Sea anemones may thrive in a high CO 2 world

Abstract

Increased seawater pCO 2 , and in turn 'ocean acidification' (OA), is predicted to profoundly impact marine ecosystem diversity and function this century. Much research has already focussed on calcifying reef-forming corals (Class: Anthozoa) that appear particularly susceptible to OA via reduced net calcification. However, here we show that OA-like conditions can simultaneously enhance the ecological success of non-calcifying anthozoans, which not only play key ecological and biogeochemical roles in present day benthic ecosystems but also represent a model organism should calcifying anthozoans exist as less calcified (soft-bodied) forms in future oceans. Increased growth (abundance and size) of the sea anemone (Anemonia viridis) population was observed along a natural CO 2 gradient at Vulcano, Italy. Both gross photosynthesis (P G) and respiration (R) increased with pCO 2 indicating that the increased growth was, at least in part, fuelled by bottom up (CO 2 stimulation) of metabolism. The increase of P G outweighed that of R and the genetic identity of the symbiotic microalgae (Symbiodinium spp.) remained unchanged (type A19) suggesting proximity to the vent site relieved CO 2 limitation of the anemones' symbiotic microalgal population. Our observa-tions of enhanced productivity with pCO 2 , which are consistent with previous reports for some calcifying corals, con-vey an increase in fitness that may enable non-calcifying anthozoans to thrive in future environments, i.e. higher seawater pCO 2 . Understanding how CO 2 -enhanced productivity of non-(and less-) calcifying anthozoans applies more widely to tropical ecosystems is a priority where such organisms can dominate benthic ecosystems, in particular following localized anthropogenic stress.

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Sea anemones may thrive in a high CO2world
DAVID J. SUGGETT*, JASON M. HALL-SPENCER†, RICCARDO RODOLFO-METALPA†,
TOBY G. BOATMAN*, ROSS PAYTON*, D. TYE PETTAY‡, VIVIENNE R. JOHNSON†, MARK
E. WARNER‡ and TRACY LAWSON*
*Coral Reef Research Unit, School of Biological Sciences, University of Essex, Colchester, CO4 3SQ, UK, †Marine Biology and
Ecology Research Centre, University of Plymouth, Plymouth, PL4 8AA, UK, ‡College of Earth, Ocean, and Environment,
University of Delaware, 700 Pilottown Road, Lewes, Delaware 19958, USA
Abstract
Increased seawater pCO2, and in turn ‘ocean acidification’ (OA), is predicted to profoundly impact marine ecosystem
diversity and function this century. Much research has already focussed on calcifying reef-forming corals (Class:
Anthozoa) that appear particularly susceptible to OA via reduced net calcification. However, here we show that
OA-like conditions can simultaneously enhance the ecological success of non-calcifying anthozoans, which not only
play key ecological and biogeochemical roles in present day benthic ecosystems but also represent a model organism
should calcifying anthozoans exist as less calcified (soft-bodied) forms in future oceans. Increased growth (abundance
and size) of the sea anemone (Anemonia viridis) population was observed along a natural CO2gradient at Vulcano,
Italy. Both gross photosynthesis (PG) and respiration (R) increased with pCO2indicating that the increased growth
was, at least in part, fuelled by bottom up (CO2stimulation) of metabolism. The increase of PGoutweighed that of R
and the genetic identity of the symbiotic microalgae (Symbiodinium spp.) remained unchanged (type A19) suggesting
proximity to the vent site relieved CO2limitation of the anemones’ symbiotic microalgal population. Our observa-
tions of enhanced productivity with pCO2, which are consistent with previous reports for some calcifying corals, con-
vey an increase in fitness that may enable non-calcifying anthozoans to thrive in future environments, i.e. higher
seawater pCO2. Understanding how CO2-enhanced productivity of non- (and less-) calcifying anthozoans applies
more widely to tropical ecosystems is a priority where such organisms can dominate benthic ecosystems, in particular
following localized anthropogenic stress.
Keywords: Cnidarian, CO2vent, Ocean acidification, Productivity, Sea anemone, Symbiodinium spp.
Received 5 April 2012 and accepted 5 June 2012
Introduction
Rising atmospheric CO2 from anthropogenic activity
will significantly increase seawater CO2 partial pres-
sure (pCO2) and result in lower ocean pH or ‘ocean
acidification’ (OA) this century (Caldeira & Wickett,
2005). Predictions suggest that by the year 2100 surface
ocean pH could be reduced by ca. 0.3–0.5 units com-
pared to present day values (IPCC, Intergovernmental
Panel on Climate Change, 2007), with profound conse-
quences for fundamental biological processes, such as
calcification and photosynthesis, and in turn the
functioningofentire marine
Hoegh-Guldberg & Bruno, 2010). The potential impact
of OA upon corals reefs has particularly received much
high profile attention. Coral reef ecosystems are charac-
terized by high productivity and diversity as a result of
primary productivity and calcification by coral cnidari-
ecosystems(e.g.
ans (Class: Anthozoa). Observations from laboratory
experiments attempting to replicate future OA scenar-
ios (e.g. Anthony et al., 2008; Edmunds et al., 2012) as
well as in situ investigations at present day naturally
high CO2 shallow water reefs (Fabricius et al., 2011;
Crook et al., 2012), generally demonstrate that calcifica-
tion and growth will be impacted by elevated CO2
(reducedpH)(seealso
However, the evidence is not entirely negative:
A growing volume of studies suggest that net calcifi-
cation rates may in fact not always be reduced under
long-term exposure to elevated CO2(Krief et al., 2010;
Rodolfo-Metalpa et al., 2010). Some calcifiers, including
coral cnidarians (Rodolfo-Metalpa et al., 2011) can
maintain intact their calcification rates at extremely low
pH levels, most likely via tissues and other organic
layers acting as a barrier to the surrounding seawater
and thus reducing dissolution. Furthermore, even
where calcification and growth rates are decreased, cal-
cifying coral cover can remain high since some species
are still able to survive in high CO2environments (e.g.
Andersson
et al.,2011).
Correspondence: David J. Suggett, tel. + 44 0 1206 872 552,
fax + 44 0 1206 872 592, e-mail: dsuggett@essex.ac.uk
© 2012 Blackwell Publishing Ltd
1
Global Change Biology (2012), doi: 10.1111/j.1365-2486.2012.02767.x

