Functional diversity in amphipods revealed by stable isotopes in an eelgrass ecosystem

Abstract

Amphipods are often dominant components of benthic marine communities and may exhibit taxon-specific differences in feeding behavior. As a result, variation in the composition of amphipod communities is an important metric for the interpretation of trophic dynamics in benthic marine ecosystems. Though previous studies of amphipod diets indicate functional diversity among taxa, few studies have examined whether these differences are detectible using time-integrated natural tracers of in situ feeding habits. We used stable isotope ratios of nitrogen (delta N-15) and carbon (delta C-13) to examine trophic structure among amphipod taxa belonging to 5 families in an eelgrass (Zostera marina) ecosystem in San Diego Bay, California. The relative contribution of sources of primary production to amphipod diets was further analyzed using a mixing model bracketed by 2 dominant sources of primary production in the system: eelgrass and algae. We detected significant differences in both delta C-13 and delta N-15 among amphipod taxa, indicating family-specific differences in feeding habits that generally agree with previous studies of amphipod diets. Hyalids fed almost exclusively on eelgrass, ischyrocerids and ampithoids tended to feed more on algae and eelgrass, respectively, and caprellids exhibited heterogeneous feeding on both algae and eelgrass. The relatively high delta N-15 value of oedicerotids suggested that this group was likely carnivorous. Our findings are in general agreement with previous descriptions of family-specific amphipod feeding behaviors, suggesting that stable isotopes are a useful tool for describing the functional roles of mesograzers in eelgrass ecosystems.

Full text

MARINE ECOLOGY PROGRESS SERIES
Mar Ecol Prog Ser
Vol. 420: 277–281, 2010
doi: 10.3354/meps08873 Published December 16
INTRODUCTION
Trophic interactions structure communities and
influence productivity across multiple trophic levels
(Paine 1980, Heck & Valentine 2007). Most studies
demonstrating such interactions in benthic marine
habitats have focused on the effects of macrograzers
(e.g. fishes, sea urchins and mollusks), whereas
smaller mesograzers (e.g. amphipods) have received
relatively less attention (Thayer et al. 1978, Duffy &
Hay 2000). Mesograzers, however, can exhibit interac-
tion strengths similar to macrograzers (Sala & Graham
2002) and may exert strong top-down effects on the
structure and productivity of benthic macrophyte
assemblages (Duffy & Hay 2000). Amphipods are par-
ticularly important in eelgrass ecosystems because
they are significant consumers of primary production
and graze preferentially on epiphytic algae, which in
turn enhances eelgrass production (Kitting et al. 1984,
Orth & Van Montfrans 1984). Determining whether
© Inter-Research 2010 · www.int-res.com*Email: nilraf@gmail.com
NOTE
Functional diversity in amphipods revealed by
stable isotopes in an eelgrass ecosystem
J. P. Farlin1,*, L. S. Lewis1, 2, T. W. Anderson1, C. T. Lai1
1Department of Biology and Coastal & Marine Institute, San Diego State University, 5500 Campanile Drive, San Diego,
California 92182-4614, USA
2Present address : Center for Marine Biodiversity and Conservation, Scripps Institution of Oceanography,
9500 Gilman Drive, La Jolla, California 92083-0202, USA
ABSTRACT: Amphipods are often dominant components of benthic marine communities and may
exhibit taxon-specific differences in feeding behavior. As a result, variation in the composition of
amphipod communities is an important metric for the interpretation of trophic dynamics in benthic
marine ecosystems. Though previous studies of amphipod diets indicate functional diversity among
taxa, few studies have examined whether these differences are detectible using time-integrated nat-
ural tracers of in situ feeding habits. We used stable isotope ratios of nitrogen (δ15N) and carbon (δ13C)
to examine trophic structure among amphipod taxa belonging to 5 families in an eelgrass (Zostera
marina) ecosystem in San Diego Bay, California. The relative contribution of sources of primary pro-
duction to amphipod diets was further analyzed using a mixing model bracketed by 2 dominant
sources of primary production in the system: eelgrass and algae. We detected significant differences
in both δ13C and δ15N among amphipod taxa, indicating family-specific differences in feeding habits
that generally agree with previous studies of amphipod diets. Hyalids fed almost exclusively on eel-
grass, ischyrocerids and ampithoids tended to feed more on algae and eelgrass, respectively, and
caprellids exhibited heterogeneous feeding on both algae and eelgrass. The relatively high δ15N
value of oedicerotids suggested that this group was likely carnivorous. Our findings are in general
agreement with previous descriptions of family-specific amphipod feeding behaviors, suggesting
that stable isotopes are a useful tool for describing the functional roles of mesograzers in eelgrass
ecosystems.
KEY WORDS: Functional diversity · Stable isotopes · Eelgrass food web · Amphipods · Amphipod
feeding
Resale or republication not permitted without written consent of the publisher

