www.sciencemag.org/content/342/6154/108/suppl/DC1

Supplementary Materials for

Surviving in a Marine Desert: The Sponge Loop Retains Resources Within Coral Reefs

Jasper M. de Goeij,* Dick van Oevelen, Mark J. A. Vermeij, Ronald Osinga, Jack J. Middelburg, Anton F. P. M. de Goeij, Wim Admiraal

*Corresponding author. E-mail: j.m.degoeij@uva.nl

Published 4 October 2013, Science 342, 108 (2013)

DOI: 10.1126/science.1241981

This PDF file includes:

Materials and Methods Figs. S1 to S3 References (32–39)

2

Materials and Methods

DOM-substrate

13C- and 15N-enriched dissolved organic matter (DOM) tracer was prepared from 50 L

axenic batch culture of the marine diatom Phaeodactylum tricornutum. This primary 5

producer was selected because it a) has a cosmopolitan distribution (32), b) is used as

natural DOM substrate in isotope-tracer studies (33), including marine sponge-feeding

studies (34), and c) can be axenically cultured in the quantities required for this laboratory

and field study. F/2 culture medium (10 bottles of 5 L), prepared with 80% 15N-NaNO3 and

100% 13C-NaHCO3 (Cambridge Isotope Laboratories), was inoculated with P. tricornutum 10

and incubated at 16°C at a 12:12 day-night cycle at NIOZ-Yerseke (35). Algae were

concentrated after 3 weeks on a 0.45 µm membrane filter (142 mm ∅) and rinsed three

times by repeated centrifugation (5 min at 1500 rpm). Cell lysis and release of DOM was

induced by addition of Milli-Q (4 h shaking). Subsequently, the suspension was filtered on

a GF/C + GF/F filter package to remove the algal cell remnants from the DOM solution. 15

Devarda’s Alloy and MgO were added (200 mg and 100 mg per 40 mL DOM solution for

48 h at RT) to remove remaining NO3- (35). The DOM solution was filtered again (GF/F)

and freeze-dried; yielding 4 g DOM dry weight. DOM-substrate contained 56% 13C and

81% 15N as measured by elemental analyzer–isotope ratio mass spectrometry (EA-IRMS; 3

replicates of 1.6-3.4 mg dry weight). Freeze-dried DOM was solubilized (2.5 mL MQ) at 20

Carmabi, Curaçao prior to core incubations and in situ food web studies.

3

Core incubations

Four encrusting sponge species, Halisarca caerulea, Haliclona implexiformis, Chondrilla

caribensis, Scopalina ruetzleri, were collected from reefs at stations Buoy #1 and Snake

Bay, and subsequently kept in aquaria with running seawater at CARMABI, Curaçao

(12°12’N, 68°56’W), where they were left to acclimatize for at least one week prior to the 5

experiments (12, 15-17). Sponge incubations were done in 1.9 L flow chambers (16, 17) for

3 h, following the addition of 80 µM DO13C and 1.6 µM DO15N (N=4 per species) (fig.

S1). Water samples to measure the concentration and 13C isotope ratio of dissolved

inorganic carbon (DIC; i.e. respiration of DOM) were taken before (t=-0.1 h), directly after

DOM addition (t=0 h), and at the end of incubation (t=3 h) in headspace vials, without 10

trapping air (± 12 mL), and were subsequently fixed with HgCl2 (15). Control incubations

(N=4) without sponges showed very limited assimilation of DOM (1.6% of sponge uptake

as measured on GF/F filter with an average pore size of 0.7 µm) and low respiration of

DOM (10.5% of sponge incubations) by bacterioplankton, indicating that the uptake and

processing of DOM was strongly dominated by sponges. Sponge tissue samples were taken 15

at the end of the incubation and freeze-dried for later isotope analysis. Water from flow

chambers was filtered (GF/F) post-incubation and analyzed for particulate organic matter

(POM) concentration and 13C/15N-enrichment to determine sponge detritus production.

Uptake of sponge detritus by reef fauna was investigated in six cores (ID 7.5 cm, 0.75 L 20

sediment in 1.5 L cores) taken from reef cavities at station Snake Bay (12) and kept in

aquaria with running seawater. Sponge detritus was collected from different specimens of

the four tested sponge species that were repeatedly fed (every 24 hours for 4 consecutive

4

days) with 13C- and 15N-enriched DOM (fig. S2). Sponges were kept in open-top plastic

cups (ID 10 cm) in the aquarium and every 24 h (after a 3-h incubation with DOM-

substrate) the sponge-derived detritus was collected by glass pipette without harming the

underlying sponge tissue (fig. S2) and subsequently stored at -20°C. Twenty mg of sponge-

derived detritus (wet weight; 0.07% 13C and 0.9% 15N above background values) was added 5

to each sediment cores (fig. S1). After 6 h, all fauna (sorted under a binocular and

consisting mainly of polychaetes and the added motile fauna) was immediately transferred

to Ag cups, freeze-dried and stored at -20°C prior to isotope analysis. During all core

incubations, the dissolved oxygen (DO) concentration (data not shown) was monitored with

an optical probe or optode (OXY-4, PreSens, Regensburg, Germany; fig. S1) to verify that 10

sufficient oxygen was present during the incubations (DO saturation was never below 74%)

and to monitor sponge pumping activity, indicated by a linear decrease in dissolved oxygen

concentration when sponges are actively pumping. Control incubations without sponges

showed that the DO concentrations did not change or slightly increased.