Page 2

Porites lutea, Fabricius et al., 2011; see also Crook et al.,
2012). Even more intriguing is the fact that coral pri-
mary productivity can often increase (or is unaffected)
while net calcification decreases/dissolution increases
for some OA scenarios (e.g. Crawley et al., 2010); conse-
quently, anthozoans can potentially remain completely
viable, but as alternative non-calcifying soft-bodied
forms under extremely high CO2/low pH (Fine &
Tchernov, 2007; see also Krief et al., 2010). However,
how such OA-driven phenotypic responses alter the
functional role of soft-bodied forms of anthozoans, or
even convey longer term ecological success, is presently
unknown. Such limited knowledge still reflects the fact
that we do not fully understand how present day non-
calcifying anthozoans, will respond to OA. Non-calcifying
anthozoans such as soft corals and anemones, play
important ecological and biogeochemical roles in reef
environments (e.g. Fitt et al., 1982; Bak & Borsboom,
1984; Muller-Parker & Davy, 2001), but much less is
known how these organisms will respond to OA
(Doherty, 2009; Towanda & Thuesen, 2012) given a
present bias in OA research towards calcifying organ-
isms (Connell & Russell, 2010).
As with reef-forming scleractinian corals, many
anemones (Class: Anthozoa, Subclass: Hexacorallia)
harbour symbiotic algae (Symbiodinium spp.) to supple-
ment their nutritional requirements. However, in
contrast to calcifying corals, anemones are fast growing
and easier to manipulate via the lack of tissue adhesion
to an underlying skeleton; thus anemones are poten-
tially model organisms for understanding the nature
with which environmental change, including OA, may
impact reef-forming cnidarians (e.g. Muller-Parker &
Davy, 2001; Weis et al., 2008). Here we present the find-
ings of the first in situ assessment of the effects of OA
on anemones whereby elevated CO2can enhance pro-
ductivity, and in turn growth, and community domi-
nance of anemones within a natural benthic ecosystem.
A major limitation for almost all OA studies to date
has been replicating the rate (decades) and biological
scale (ecosystems) at which OA operates. However,
natural CO2vents at coastal sub-tropical rocky shores
(Hall-Spencer et al., 2008; Kroeker et al., 2011; Meron
et al., 2012) and tropical coral reefs (Fabricius et al.,
2011) provide unique experimental settings to evaluate
relatively long term changes in pCO2(pH) across many
biological and spatial scales. The few studies conducted
to date at CO2 vent sites have generally revealed a
decrease in calcifying invertebrate and macroalgal
abundance and increased contribution of non-calcifying
macroalgae and/or seagrass to overall benthic cover
(Hall-Spencer
et al.,2008;
suchchangesagaincorrespond
CO2(lower pH) induced reduction of calcification and/
Fabricius
et al.,
the
2011);
elevatedto
or
invertebrates (including gastropods and hermatypic
corals; Hall-Spencer et al., 2008; Rodolfo-Metalpa et al.,
2011).
We present novel observations across a natural
coastal CO2gradient from a shallow cold vent system
(Vulcano, Italy) (Johnson et al., 2011, 2012) supporting
previous observations from other vent sites that certain
invertebrates increase in abundance with increasing
pCO2/decreasing pH (Cigliano et al., 2010; Kroeker
et al., 2011). Here, anemone (Anemonia viridis) abun-
dance increased with pCO2, and dominated the inverte-
brate community at high pCO2conditions. We tested the
hypothesis that their increased dominance was driven
(bottom up) via enhanced anemone productivity. Physi-
ological analyses demonstrated an increase of gross
maximum photosynthesis (Pmax
(R) (but with Pmax
endosymbiont abundance (but unchanged diversity)
with increasing pCO2. Our observations are the first to
show that non-calcifying anthozoans can be actively
selected for under OA conditions and thus it is essential
that greater focus is given to better understand how this
previously neglected group will contribute to ecosystem
scale productivity and nutrient cycling in future oceans.
enhancedshelldissolutioninthedominant
G) and respiration rates
G> R) as well as increased dinoflagellate
Materials and methods
Sample site environment and benthic community
analyses
Data collection was conducted in the sublittoral of North Vul-
cano Island (38° 25′ N, 14° 57′ E) ca. 25 km North East of Sicily,
11th to 26th May 2011. Several vents naturally release CO2in
coastal waters here as a result of the close proximity to Vul-
cano’s active volcano (described previously, Johnson et al.,
2011, 2012). As with recent investigations from this site (John-
son et al., 2012) we selected three reference sites (R1-3) away
from the vents, and hence representative of ‘present day’
pCO2conditions, and three sites (S1-3) with increasing prox-
imity to the vents; together these six sites provided a gradient
of decreasing pH (increasing pCO2) from ca. 8.2 (365 latm) at
to 7.6 (1425 latm) (see Table 1). Full details of the carbonate
chemistry (and associated methodology) for these six sites are
given in Johnson et al. (2012). Variability in the carbonate
system also increases with proximity to the vents (Table 1), a
factor that is also discussed further in Johnson et al. (2012).
All sample sites were shallow (1–2m) and investigated
between 10:00 and 12:00 local time. Water temperature was
measured using a HOBO®logger (Tempcon, USA) throughout
the sampling period and was constant across all sites at ca.
20.6–21.4 °C (data not shown). Light attenuation (Kd[PAR],
m?1) within the upper 1–2m was measured via the light
sensor of a Diving-PAM (Pulse Amplitude Modulated) fluo-
rometer (Walz GmbH, Germany) as described previously by
Hennige et al. (2008), and also remained constant between
© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02767.x
2 D. J. SUGGETT et al.