Mar Ecol Prog Ser 420: 277–281, 2010
amphipods are grazing on an algal-based or eelgrass-
based (living or detrital tissue) food source has implica-
tions on their net (positive, negative, or neutral) effects
on ecosystem primary production.
In studies of marine food webs, amphipods are often
grouped into a single trophic guild based on the
assumption that they are functionally redundant
(Thayer et al. 1978, Kitting et al. 1984, Bell 1991, Fre-
driksen 2003, Nagelkerken et al. 2006, Davenport &
Anderson 2007). This assumption, however, has been
challenged due to contrary evidence provided by feed-
ing assays and gut content analyses (Poore 1994, Duffy
& Harvilicz 2001). Though such studies provide valu-
able insight into feeding behaviors of amphipods, they
are limited to observing individual grazing behaviors
as snapshots in time (e.g. gut contents) or under less
realistic conditions (e.g. laboratory assays and meso-
cosms).
The diversity of amphipod feeding behaviors in the
wild remains an important gap in our knowledge of
benthic ecosystems (Duffy & Hay 1991) and is crucial
to our understanding of food web dynamics in these
systems (Poore et al. 2008). One method of exploring
the feeding history and trophic diversity of wild organ-
isms is through the use of stable isotope ratios of car-
bon (13C/12C) and nitrogen (15N/14N), whereby carbon
is used to trace primary production and nitrogen to
infer trophic level (Peterson & Fry 1987). Our objective
was to use stable isotopes to examine the feeding his-
tory of 5 amphipod taxa, each within a different family
(Ampithoidae, Caprellidae, Hyalidae, Ischyroceridae,
and Oedicerotidae) within an eelgrass (Zostera marina)
ecosystem in San Diego, California. We selected these
5 amphipod families because they were common in the
study area (Lewis 2009), were readily identified using
taxonomic keys (e.g. Chapman 2007), and were
expected to exhibit different modes of feeding based
on previous research and jaw morphology (Chapman
2007). We then examined whether our findings based
on stable isotope ratio analyses agreed with previous
descriptions of family-specific feeding behaviors.
MATERIALS AND METHODS
Study area and collection. Amphipods, filamentous
epiphytic algae, eelgrass, and eelgrass detritus were
collected haphazardly during low tide from ca. 100 m2
of eelgrass habitat adjacent to Shelter Island in San
Diego Bay, California. This site was chosen to take
advantage of the existing infrastructure associated
with ongoing research (Lewis 2009). At low tide,
researchers waded into the eelgrass bed to a depth of
approximately 0.5 m prior to collecting samples. Eel-
grass samples, containing epiphytic algae and epi-
fauna, were pulled by hand and immediately placed in
500 μm mesh bags. The benthos was then skimmed to
collect detritus and benthic amphipods. A total of 9
grab samples and 8 benthic samples were collected on
18 October and 13 November, 2008. All samples were
immediately placed on ice and transported to the labo-
ratory for processing. In the lab, epiphytic algae and
fresh young eelgrass leaves were isolated from grab
samples. The remaining eelgrass samples were thor-
oughly rinsed with deionized water over a 500 μm
sieve to isolate amphipods from other material. Benthic
samples were also rinsed over a 500 μm sieve, and
amphipods and detrital (i.e. dead and decaying) eel-
grass were removed and isolated. All samples were
frozen (–20°C) until preparation for stable isotope
analysis. We intended to sample macroalgae, but none
were present in the eelgrass bed during sampling. Epi-
phytic filamentous algae were analyzed and inter-
preted as a general ‘algal signature’ on the basis of the
assumption that macroalgae and filamentous algae
have similarly depleted δ13C values (approximately
10‰ less) than those of eelgrass (Currin et al. 1995,
Moncreiff & Sullivan 2001).
Amphipod identification. Amphipods were sorted to
family according to Chapman (2007). We used this tax-
onomic category because identification of amphipods
to genus and species was not feasible for all sexes and
stages of specimens used in our study (Chapman
2007). We later identified the numerically dominant
genus within each family (Table 1) by further examin-
ing identifiable voucher specimens with taxonomists
from the San Diego City Metropolitan Waste Water
District.
Sample preparation and stable isotope analysis.
Samples were thawed, sorted in clean Petri dishes, and
then rinsed with deionized water to remove residual
salt water. Amphipods were sorted to family and indi-
viduals were pooled (n ≈5 to 45 per replicate sample;
Table 1) to attain adequate material for isotope analy-
ses (1.0 to 2.0 mg dry wt). Young eelgrass leaves, detri-
tal eelgrass, and epiphytic algae were cleaned of all
fouling material prior to processing and analysis. All
samples were placed in a drying oven at 60°C for 48 h,
homogenized, placed into tin capsules and weighed.
Isotopic ratios were measured by combustion using
continuous-flow isotope-ratio mass spectrometry at
San Diego State University’s (SDSU) Ecology Analyti-
cal Facility (Thermo Scientific DeltaPlus Advantage
IRMS, n = 66) and the Stable Isotope Facility at the
University of California, Davis (PDZ Europa 20-20
IRMS, n = 33). Stable carbon and nitrogen isotope
ratios of a sample were measured against laboratory
reference materials selected to be compositionally sim-
ilar to the samples being analyzed in both analytical
laboratories. At SDSU, laboratory reference materials
278

