Electronic supplementary material: detailed methods
Overview
Study site and species
Three binary-style feeding preference trials were conducted using the surf parrotfish,
Scarus rivulatus, between May and August 2014, at Orpheus Island Research Station (OIRS;
18°32’ S, 146°20’ E). Orpheus Island is located on the inner-shelf of the Great Barrier Reef
and lies close to the boundary of the inshore terrigenous sediment wedge (Woolfe et al. 2000;
Larcombe et al. 2001). 1) Scarus rivulatus is the most abundant grazing herbivore on
Orpheus Island (Fig. 1a, b), 2) when housed in schools of 3, S. rivulatus of this size readily
acclimated to captive conditions, and 3) fishes in this size class feed as adults, by scraping
EAMs from the reef surface; smaller juveniles crop individual algal filaments (Bonaldo and
Bellwood 2008).
Underwater visual censuses
To initially quantify the abundance of S. rivulatus and compare it to other herbivorous
species on Orpheus Island, the abundance and biomass of roving herbivores was estimated
using underwater visual censuses. Each census consisted of a 10 min timed swim on the reef
crest/outer flat of Pioneer Bay on SCUBA (following Fox and Bellwood 2007). Twenty-four
replicate transects were swum in Pioneer Bay, each separated by a minimum of 10 m. This
design essentially resulted in a survey spanning the entire bay. Fishes were categorised into
2.5 cm size classes <10 cm and 5 cm size classes >10 cm. To supplement the abundance data,
the size-class data were used to estimate biomass of each species (following Fox and
Bellwood 2007).
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Sediment sampling
Sediment collection
The three feeding preference trials used in the study assessed the effects of differing
sediment source (terrigenous or reefal), grain size (fine or coarse), and organic content (high
or low) on grazing by S. rivulatus. This required the production of six different sediment
treatments, each replicating the mass and, where applicable, the grain size distribution of
benthic sediments from the reef flat of Orpheus Island. To quantify the mass and grain size
distribution of sediments bound in EAMs at Orpheus Island, EAM sediments were collected
from four reef flat sites (>400 m apart) on the leeward side of the island. Sediments were
collected from flat, low-complexity EAM-covered surfaces using a submersible 12 V
electronic vacuum sampler (adapted from Kramer et al. 2012). Eight replicate samples were
collected at each site from 87 mm diameter circular quadrats placed randomly along a 50 m
transect (32 sediment samples in total). Of the eight replicate samples from each site, five
were randomly selected for mass analysis and three for particle size analysis, and were
processed as described below.
Sediment processing: mass analysis
Five samples from each site were selected for mass analysis. We used a series of three
processes to sequentially determine the mass of all particulates, all inorganic sediments, and
silicates in each sample.
Initially, total particulate mass (including all sediments and organic material) was
determined by rinsing samples three times with fresh water to remove salts (leaving at least 3
h for samples to settle between rinsing) then drying them to constant weight at 60 ºC.
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Inorganic sediment mass was determined by bleaching the samples with 30%
hydrogen peroxide (H2O2) for a minimum of 7 d following Cortés and Risk (1985; hereafter
treatment with hydrogen peroxide is termed ‘bleaching’). Samples were then dried and reweighed. The bleaching removed organic material in the samples through oxidation
(Lavkulich and Wiens 1970; Cortés and Risk 1985). Bleached sediment mass therefore
represents the inorganic sediment only, and:
organic material mass = total particulate mass – inorganic sediment mass.
To determine the mass of silicates following bleaching, the sediments were acidified with 5%
hydrochloric acid (HCl) until no bubbles evolved within a 24 h period (following BrownSaracino et al. 2006). Sediments were rinsed to remove any salts formed, then dried and reweighed to give a final mass. The acid dissolved the carbonate sediments, thus the final mass
represents that of the silicates in the sediment (Brown-Saracino et al. 2006) and:
carbonate material mass = inorganic sediment mass – silicate mass.
Sediment processing: particle size analysis
The remaining three samples from each site were used to assess the particle size
distribution of samples. These samples were subjected to the same sequence of processes but
at each stage sediment particle size was measured using a laser particle analyser (LPA;
Mastersizer 2000). LPA sampling was conducted on separate samples as it results in loss of
material which cannot be accounted for in the mass analysis. Following processing and LPA
analyses we could determine the particle size distributions of the organic material, the
inorganic sediments, and the silicates, using the comparisons described above.
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Sediment properties
Mass and particle size analysis of Orpheus Island sediments indicated that the EAM
of the reef flat of Orpheus Island contained 1.19 ± 0.20 kg m-2 (mean ± SE) of particulate
matter. On average 84.79 ± 1.41% of the particulate material was composed of carbonates,
while only 14.73 ± 1.34% was silicates and 2.67 ± 0.34% organic material. Silicate sediments
were predominantly silts (<63 µm diameter; 89.11% by volume), whereas inorganic
sediments (carbonates and silicates) were predominantly sands (>125 µm; 52.18% by
volume; Fig. S1a).