Page 3

Table 1
Chemical and biological characteristics measured from both reference (R) and elevated CO2(S) sites at Vulcano: (i) Median (min-max) pH (NBS scale) and pCO2(latm)
taken from Johnson et al. (2012) (see source reference for full carbonate chemistry; note, however, that data are taken from throughout Sept 2009–2011 to demonstrate the long-
term intra-site variability relevant to ecological scale processes); and (ii) Mean (± standard error) of anemone pedal disc (PD) diameter, Symbiodinium cell concentration per unit
anemone tentacle, and parameters describing anemone productivity from across the study sites: PAM-fluorescence derived light intensity of saturated productivity (EK, Eq. 1),
light-limited and light-saturated electron transfer rate (a and ETRmax, Eq 2) and DTCO2derived respiration and maximum gross photosynthesis rate (R and Pmax
G); also shown is
the gross photosynthesis-to-respiration ration (Pmax
G: R). In all cases n = 5 for each site except anemone PD diameter where n = 37 (R1), 32 (R2), 30 (R3), 51 (S1), 59 (S2), 177 (S3).
Note that for Tukey HSD post-hoc ANOVA groupings: sites that are not significantly different from one another are grouped within square brackets; each set of square brackets
indicates significantly different groups of species whereas hyphens indicate overlapping groupings
Site
ANOVA
Post-hoc
grouping
R1
R2
R3
S1
S2
S3
pHNBS
8.17
(8.35–8.06)
8.18
(8.29–8.08)
8.18
(8.29–8.10)
8.08
(8.22–7.76)
7.71
(8.10–7.07)
7.66
(8.24–6.80)
pCO2(latm)
388
(241–513)
365
(274–471)
364
(272–446)
510
(355–1119)
1244
(474–5628)
1428
(337–10730)
Anemone PD
diameter (mm)
21.41
(1.12)
22.22
(1.24)
22.81
(1.03)
26.56
(1.23)
30.51
(1.36)
28.81
(0.78)
F5,380= 11.09
[R1–R2–R3]
–[S1]–[S2
–S3]
Symbiodinium
concentration
(cells 9 105cm?2)
3.76
(0.206)
2.92
(0.281)
3.92
(0.432)
8.25
(0.922)
13.05
(2.298)
15.28
(3.459)
F5,24= 22.26
[R1–R2–R3],
[S1]–[S2–S3]
EK(lmol photons
m?2s?1)
466.1
(21.81)
481.6
(18.17)
472.0
(17.64)
458.4
(23.16)
469.3
(16.53)
474.7
(23.73)
F5,24= 1.59
NS
a (mol electrons
[mol photons]?1)
0.481
(0.008)
0.441
(0.029)
0.499
(0.011)
0.601
(0.014)
0.640
(0.006)
0.706
(0.025)
F5,24= 33.71
[R1–R2–R3]
–[S1–S2]
–[S3]
ETRmax(lmol
electrons m?2s?1)
226.9
(16.30)
212.9
(15.12)
248.0
(11.01)
272.5
(16.73)
302.1
(8.24)
319.2
(13.27)
F5,24= 28.22
[R1–R2]
–[R3]–[S1]
–[S2–S3]
Pmax
G
(lmol CO2g?1h?1)
0.239
(0.013)
0.236
(0.014)
0.240
(0.016)
0.269
(0.014)
0.311
(0.012)
0.423
(0.020)
F5,24= 25.09
[R1–R2–R3]
–[S1]–[S2],
[S3]
R (lmol CO2g?1h?1)
0.216
(0.011)
0.217
(0.008)
0.219
(0.015)
0.236
(0.010)
0.248
(0.013)
0.326
(0.019)
F5,24= 10.59
[R1–R2–R3]
–[S1–S2],
[S3]
Pmax
G: R (mol CO2
[mol CO2]?1)
1.102
(0.027)
1.082
(0.029)
1.098
(0.034)
1.192
(0.023)
1.254
(0.020)
1.301
(0.014)
F5,24= 11.64
[R1–R2–R3],
[S1–S2],
[S3]
© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02767.x
CO2ENHANCES ANEMONE ECOLOGICAL SUCCESS
3