Farlin et al.: Stable isotopes of amphipods
were calibrated against NIST standard reference
materials USGS40 and USGS41. Delta (δ) values for C
and N were calculated using standard delta notation:
(1)
where Ris the molar ratio of the heavy to light isotopes.
Carbon and nitrogen isotope ratios are reported rela-
tive to the Vienna PDB scale (Coplen et al. 2006) and
atmospheric N2(Mariotti 1983), respectively.
A 2-source mixing model was used to estimate
dietary composition of amphipod families using the
equation:
(2)
where fis the fraction of the diet derived from algae.
The average δ13C values of producers used in the cal-
culation were –9.2 ± 1.35‰ for eelgrass (aver-
age of live and detrital eelgrass) and –17.2 ±
4.5‰ for algae. Trophic enrichment was cal-
culated as the mean difference between δ15N
values of each amphipod family and the aver-
age δ15N value of primary producers (10.7 ±
1.7‰).
Statistical analysis. Outliers greater than
1.5 ×IQR (inter-quartile range) were identi-
fied and removed prior to analyses, resulting
in less than 3% exclusion of data in analyses.
Differences among amphipod families in δ13C
and δ15N values were examined using 1-way
ANOVA. When significant, pairwise multiple
comparisons among amphipod taxa were
conducted using Tukey’s HSD test. All
statistical analyses were conducted using
SYSTAT 12.01.02. We used ‘IsoError’ (Phillips
& Gregg 2001) and a 2-source mixing model
to estimate the proportion of the diet of the
amphipod families derived from eelgrass- vs.
algae-based carbon sources. Errors were
propagated and reported as 95 % confidence intervals
for the mixing-model results using IsoError software.
RESULTS
Carbon isotope signatures of eelgrass (mean of live
and detrital) and algae differed by 8 ‰ and bracketed
all amphipod δ13C values (Fig. 1). Significant differ-
ences in δ13C were detected among amphipod taxa
(F4,45 = 5.7, p = 0.001), with hyalids showing a signifi-
cantly higher δ13C than all other families. Oedicerotids
were enriched in δ15N relative to hyalids (F4,45 = 4.055,
p = 0.007) and appeared to be partially carnivorous as
suggested by their higher, but slightly overlapping,
δ15N values (Fig. 1) and greater trophic enrichment
(Table 1). Caprellids, ischyrocerids, and ampithoids all
exhibited intermediate δ13C and δ15N values (Fig. 1,
fCC
C
=−
−
δδ
δδ
13 13
13
amphipods eelgrass
algae 113Ceelgrass
δ= −
⎛
⎝
⎜⎞
⎠
⎟×
R
R
sample
standard
1 1000
279
Family Genus δ15N δ13C Reps Ind./rep. % algae δ15N enrichment
Ampithoidae Ampithoe 12.4 ± 0.5 –12.4 ± 1.0 10 11.5 39.2 ± 16% 1.7
Caprellidae Caprella 11.6 ± 0.3 –13.2 ± 0.5 10 12.9 49.7 ± 13% 0.9
Hyalidae Protohyale 11.5 ± 0.4 –9.7 ± 0.3 11 16.5 5.2 ± 6 % 0.8
Ischyroceridae Erichthonius 12.4 ± 0.2 –14.0 ± 0.9 10 42.3 59.8 ± 18% 1.7
Oedicerotidae Hartmanodes 14.0 ± 0.7 –12.1 ± 1.3 4 4.5 35.9 ± 19% 3.3
Primary producers
Live eelgrass 10.7 ± 0.5 –8.3 ± 0.4 8 1
Detrital eelgrass 10.5 ± 0.4 –10.2 ± 0.2 8 1
Filamentous algae 11.0 ± 0.5 –17.2 ± 1.8 6 >100
Table 1. Isotopic ratios of amphipod groups and primary producers from an eelgrass bed in San Diego Bay, California. Also shown
are no. of replicates, average no. of individuals per replicate, proportion of algae in the diet (± 95 % CI) and trophic enrichment
of amphipods
Fig. 1. Mean stable isotope ratios (±1 SE) of amphipod families (filled
symbols), and primary producers (open symbols) from an eelgrass bed in
San Diego Bay, California