Sediment treatment selection
The mean sediment load (1.19 kg m-2) and grain size distribution (Fig. S1) of the reef
flat of Orpheus Island were used as a baseline for experimental sediment loads. This baseline
was selected because 120–170 mm TL S. rivulatus are most abundant and display their
highest grazing rates on the reef flat at Orpheus Island (Fox and Bellwood 2007).
To create the sediment treatments for the sediment source preference trials, siliceous
terrigenous sediments were collected from Ross River estuary, Townsville (19°18’ S,
146°48’ E) and reefal carbonate sediments from Lizard Island lagoon (14°42’ S, 145°27’ E).
Sediments were bleached for 7 d, as per sediment analysis (following Cortés and Risk
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Fig. S1. a Grain size distribution of benthic sediments (inorganic sediment) from reef flat
EAMs on the leeward coast of Orpheus Island. These values were used as the standard
particle size distribution for the sediment source preference trails. b Fine and coarse
sediment treatments were defined as the upper and lower halves of the total volume of
sediment. c and d represent the high and low organic treatments respectively, in which 14%
and 2% of the two finest size classes were replaced with equivalent portions of organic
material. All trials used the mass of sediment found on the reef flat at Orpheus Island as a
standard treatment (1.19 kg m-2).
1985) then dried and sieved into grain size fractions. This process removed organic material
while leaving no chemical residue (Cortés and Risk 1985). Hydrogen peroxide was selected
rather than sodium hypochlorite in trials to avoid olfactory residues in the sediment, i.e. to
avoid the smell of bleach. The treated fractions from each location were then combined
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separately to form two treatments: terrigenous (Ross River) and reefal (Lizard Island), with a
grain size distribution matching that of Orpheus Island reef flat (Fig. S1a).
For grain size preference trials, sediments were collected from the reef at Orpheus
Island and were bleached and sieved as above. The sediments were then mixed to form two
treatments with contrasting grain size distributions: fine (<63–250 µm) and coarse (>125–
1000 µm; Fig. S1b). The treatments replicated the finer 50% and the coarser 50% of the
sediment grain size distributions (by percentage volume) of the Orpheus Island reef flat, as
determined by grain size analysis.
Sediments for the organic load preference trials were also collected from the reef at
Orpheus Island. Sediments were processed as above with a grain size distribution matching
Orpheus Island reef flat, but with the addition of organic material (Hikari Marine A) to form
two treatments: high and low organic load (14 and 2% organic material, respectively; Fig.
S1c, d). These values are representative of the mean organic content in sediments on the reef
crest of Lizard Island (a mid-shelf reef, 14%; Purcell and Bellwood 2001) and the reef flat of
Orpheus Island (2%; as described above). The organic material was selected to approximate
the nutritional composition of detrital aggregates (Tenore 1981). The material was ground,
sieved and used to replace proportions of the fine sediment fractions (<125 µm), matching
high-nutrient detrital aggregates found in reef EAMs (Wilson et al. 2003).
Feeding preference trials
Scarus rivulatus used in the feeding preference trials were collected from Pioneer
Bay, Orpheus Island, using barrier nets while on SCUBA. Fish were randomly allocated into
schools of three and housed in 45 L aquaria. Eighteen schools (54 fish) were used as
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replicates for each of the sediment source and grain size trials, and 11 schools (33 fish) were
used as replicates for the organic load trials. Schools were independent replicates and only
exposed to a trial once. The feeding surfaces used in preference trials were prepared from
flat, EAM-covered pieces of coral rubble, collected from the reef flat of Orpheus Island. To
control for size, the surfaces were shaped into 50 cm2 circular pieces using a chisel and
mallet. All preference trials followed the same experimental procedure.
For each trial, two prepared feeding surfaces were placed into one end of an aquarium
containing three fish. Once placed into a tank, the feeding surfaces were immediately covered
with a PVC pipe, to prevent feeding, and a sediment treatment was randomly assigned to
each. The sediment treatments were then applied to each feeding surface by pouring the
wetted sediment into the pipe. The fish and sediments were then left to starve and settle,
respectively, for 24 h. After this, a video camera (GoPro) was placed into the aquarium to
record feeding preferences, and the PVC pipes removed. The feeding behaviour of the S.
rivulatus schools was then filmed for a minimum of 3 h.
The video footage was analysed to quantify the bite rates of S. rivulatus schools on
each feeding surface. The initial five minutes of the recording were discarded to account for
acclimation of the fish after the removal of the pipes and placement of the camera, and to
ensure that all fishes had sampled both feeding surfaces before the bite rate was recorded,.
The numbers of bites taken on each surface for the next 30 min were recorded. This
observation period was selected due to a significant decrease in bite rate by S. rivulatus over
time. The location of the first bite taken by any fish from each school and of each individual
in a school was also recorded immediately after pipe removal. Bite rates and first bites by
each individual were compared between sediment treatments using paired t-tests on
untransformed data while the first bites taken by any fish were compared with Wilcoxon
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matched-pairs signed-rank tests. All values are presented as means ± standard error unless
otherwise noted.
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