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sites, at 0.27–0.33 m?1. Therefore, temperature and light avail-
ability were not further considered as environmental factors
significantly contributing to any ecological/physiological dif-
ferences between sites.
Benthic assemblages for each of the six sites were assessed
using a visual census method ca. 0.0–0.3m below low water. A
quadrat measuring 0.25 m2was randomly placed for quantify-
ing sea urchin (Paracentrotus lividus, Arbacia lixula) and anem-
one (A. viridis and Actinia equina) abundance; a total of 20
separate counts (per 0.25 m2) were made at each site. Continu-
ous-line intercept transects (n = 3 per site) of 20 m were fur-
ther used for general characterization of the relative (%) cover
of the major benthic groups (canopy forming macroalgae, e.g.,
Sargassum vulgare, Cystoseira spp., seagrass, sessile inverte-
brates etc.). Pedal disc size of all anemones located up to
0.5 m either side of the transect (to yield a 20 m2belt transect)
were subsequently measured with vernier callipers (precision,
0.1 mm); dimensions reported here are the mean length and
width.
Anemone productivity
Anemones (n = 5) were randomly collected from each site and
returned to the laboratory for subsequent examination of pro-
ductivity via two complementary approaches; specimens from
each site were examined on consecutive days: Active chloro-
phyll a fluorescence rapid light curves (RLCs) initially pro-
vided estimates of photosynthetic activity (as the electron
transfer rate, ETR) whilst TCO2-drift measurements in the
light and dark subsequently provided corresponding rates of
primary production and respiration. All anemones were ini-
tially blotted dry and wet-weighed (Technico-PW01 balance,
± 0.01g) and then maintained in aquaria with seawater filled
from the corresponding sample site. Specimens were returned
to the original site of collection to minimize the length of time
ex situ post examination.
Rapid light curves, RLCs were collected using a diving-
PAM programmed according to settings described previously
(Hennige et al., 2008; Suggett et al., 2012) to deliver nine light
steps of 20 s duration with increasing intensity from 0 to ca.
3000 lmol photons m?2s?1. Optimum instrument sensitivity
(gain) and induction intensity (saturation width and intensity)
were verified prior to each RLC and all anemones were dark
acclimated for ca. 1–2 h prior to examination. Each light step
yielded a measure of the minimum and maximum fluores-
cence yield (F′ and Fm′, respectively, instrument units) from
which the photochemical efficiency ([Fm′-F′]/Fm′ = Fq′/Fm′,
dimensionless) could then be determined. An equation
describing the light-dependency of the photochemical effi-
ciency (Hennige et al., 2008) was then fit using least squares
non-linear regression to the RLC measures of Fq′/Fm′,
F0
q=F0
m¼
F0
q=F0ðmaxÞ
m
EK
??
1 ? exp ?E=EK
ðÞ
??
hi
=E
ð1Þ
where E is the light intensity for each light step (lmol pho-
tons m?2s?1), Ekis the saturation light intensity (lmol pho-
tons m?2s?1) and
Fq′/Fm′(max)
photochemical efficiency (dimensionless). Mean values for Ek
isthemaximumPSII
(Table 1) and Fq′/Fm′(max)(data not shown) were not different
between sites suggesting a uniform extent of photoacclimation
(e.g. Suggett et al., 2009, 2012), and thus confirming that differ-
ences in light availability across the sites/sampling period
were negligible.
Photosynthetic electron transport rate (ETR, lmol elec-
trons m?2s?1) was subsequently determined for each light
step from the product of E, Fq′/Fm′ and a constant factor of 0.5
(Hennige et al., 2008); this factor accounts for assumptions that
only 50% of all absorbed photons are utilized by PSII and that
the quantum yield electron transfer of a trapped photon
within a reaction centre is 1 mol electron [mol photon]?1(see
Suggett et al., 2011, 2012). In this case ETRs do not include
quantitative information as to the extent of light absorbed and
are therefore relative. ETR values from each RLC were fit to a
modified equation describing the light-dependency of photo-
synthesis (see Hennige et al., 2008) using non-linear least
squares regression,
ETR ¼ ETRmax1 ? exp ?aE=ETRmax
ðÞðÞð2Þ
where a (mol electrons [mol photons]
(lmol electrons m?2s?1) describe the light-limited and light-
saturated electron transfer rate respectively.
Anemones weresubsequently
5 9 100 mL glass Duran bottles filled with seawater from the
corresponding site for TCO2drift determinations. Two addi-
tional bottles were filled with seawater alone to provide a
simultaneous control of any TCO2drift induced by activity
other than the anemones since the seawater was unfiltered.
Lids for the bottles were pre drilled to fit the pH and tempera-
ture probes (Hydrocheck CD7000; WPA Ltd. Cambridge, UK;
pH ± 0.01 and °C ± 0.1) and set to log every 15 min over a
90 min incubation period; the pH sensor was calibrated daily
using pre-made NISTbuffer solutions (pH 4.0 and 7.0, Hanna
Instruments, Leighton Buzzard, UK) to yield pH total scale
(pHT) measurements. All bottles were placed in a makeshift
water bath where water was changed every 15 min and mixed
with additional refrigerated water (4 ºC) to maintain the
temperature within 1–2ºC of that in situ at the time of sampling
(~21 °C). All vessels were incubated outdoors (under natural
light), and covered with neutral density filter to provide
intensities similar to those in situ (ca. 600–1000 lmol pho-
tons m?2s?1at noon local time), to determine maximum net
photosynthesis rates (e.g. Anthony et al., 2008). Incubations
were performed between 12:00 and 13:30 (local time), and sub-
sequently repeated in darkness after sunset (20:00–21:30, local
time) to determine corresponding respiration rates.
Measurements of temperature, salinity, pHT along with
those for total alkalinity (TA) for each site (see Johnson et al.,
2012), were used to calculate the total concentration of inor-
ganic carbon (= TCO2, lmol CO2L?1, as the sum of free CO2,
HCO3?and CO32?) via CO2SYS software (Lewis & Wallace,
1998) using the constants of Roy et al. (1993) and Dickson
(1990) for KSO4. The concentration of TCO2was determined at
the start and end of each incubation and we assumed that TA
did not significantly vary during the drifts since the dominant
biology, the anemones, are non-calcifying. Rates of maximum
?1) and ETRmax
transferredtooneof
© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02767.x
4 D. J. SUGGETT et al.