15

In situ food web study

Two cavities (75 L and 125 L; 15 m water depth) were selected at station Snake Bay (12).

Glass funnels (~300 cm2) were placed in the cavity, close to the sponge-covered cavity wall

to collect sponge-derived detritus (fig. S3A). Cavities were temporarily isolated from the

surrounding reef water (12, 14) for two consecutive 3 h periods prior to the injection of 40 20

µM DO13C and 0.8 µM DO15N (fig. S3, A and B). The DOM substrate was syringe-

injected through three tubes placed at different positions in the cavity. Samples for isotope

analysis were taken at t=0 (background), 6 (first sampling after DOM addition), 23, 30 and

5

51 h and comprised of sponge tissue (cut from different sponge species within the cavity),

sponge detritus (collected in the funnels during the period prior to the sampling event),

surface sediment (collected with a teaspoon from the upper millimeters of sediment at

different locations in the cavity), motile fauna (when found during sampling), non-sponge

filter-feeders (a mixed sample of mainly tunicates, bryozoans and (soft) corals at different 5

locations within the cavity) and bacterioplankton (GF/F filtered water (1 L) at t=6).

Samples were freeze-dried and stored at -20°C prior to isotope analysis.

Sample processing and data analysis

All DIC, sediment, POM and tissue samples were transported to the laboratory at NIOZ-10

Yerseke (The Netherlands), where they were homogenized and transferred to Ag cups. The

samples were then initially acidified with 20 µL of 2.5% HCl until effervescence ceased

(visually verified), and subsequently acidified with 20 µL of 25% HCl to ensure complete

inorganic carbon removal (36). The samples were measured simultaneously for 13C/12C and

15N/14N by elemental analyzer–isotope ratio mass spectrometry (EA-IRMS) (37). For DIC 15

analysis, a 3-mL headspace within each vial was created by He flushing. Samples where

then acidified (0.1 mL H3PO4 mL-1 sample) and a gas-sample was taken from the

headspace for subsequent measurement of 13C-DIC and DIC concentration on the EA-

IRMS (38, 39). Background δ13C/δ15N values (N=3) from sampled organisms on the reef

were defined at -19.3/5.4‰ (sponge), -19.2/7.5‰ (other filter feeders), -17.5/7.3‰ (sponge 20

detritus), and -16/5‰ (infauna and motile fauna). Isotope data from the core incubations

are corrected for enrichment of substrate (DOM or sponge-detritus) and expressed as

6

incorporated tracer per unit biomass. Isotope data from the field experiment are presented

in ∆δ-notation (‰ enrichment above background values).

The quantitative estimation of organic carbon fluxes shown in Figure 3 were based on, or

derived from, several studies (2, 5-7, 11, 12) on both Caribbean and Indo-Pacific reefs. The 5

carbon flux into the microbial loop (5-50 mmol C m-2 d-1) was estimated from DOC

degradation experiments – 0.72-3.12 µmol L-1 d-1 (11) and 0.4-0.9 µmol L-1 d-1 (12) –,

projecting a 1-m2 reef area with an average overlying water column of 15 m – 15 m3 per

projected m-2. The availability of organic carbon from the sponge loop to the fauna (10-84

mmol C m-2 d-1) was derived from this study (11-24% conversion efficiency), whereas the 10

bacterial growth efficiency (6-32%, (11)) was used to estimate the availability of microbial

organic carbon to fauna (0.3-16 mmol C m-2 d-1).

7

Fig. S1

Fig. S1. Schematic view of experimental set-up of core incubation with sponges (left;

see also Fig. 1A-B) and sponge-derived detritus (right; see also Fig. 1C). Detritus for

the sediment (+/- motile fauna) was collected after repeated feeding with 13C- and 15N-5

labelled DOM substrate.

8

Fig. S2.

Fig. S2. The encrusting marine sponge Halisarca caerulea, one of the four tested

common coral reef sponge species tested in this study, in the aquarium after

harvesting from the reef. White arrows (left) indicate the oscula, i.e. outflow openings of 5

the sponge. After 24 hours in the aquarium, the sponge produced large amounts of

brownish fluffy material, i.e. the sponge-derived detritus, as indicated by the black arrows

(right).

10

9

Fig. S3.

Fig. S3. Coral reef field experiment setup. (A) Glass funnels (4 x ~75 cm2) were placed

inside the cavity (volume of approximately 75 L). (B) Syringes with 13C- and 15N-labelled

DOM substrate were prepared. (C) During 2 consecutive incubation periods of 3 h, water 5

exchange between the cavity and the surrounding coral reef water was restricted with a

tightly woven cotton cloth (12, 14). Black arrows indicated the 3 syringes (connected to

tubing leading to different parts of the cavity to obtain an even distribution of DOM-

substrate) with which the DOM-substrate was injected.

10

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Surviving in a marine desert: the sponge loop retains resources within coral reefsde.Goeij.SM.cover.page.pdfSurviving in a Marine Desert: The Sponge Loop Retains Resources Within Coral Reefs

de.Goeij.SM.refs.pdfReferences and Notes