73) 3 C
Isopoda N. borealisYN 7.04 (0.35) -19.76 (0.80) 5.24 (0.17) 3 OMN
Decapoda Decapoda larvae IIY 7.27 -16.65 8.46 4 UNK
Decapoda Decapoda larvae IN 7.35 -17.82 4.55 4 UNK
Cnidaria HydromedusaeN 6.88 (1.33) -18.89 (1.89) 7.35 (3.34) 4 C
Copepoda C. helgolandicusYN 8.79 (1.25) -18.00 (0.91) 7.41 (1.37) 4 OMN
Pteropoda Clione sp.N 9.25 (0.41) -18.60 (0.62) 5.13 (0.53) 4 C
Y= taxa collected and analysed during cascading events
N= taxa collected and analysed during no-cascading events
Table 2. Isotopic values of potential food sources for deep-sea zooplankton in the Bari Canyon and
adjacent slope.

δ13C δ15N
POM from -25.0‰ to -20.0‰ from +3.0‰ to +9.2‰ Faganeli et al. (2009)
POM from -25.8‰ to -20.3‰ from -6.6‰ to +18.1‰ Berto et al. (2013)
OM from -27.8‰ to -25.0‰ from +1.5‰ to +6.8‰ Langone et al. (2016)
aged-OM from -27.8‰ to -25.0‰ from -0.8‰ to +1.2‰ Langone et al. (2016)
Table 3. Results of PERMANOVA pair-wise tests comparing isotopic ratios of postulated feeding
groups based on 9999 permutations. Ff = Filter feeders; OMN = Omnivores; C = Carnivores; UNK
= Taxon with unknown feeding habits.

Groups t
Ff, OMN 1.4ns
Ff, C 3.57**
Ff, UNK 3.7**
OMN, C 1.93*
ns
OMN, UNK 1.90
C, UNK 1.42ns
*=p < 0.05; **=p < 0.01; ns = not significant
Table 4. Main results of two-way PERMANOVA tests comparing isotopic ratios of zooplankton in
periods with or without cascading and among sites based on the Bray-Curtis dissimilarities, and of
the pairwise comparisons based on factor “site”.

Source df MS Pseudo-F P
cascading 1 32.96 7.07 0.005
site 2 16.24 3.48 0.019
cascadingXsite 2 1.13 0.24 0.88
Residuals 107 4.66
Total 112
Pair wise “site” t
EE vs. BB 2.34 0.013
FF vs. BB 0.42 0.797
EE vs. FF 2.44 0.008
Table 5. Results of two-way univariate PERMANOVA (cascading and site) carried out separately
for 15N and 13C, based on the Euclidean dissimilarities. The pairwise comparison for factor “site”
for 15N values is also provided.

15N 13C
Source df MS Pseudo-F Source df MS Pseudo-F
cascading 1 27 8.02** cascading 1 5.96 4.62*
site 2 14.03 4.16 * site 2 2.21 1.71 ns
cascadingXsite 2 0.67 0.20ns cascadingXsite 2 0.46 0.36ns
Residuals 107 3.37 Residuals 107 1.29
Total 112 Total 112
Pairwise for factor “site” t p
BB vs. EE 2.69 0.009
BB vs. FF 0.25 0.808
EE vs. FF 2.71 0.009
*=p < 0.05; **=p < 0.01; ns = not significant
Table 6. Results of GLM models carried out on 13C values and environmental variables for the
whole sampling area: Riv2 and Riv3= river discharge recorded two and three months before
sampling, respectively; S = salinity recorded on the bottom.

type of
Df Deviance Resid. Df Resid. Dev F correlation
NULL 176 372.67
Riv2 1 50.43 175 322.24 299.62*** +
Riv3 1 18.51 174 303.73 109.99** +
S 1 12.55 173 291.18 74.55** +
AIC 600.42
explained deviance 21.87%
**=p < 0.01; ***=p < 0.001
Figure captions

Figure 1. Map of the study area with the location of the mooring stations: mooring FF (733 m water
depth) located on the open slope, mooring EE (1194 m water depth) in the deepest part of the basin,
and mooring BB (606 m water depth) in the northern channel of the Bari Canyon system. Insertion
of a sediment trap position near the seafloor (at 35 meters above the bottom), collecting both
vertical and advective flow is also shown. Multibeam bathymetry data by Trincardi et al. (2014) and
Foglini et al. (2016).

Figure 2. Graphical representation of the trophic groups, based on their isotopic composition of
δ13C and δ15N, as identified by a) hierarchical clustering (Euclidean distance of untransformed data
subjected to pair-averaged grouping) for 27 macrozooplankton taxa and b) nMDS plots, here the
results of the cluster analysis are superimposed on the figure, showing 3 main groupings at 2.6
(Euclidean) distance (dashed line). Roman numerals at the tree branches identify groups of taxa
belonging to different trophic guilds.