Page 5

net
lmol CO2g?1h?1) were subsequently calculated as,
photosynthesis andrespiration(Pmax
G
and R,
Pmax
N ðRÞ ¼
dTCO2ðsampleÞ ? dTCO2ðcontrolÞðÞm
½?= dTw
ðÞð3Þ
where v and w are the water volume (L) and anemone wet
weight (g) while DT and DTCO2are the difference of time (h)
and TCO2(lmol CO2L?1) at the start and end of incubation;
the mean of both DTCO2(control) values was subtracted from
each DTCO2(sample). Respiration rates were multiplied by a
factor of ?1 to yield positive values. Maximum gross photo-
synthesis rates (Pmax
lated as Pmax
N
+ R.
G, lmol CO2g?1h?1) were finally calcu-
G, = Pmax
Microalgal symbiont characteristics
Cellular density and the genetic characterization by ITS-2
(internal transcribed spacer region-2) of the anemone’s mic-
roalgal endosymbiont (Symbidinium spp.) were determined
to support interpretation of any potential changes in anem-
one productivity. Small tentacle samples were randomly
removed from each anemone used to determine productiv-
ity. Tentacle surface area (SAT, cm?2) of the sample was
measured again using vernier callipers prior to storage in
glutaraldehyde (1%).Each
ground in hand-held, glass tissue-homogenizers in water
(0.6 mL = VT) and an aliquot transferred to a haemocytom-
eter for cell counts (eight counts per sample) using light
microscopy. Symbiodinium density (cells cm?2) was thus
determined as,
tentaclesamplewas later
Cells cm?2¼ cells mL?1? ðVT=SATÞ
4
Nucleic acid extractions were conducted using a modi-
fied Promega Wizard genomic DNA extraction protocol (fol-
lowing LaJeunesse et al., 2003). Symbiont
characterized by denaturing gradient gel electrophoresis
(DGGE) fingerprinting of the partial 5.8S and internal tran-
scribed spacer (ITS) region 2 (LaJeunesse, 2002); this region
was amplified using a touch-down thermal cycle profile
with the primers ‘ITS2clamp’ and ‘ITSintfor2’ (LaJeunesse
& Trench, 2000), and the PCR products resolved on dena-
turing gels (45–80% of 7M urea & 40% formamide) using
a CBScientific system (Del Mar, CA, USA) for 16 h at 115
volts.Thedominantband
excised, re-amplified and cycle sequenced to provide the
ITS2 sequence that dominated the symbiont’s genome.
identity was
of theDGGE profilewas
Results
Benthic community structure and anemone distribution
Total macroalgal cover (canopy forming macroalgae, as
a % of total benthic cover) during the anemone and
urchin surveys wasgenerally
elevated CO2sites, ca. 40–60%, than at the reference
higher forthe
sites, ca. 20–30% (Fig. 1). However, cover at the site
with the highest pCO2(S3) was not significantly differ-
ent from that at R3 (see Fig. 1 legend for ANOVA results).
Increased macroalgal cover was accompanied by a
change in dominance from both calcifying and non-
calcifying species at the reference sites (not shown) to
fleshy macroalgal cover at the high CO2sites (phaeo-
phytes, Johnson et al., 2012).
Reciprocal changes were observed between the sea
urchin (Arbacia lixula and P. lividus) and anemone
(A. viridis) population abundances for the reference vs.
high CO2sites. Both sea urchin species exhibited con-
sistent abundances, ca. 1–2 m?2(P. lividus) and 4–
6 m?2(A. lixula), across the reference sites but were
completely absent from any of the higher CO2sites (see
also Johnson et al., 2012). In contrast, A. viridis abun-
dance remained consistent (of ca. 10 m?2) across the
reference sites but significantly increased to ca. 20 and
40 m?2at sites S1-S2 and S3 respectively (see Fig. 1). In
addition to abundance, A. viridis size (pedal disc)
increased from ca. 21–23 mm at the reference sites to
Fig. 1 Mean (± standard error) of (i) Canopy forming macroal-
gal cover (% of total benthic cover) and (ii) Sea urchin (Arbacia
lixula and Paracentrotus lividus) and anemone (Anemonia viridis)
abundance (no. per m2) from the reference (R) and elevated CO2
(S) sites at Vulcano; note that the median pCO2(Table 1) for
each site is given in parentheses below each site. Macroalgal
cover was collected from n = 6 continuous-line intercept tran-
sects per site; note that significant differences were assessed
using ANOVA after arcsin transforming the % (proportion) cover
data: F5,30= 36.88 with sites grouped via the post hoc Tukey test
(as described in the legend for Table 1) as [R1–R2]–[R3–S3]–[S1–
S2]. Urchin and anemone abundance data were collected from
20 quadrat counts per site. ANOVAs upon the untransformed
count data were not significant for the two urchin species; how-
ever, the
grouped as [R1–R2–R3]–[S1–S2], [S3].
ANOVA for A. viridis was F5,114= 23.49 with sites
© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02767.x
CO2ENHANCES ANEMONE ECOLOGICAL SUCCESS
5

Page 6

ca. 27–31 mm at the higher CO2 sites (Table 1). The
largest anemones were observed at sites S2–S3.
Anemone productivity
Light-limited (a) and light saturated ETRs (ETRmax) as
well as the maximum gross photosynthesis rate (Pmax
generally increased from the reference to the high CO2
sites (Table 1; also Fig. 2a). Values for a, ETRmaxand
Pmax
G
were ca. 0.4–0.5 mol electrons [mol photons]?1,
210–250 lmol electrons m?2s?1and 0.22 lmol CO2
g?1h?1, for the ambient pCO2 reference sites (R1-3,
Table 1); as with anemone size (above), values for these
parametersdescribingprimary
increased by ca.10–20% at S1 but were not statistically
G)
productivityall
different from those at R3. Highest values of a, ETRmax
and Pmax
G
were observed at the highest pCO2 sites
(S2–S3), ca. 0.65–0.70 mol electrons [mol photons]?1,
300–320 lmol electrons m?2s?1
CO2g?1h?1.
Both measures of primary productivity, ETRmaxand
Pmax
G, increased in proportion with pCO2across the ref-
erence (R1–R3) sites, S1 and S2 (Fig. 2a); thus the yield
of CO2assimilation from PSII photosynthetic activity
washighlyconservedas
decreased). However, the increase of Pmax
exceeded that of ETRmax(ca. 10%) between S2 and S3
indicating an increased yield of CO2assimilation from
PSII photosynthetic activity for the site with the highest
pCO2.
Respiration rates also increased with pCO2, i.e. from
ca. 0.22 lmol CO2g?1h?1(R1-3) to ca. 0.24–0.33 lmol
CO2g?1h?1(S1–S3) (Table 1); however, the increase of
R was less than that of Pmax
increased from ca. 1.1 at the reference sites to ca. 1.2–
1.3 mol: mol from S1 to S3 (Table 2) and hence with
increasing pCO2(Fig. 2b). Both increases of Pmax
Pmax
G:R suggest that greater anemone size/abundance
with pCO2may be driven, at least in part, by enhanced
metabolic activity, and notably from increased primary
productivity over respiration.
and 0.31–0.42 lmol
pCO2
increased (pH
G
(ca. 40%)
G
such that the Pmax
G: R
G
and
Microalgal symbiont characteristics
Symbiodinum populations across all sites/anemones
were identified to be the same ITS2 ‘type’ of clade A.
This symbiont, designated here as A19, was previously
isolated from the Mediterranean (Genbank accession
449046) and belongs to a lineage within clade A previ-
ously described as A′ that appears geographically
restricted, yet regionally abundant, to the temperate
locations of the Mediterranean and the north-eastern
Atlantic (Savage et al., 2002; Visram et al., 2006). Thus,
acclimation via a single Symbiodinium type regulates
the enhanced primary productivity with lower pH/
higher CO2.
Cell density (per cm?2tentacle) was constant with
tentacles containing ca. 3–4 x105cells cm?2for the refer-
ence sites R1–R3 but increased with pCO2 to 13–
15 9 105cells cm?2by S2–S3 (Table 2); cells contained
ca. 20% more dividing cells at S3 compared to sites
R1–R3 (mean ± standard
1.71 ± 0.12% for R1–R3 vs. 2.08 ± 0.18% for S3, t-test,
P < 0.05; not shown). Overall, the changes of Symbiodi-
nium cell density were linearly correlated with those of
ETRmax
(and to a lesser extent Pmax
(r2= 0.813) (see Fig. 2a) across all sites. As such, the
increased ETRmax(and Pmax
G) with pCO2/decreasing pH
was predominantly driven by increased Symbiodinium
errormitotic indexof
G), r2= 0.892
(a)
(b)
Fig. 2 Relationships of (a) the maximum rate of gross produc-
tivity (Pmax
fer rate (ETRmax, lmol electrons m?2s?1) and Symbiodinium cell
density (cell 9 105cm?2tentacle) for the reference (R) and ele-
vated CO2 (S) sites at Vulcano. Note that each point is the
mean ± standard error of all replicates (and also that data for all
of the reference sites, R1–R3 has been pooled for figure clarity);
Bartlett’s type II regression equations (and correlation coeffi-
cients) for these relationships were: ETRmax= [(Pmax
115.2 (r2= 0.763, n = 30, P < 0.001); also ETRmax= [(cells 9 105)
? 7.542] + 204.3 (r2= 0.892, n = 30, P < 0.001); Pmax
105) ? 0.191] + 16.73 (r2= 0.813, n = 30, P < 0.001); and (b) the
mean (± standard) ratio of the maximum gross photosynthesis
to respiration rate (Pmax
G:R, mol CO2fixed: mol CO2released) vs.
the median pCO2and pH (see Table 1) across all sites.
G, lmol CO2g?1h?1) vs. the maximum electron trans-
G? 518.1] +
G
= [(cells 9
© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02767.x
6 D. J. SUGGETT et al.