Mar Ecol Prog Ser 420: 277–281, 2010
Table 1). The mixing model indicated that eelgrass
production (live or detrital) constituted 94.8% and
60.8% of the diet of hyalids and ampithoids, respec-
tively. Algal production constituted 59.8% of the diet
for ischyrocerids, whereas the mixing model indicated
a relatively equal contribution of algae and eelgrass to
the diet of caprellids.
DISCUSSION
We employed stable isotopes to explore time-
integrated differences in the diets of amphipod taxa in
situ. Our findings are in general agreement with previ-
ous descriptions of family-specific feeding habits
(Brawley & Fei 1987, Dewey 1970, Dixon & Moore
1997, Poore 1994, Duffy & Harvilicz 2001, Chapman
2007, Poore et al. 2008). For example, intermediate
δ13C signatures for ampithoids, caprellids and ischyro-
cerids agree with previous reports of heterogeneous
feeding behaviors (Brawley & Fei 1987, Dewey 1970,
Duffy & Harvilicz 2001), with our mixing model deter-
mining that ampithoids consume more eelgrass and
ischyrocerids favor microalgae. Hyalids have previ-
ously been described as feeding on brown algae (Poore
2004). Our δ13C measurements suggest that hyalids
were supported by eelgrass-derived carbon, which
was in agreement with a macrophagous diet, but not in
agreement with the source of carbon. Based on jaw
morphology (adapted for consuming macrophytes),
consumption of eelgrass in the absence of macroalgae
in our study is a likely alternative feeding response.
Oedicerotids are known to feed as predators on
meiobenthic animals such as harpacticoid copepods
(Yu & Suh 2002), consistent with the enriched δ15N val-
ues observed in our study. Assuming these oedicero-
tids were mostly carnivorous, our data suggest
ca. 1.2‰ trophic enrichment, in agreement with the
mean δ15N enrichment (1.0 to 1.5 ‰) observed in simi-
lar ammonotelic marine crustaceans (McCutchan et al.
2003, Vanderklift & Ponsard 2003).
Stable isotope analysis has been widely used for
tracing the flow of carbon and nitrogen through food-
webs (Fry 2006). In light of its utility and common use
in ecology, it is surprising that so few studies have used
this tool to explore the functional roles of amphipods in
nature. Gut contents of amphipods are often highly
degraded and difficult to evaluate, and although feed-
ing assays provide excellent information on what
organisms prefer to eat in a laboratory, they are limited
by the less realistic settings and reduced variety of
food items offered (Duffy & Hay 1991). By using stable
isotopes, we were able to determine the relative
importance of algal versus eelgrass production to feed-
ing in amphipods, and their trophic position, in nature.
Furthermore, these signatures provide time-integrated
diet histories (e.g. weeks to months) rather than instant
snapshots in time (Kaufman et al. 2008). Though iso-
topic turnover can be influenced by temperature and
several other factors (McIntyre & Flecker 2006), stable
isotopes have been shown to be fairly conserved
throughout adult invertebrate life cycles (Fry & Arnold
1982).
Our conclusions regarding the relative importance of
eelgrass- versus algae-derived carbon and trophic
position among amphipod taxa are likely robust and
informative. We have demonstrated that stable iso-
topes can be used to assess the function of amphipods
with respect to the dominant trophic pathways used
while in their natural habitat. Such information is criti-
cal to our understanding of trophic dynamics in eel-
grass ecosystems, for example, of how changes in top-
down and bottom-up forcing are transmitted across
trophic levels. In addition, by scaling studies such as
ours to include a more comprehensive food web, ecol-
ogists can assess the relative importance of eelgrass
and algal production in eelgrass ecosystems and how
this importance may vary in space or time, or as a con-
sequence of environmental change.
Acknowledgements. This research was supported by a grant
from the Unified Port of San Diego, and we thank E. Maher
and D. Merck. We also thank L. Thurn at the San Diego State
University Ecology Analytical Lab for assistance with stable
isotope analysis and R. Velarde and E. Moore at the San
Diego City Metropolitan Waste Water District for assistance
with identification of voucher specimens. A. Bohonak and
P. Canning provided constructive comments on the manu-
script, along with the anonymous reviewers whose comments
improved the manuscript. This is Contribution No. 6 of the
Coastal and Marine Institute Laboratory, San Diego State
University.
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Editorial responsibility: Joseph Pawlik,
Wilmington, North Carolina, USA
Submitted: December 28, 2009; Accepted: October 13, 2010
Proofs received from author(s): November 30, 2010
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