Figure 3. Scatterplot of mean δ15N (‰) vs. δ13C (‰) values of deep-sea zooplanktonic taxa
collected in the Bari Canyon and the adjacent slope analyzed by period (i.e. cascading vs. no
cascading).

Figure 4. Temporal trends of a) potential temperature (°C) recorded at moorings FF, EE and BB
from March 2012 to October 2012 and b) potential temperature (°C) recorded at mooring BB from
March 2010 to October 2012.

Figure 5. Surface chlorophyll-a concentrations (mg/m3) at the three stations (BB, FF and EE) from
December 2009 to January 2013.

Figure 6. Averaged monthly Po river discharge (m3 s-1) from December 2009 to November 2012.

Figure 7. Results of stable isotope analysis in R (95, 75 and 50% credibility intervals) showing
estimated source contributions for zooplankton communities at BB, FF and EE site. POM_NA:
mean POM signatures from the North Adriatic from Fagameli et al. 2009 and Berto et al 2013;
marine_phyto: mean isotopic signatures of marine phytoplankton recorded in May by Harmelin-
Vivien et al., 2008; soil_OM and aged_OM = isotopic signatures of soil-derived OM and aged
POM as in Langone et al., 2016.

Figure 8. Scatterplot of mean δ15N (‰) vs. δ13C (‰) values of a) the different taxa grouped for
each trophic level and b) the major trophic groups, as obtained by cluster analysis for zooplankton
(For the definition of the areas see Langone et al., 2016).
Figure 1.

1 Fig. 2

2 a)

4 b)

2D Stress: 0,01 feeding guild

Clio sp. FF
OMN
Decapoda larvae II C
UNK
Vibilia sp. Distance
Decapoda larvae I
Lensia sp. 1.7
Euphausiacea larvae 2.6
Hydromedusae
C. helgolandicus
Brachyura larvae Acartia sp.
Siphonophora Pleuromamma sp.
C. typicus
Clione sp. C. appendiculata

Cyclothone sp.
Euphausia sp.
Sagittoidea
H. galba Euchirella sp.
N. borealisN. megalops Ostracoda
Heterorhabdus sp.
Euchaeta sp. Haloptilus sp.
Paracalanus sp.

6
Figure 3.

12

10

8
δ15N

no cascading
2
cascading

0
-24 -23 -22 -21 -20 -19 -18 -17 -16 -15 -14
δ13C

Figure 4.

14
Mooring FF
13.6
13.2
12.8

Potential Temperature, °C
12.4
12
Mooring EE 13.6
13.2
12.8
12.4
12
Mooring BB 13.6
13.2
12.8
12.4
a)
12
Jul-12
Apr-12

Jun-12

Aug-12

Sep-12

Nov-12
Mar-12

May-12

Oct-12

14
Potential temperature, °C

13.6

Mooring BB 13.2

12.8

12.4
b)
12
Mar-10

Sep-10

Mar-11

Sep-11

Mar-12

Sep-12


Chl a (mg/m3)
Figure 5.

0.4

0.0
0.1
0.2
0.3
0.5
0.6
0.7
0.8
Dec09

Feb10

Apr10

June/July10

Sept10

Nov10

Jan11

Mar11

May11

Aug11

Nov11

Jan12

Mar12

May12

Aug12

Oct12

Dec12
FF
EE
BB
Figure 6.

Figure 7.

1 Figure 8.

10
PO RIVER DISCHARGE
TROPHIC LEVEL 2
8 TROPHIC LEVEL 3
TROPHIC LEVEL 4

6 marine
soil phytoplankton
δ15Ν

derived
OM
4

aged OM a)
0
-28 -26 -24 -22 -20 -18 -16

3 δ13C

4
10
PO RIVER DISCHARGE
FILTER FEEDERS
OMNIVORES
CARNIVORES
8 UNKNOWN
MUCUS FEEDERS

6 marine
soil phytoplankton
δ15Ν

derived
OM
4

aged OM b)
0
-28 -26 -24 -22 -20 -18 -16

5 δ13C
6
7

8
Highlights

 Deep-sea zooplankton represent a key component of deep-sea ecosystems, linking POM to higher trophic
levels.
 Based on stable isotopes of N and C, the deep-sea zooplankton food web is organized in 3 trophic levels
from POM feeders to ultra-specialized carnivores.
 Dense Shelf Water Cascading likely influences the trophic dynamics of deep-sea zooplankton in the
whole area.
 Notwithstanding, deep-sea zooplankton seemed to rely mainly on vertical fluxes from surface primary
production.