Page 7

cell density as opposed to increased Symbiodinium pro-
ductivity per cell.
Discussion
Anemones in present day benthic systems promote bio-
diversity by supporting mutualisms and numerous pre-
dators (e.g. Holbrook & Schmitt, 2005), reducing
macro- and filamentous-algal recruitment (Taylor &
Littler, 1982) and overgrowth (Bak & Borsboom, 1984),
and maintain primary productivity rates that can be as
high as that of algae (Fitt et al., 1982). Thus, the
enhanced metabolic activity (productivity) and, in turn
growth (size and abundance), of anemones with
increasing pCO2/reduced pH suggest that OA could
alter the role of anthozoans in benthic communities
with fundamental implications to ecosystem function.
At Vulcano, concomitant increases in anemone abun-
dance with pCO2corresponded with enhanced PG, i.e. a
response comparable to that of the macroalgae at this
site (Johnson et al., 2012). Although the increases in
anemone abundance were perhaps not as pronounced
as for the microalgae, the increase in PG(also>R) would
suggest that under OA, anemones could contribute sig-
nificantly to driving the ecosystem metabolism towards
greater net autotrophy and CO2sequestration. Such a
biogeochemical response is potentially important in off-
setting OA impacts to reef environments (Anthony
et al., 2011), but this comes at a cost to the diversity of
species and function.
Seawater acidification has been shown to reduce eco-
logical diversity (e.g. Hall-Spencer et al., 2008; Fabricius
et al., 2011), in particular where enhanced dissolution
impedes growth of calcifying invertebrates (Rodolfo-
Metalpa et al., 2011) and therefore limits their ability to
keep the biomass of algae, or indeed other space com-
petitors, in check. As non-calcifiers that supplement
heterotrophy with autotrophy (although our higher val-
ues of PGover R would suggest autotrophy dominates),
anemones are already at an advantage over other inver-
tebrates to lower pH conditions. Calcifying inverte-
brates that can compete under lower pH conditions
inevitably exhibit reduced growth rates (Fabricius et al.,
2011) as more energy is likely redirected to maintain
calcification (e.g. Krief et al., 2010); however, in the case
of the anemones their success via elevated productivity
may be further exacerbated by their capacity to com-
pete with algae for space via chemical deterrents (Bak
& Borsboom, 1984). Indeed, a decrease in macroalgal
cover was observed at the highest CO2site (S3), com-
pared to S1 and S2, where the corresponding anemone
abundance was almost a factor of 2 greater at S3 than at
S1–S2. Anthozoans are well known for their aggressive
control of space, perhaps giving soft-bodied anthozoan
forms a competitive edge should they persist under
OA; such enhanced fitness could indeed be crucial
where elevated CO2already enhances algal competition
(Diaz-Pulido et al., 2011; Johnson et al., 2012).
Of course understanding the driving mechanisms
behind enhanced competition under OA also requires
knowledge of whether or not anemone predation is also
affected. Anemones are predated by a huge diversity of
organisms, including crustaceans (decapods), molluscs
(gastrapods), echinoderms (starfish) (e.g. Ottaway,
1977), which are all likely impacted at some stage of
their life history by ocean acidification (e.g. Andersson
et al., 2011). Unfortunately, little is known as to the
predator-prey dynamics of Mediterranean A. viridis
populations and thus we cannot presently address
this issue; instead we focus on the enhanced PG,
which clearly provides evidence for some fundamental
role of OA in enhancing anemone success from the
bottom up.
Regulation of anemone productivity by environmen-
tal factors such as light availability (e.g. Bythell et al.,
1997; Muller-Parker & Davy, 2001) is reasonably well
understood; however, the role of CO2(pH) has been
largely neglected. Some OA-focussed lab experiments
have been recently performed on temperate anemones
of the temperate genus Anthopleura, but with contrast-
ing results between studies/species: An increase of PG
and R (but PG>R) as well as more abundant (and larger)
Symbiodinium cells with decreasing pH were evident for
Anthopleura elegantissima (Towanda & Thuesen, 2012),
i.e. results consistent with our observations at Vulcano
for A. viridis. However, only small increases of Symbi-
odinium cell density and an increase of R exceeding that
of PGwere observed at lower pH for A. aureoradiata
(Doherty, 2009). Unfortunately, neither experiment was
performed for long enough to determine whether either
of these OA responses was conveyed into longer term
differences in anemone fitness, such as growth and
reproduction.
The underlying reason for these contrasting experi-
mental results is not clear, and may simply reflect spe-
cies-specific differences, but could equally reflect
differences in conditions other than CO2availability;
for example, although both species were provided with
similar feeding regimes (every 4–5 days), A. elegantiss-
ima and A. aureoradiata were maintained under temper-
ature and light conditions of 12ºC and 660 lmol
photons m?2s?1(14 : 10 L : D) and 16ºC and 275 lmol
photons m?2s?1(12 : 12 L : D) respectively. Both light
and temperature moderate the extent of CO2uptake
(and hence the likely responses observed to OA, e.g.
Rodolfo-Metalpa et al., 2011) but it is not possible to
determine which may be contributing to the contrasting
responses from the data currently available.
© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02767.x
CO2ENHANCES ANEMONE ECOLOGICAL SUCCESS
7

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Our observations from Vulcano and those of Towan-
da & Thuesen (2012) and Doherty (2009) would suggest
that Symbiodinium productivity in anemones is limited
by inorganic carbon (iC) availability. As with other
symbiotic cnidarians, Symbiodinium in anemones are
separated from the external inorganic carbon supply by
several membranes and thus limited by the supply of
iC (e.g. Benazet-Tambutte et al., 1996; Muscatine et al.,
1998; Davy & Cook, 2001; see also Brownlee, 2009). To
overcome iC limitation the host cnidarian employs
external (likely membrane-bound) carbonic anhydrase
to convert HCO3- to CO2and enhance the iC supply
from seawater to the Symbiodinium cells (e.g. Ganot
et al. (2011) for A. viridis; see also Weis (1993), Weis &
Reynolds, 1999). Increased iC availability promotes
autotrophy (PG), which in turn would increase the met-
abolic exchange with the host (Brownlee, 2009) and
host respiration (Harland & Davies, 1995). Such a
response would be consistent with the greater OA-
induced increase of PGover R; that said, changes of PG
may not fully account for those of R, in particular
where PGdoes not fully meet the metabolic demands of
the host (Davy et al., 1996). Although previous observa-
tions from the anemone A. elegantissima (Towanda &
Thuesen, 2012) suggest that the host receives more of
their respiratory carbon from PGunder OA, we cannot
presently discount that differences in food supply (par-
ticulate organic carbon, POC) may also exist between
reference and elevated sites to explain the differences
of R, and thus account for the relative role of iC vs.
POC availability upon elevated growth.
Importantly the increased PG with pCO2 observed
here corresponded with enhanced Symbiodinium cell
density whereas ETRmaxand Pmax
tively constant. Symbiodinium can invest into enhanced
growth (over cell-specific productivity) under OA
(Brading et al., 2011) but such a response does not seem
consistent with previous OA observations from calcify-
ing anthozoans (e.g. Crawley et al., 2010; Krief et al.,
2010; Meron et al., 2012) (see below). However, Symbi-
odinium cells are often nutrient-limited within (anem-
one) hosts, and the host environment likely setting the
upper limit on the rate of Symbiodinium cell-cycle pro-
gression (Smith & Muscatine, 1999; see also Muller-
Parker & Davy, 2001). Thus, increases in cell density
can occur where densities are simply not optimum (at
‘steady state’ levels) and environmental conditions
become more favourable for growth (Jones & Yellow-
less, 1997; Muscatine et al., 1998); as such, Symbiodinium
cell densities at the lowest CO2sites could be lower
than required for optimum growth. It is important to
also note that our approach for normalizing Symbiodinium
cell density to tentacle SA may influence how changes
of pCO2drive those of cell density. Specifically, tissue
G
cell?1remained rela-
protein content per unit area appears to increase with
pCO2 for both anemones (Towanda & Thuesen, 2012)
and scleractinian corals (Meron et al., 2012). Therefore,
our observations of increased cells cm?2with pCO2may
reflect the capacity of increased tissue content (per cm?2
tentacle)to simply harbour
Symbiodinium cell densities ultimately are increased with
pCO2.
An increase of PGwith CO2, i.e. ‘CO2enrichment’, is
somewhat analogous to that expected from addition of
other inorganic nutrients essential for Symbiodinium
growth (Weis 1933); for example, NH4+additions have
been repeatedly shown to increase Symbiodinium cell
number and productivity by anemones (e.g. Cook et al.,
1988; Muscatine et al., 1998, Smith & Muscatine, 1999)
suggesting that mutualistic associations occur between
anemones and other organisms that excrete NH4+, e.g.
anemone fish in tropical systems, provide nutritional
benefits that can promote anemone growth (e.g.
Holbrook & Schmitt, 2005). In fact under extreme nutri-
ent addition events, e.g. eutrophication of tropical reef
systems, phase shifts dominated by enhanced anemone
abundance can occur (Tkachenko et al., 2007). Such
responses raise a critical point here:
Stimulation of productivity by inorganic carbon (OA)
would require that nutrients such as nitrate and phos-
phate are available to build additional organic skele-
tons and thus ‘fuel’ the benefits of CO2availability into
growth; indeed, reports do exist of high NH4+excretion
rates by temperate anemones (Jensen & Muller-Parker,
1994), suggesting higher intrinsic nutrient availability/
storage inherent to temperate cnidaria (see also Davy
et al., 2006) could support elevated PGunder OA, but
such reports are rare. Stimulated growth of anemones
can occur under increased nutrient availability without
reductions of seawater pH (Tkachenko et al., 2007). In
this case the actual benefit to the host, in terms of car-
bon translocated (and hence R), may not increase unless
more CO2is available (Davy & Cook, 2001). Our data
does provide some evidence that nutrient availability
may be enhanced at the lowest pH site since ETRmax
becomes more closely coupled with Pmax
such a response is typical of stress relief, where elec-
trons would otherwise be funnelled into alternative
acceptors and/or cyclic flow (e.g. Suggett et al., 2011).
So are the OA responses we observe at Vulcano
potentially influenced by nutrient availability?
At present we cannot determine whether changes in
particulate organic nutrient supply occur along the CO2
gradient; however, some data were collected for dis-
solved inorganic nutrients (nitrate plus nitrite, DIN, as
well as phosphate, DIP) but not NH4+(L. Al-Moosawi,
Personal communication) from surface samples during
May 2011: DIP concentrations remained undetectable
more cells. Evenso
G
(see Fig. 2);
© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02767.x
8 D. J. SUGGETT et al.

Page 9

(<10 nM) across the pH gradient. DIN concentrations
also remained low (ca. 0.2 lM) for waters with a pH of
8.2–8.0, but did increase from ca. 0.2–0.8 lM as pH
decreased from 7.6 to 7.2. Thus, it is possible that DIN
concentrations are elevated periodically for S3 and, to a
lesser extent, S2 (as per the pH range for these sites,
Table 1). Even so, it is important to point out that (i)
these elevated DIN concentrations at the lower pH are
still somewhat lowto
enhanced productivity here, (ii) a CO2response is still
observed for S1 where DIN concentrations are the same
as for the reference sites, and (iii) the greater increase of
Pmax
G
over ETRmax, potentially indicative of stress relief
(possibly from DIN limitation) is only observed for S3.
Thus, data would suggest that the primary response is
to pCO2but could be moderated by periodically ele-
vated nitrogen availability at the high(est) CO2sites;
further verification is required and with a more detailed
understanding of pCO2and DIN (co)-variability. The
previous laboratory studies examining the response of
anemones to OA (Doherty, 2009; Towanda & Thuesen,
2012) do not report DIN (DIP) and therefore it is unclear
whether or not nutrient availability may also in part
explain their somewhat contrasting observations.
Ocean acidification-induced stimulation of PGfrom
anemones would be generally consistent with observa-
tions from other cnidarians. Experiments simulating
OA have frequently demonstrated that hermatypic (cal-
cifying) corals exhibit increased primary productivity
rates (PNand/or PG) with decreasing pH (e.g. Langdon
& Atkinson, 2005; Crawley et al., 2010; Krief et al., 2010;
Rodolfo-Metalpa et al., 2010). However, these increases
may often be relatively small, ca. 10–20% increases with
a twofold increase in pCO2 (Rodolfo-Metalpa et al.,
2010) and/or, restricted to only moderate changes in
pCO2(Crawley et al., 2010); indeed some experiments
have shown that PGand/or PNis either unchanged or
can even decrease with increasing pCO2(e.g. Schneider
& Erez, 2006). Such diversity of responses is perhaps
not surprising since other factors likely regulating pro-
ductivity, e.g. light availability (see Langdon & Atkin-
son, 2005) and the role of calcification in modifying DIC
availability (Schneider & Erez, 2006; Krief et al., 2010),
are again not well accounted for between experiments.
Furthermore, reconciling these contrasting responses is
confounded by a lack of knowledge of Symbiodinium
genetic identity, which is almost never reported from
coral OA studies but can fundamentally determine
whether or not PGis likely to respond to OA (Brading
et al., 2011). Indeed, differences between Symbiodini-
um spp. may also explain why the OA response for
anemones, i.e. enhanced PGvia increased cell density
over productivity cell?1, appears to differ from that
previously observed for corals.
sustainthesubstantially
At Vulcano, enhanced PGobserved for the anemones
was driven by increased growth (but not PGcell?1) of a
specific ITS2 type (A19) with CO2. Such a response of
increased growth with pCO2 has been observed for
another clade A-type, isolated from a tropical anemone
(A13, Condylactis gigantea) (Brading et al., 2011); this
same study also demonstrated enhanced PGcell?1for
an A-type originally isolated from the scleractinian
coral Montastrea faveolata, further suggesting that some
clade A Symbiodinium may be particularly affected by
OA. Thus, OA enhancement of productivity for anemo-
nes (and other non-calcifying cnidarians) may be
expected where Symbiodinium within clade A domi-
nates, such as in Mediterranean and/or European
waters (Savage et al., 2002; Visram et al., 2006). Conser-
vation of a single Symbiodinium type across natural
pCO2gradients is consistent with recent reports from
scleractinian corals (Balanophyllia europaea and Cladocora
caespitosa) at another Mediterranean vent site (Ischia,
Meron et al., 2012); although whether or not this single
type also exhibited enhanced PGwith pCO2at Ischia is
presently not known. The experiments by Fine & Tcher-
nov (2007) showing viable cnidarian growth under
extremely low pH have only been demonstrated on
Mediterranean corals to date.
Whetheror notstrong
responses of anthozoan productivity go beyond har-
bouring A-types in the Mediterranean is currently
unknown; however, OA-induced stimulation of anem-
one productivity occurs with Symbiodinium from clade
B as well (A. elegantissima, Towanda & Thuesen, 2012).
Even so, the strong OA response observed with Symbi-
odinium A19 at Vulcano could imply that OA enhanced
productivity would perhaps not be as prominent in
tropical reefs where anemones and other cnidarians,
such as hard and soft corals, harbour genetically differ-
ent symbionts within clade A in addition to those from
other clades (e.g. Finney et al., 2010; LaJeunesse et al.,
2010). Strong geographical and temporal delineations
of Symbiodinium diversity do exist for anemone popula-
tions (Venn et al., 2009; Sanders & Palumbi, 2011).
Thus, further understanding how OA responses in
cnidarians are moderated by symbiont identity will be
key, in particular where specific phylotypes may fur-
ther determine how cnidarians can respond to addi-
tional stressors, such as anomalous temperatures.
Overall, our data show that productivity and in turn
growth of anthozoans can be substantially up-regulated
by OA-like conditions; these factors appear to contrib-
ute, at least in part, to a bottom-up enhancement of
anemone abundance in Vulcano’s benthic community.
Similar OA-induced enhancement of (non-calcifying)
cnidarian abundance from other present day CO2vent
sites has not been previously observed (Hall-Spencer
Symbiodinium
driven
© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02767.x
CO2ENHANCES ANEMONE ECOLOGICAL SUCCESS
9

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et al., 2008; Fabricius et al., 2011) but may represent a
complex mix of factors regulating the responses; nota-
bly (i)ecological interactions
enhanced PG results in increased net growth/abun-
dance (e.g. enhanced predation), (ii) interactive influ-
ences of other environmental conditions and (iii)
Symbiodinium identity. How the responses we currently
observe at Vulcano translate to broader (ecosystem)-
level responses by non-calcifying cnidarians under OA
cannot currently be determined but is an obvious prior-
ity: Non-calcifying cnidarians, such as soft corals and
anemones, can play key ecological and biogeochemical
roles and often (re)populate reefs that have been
impacted by local stressors (Tkachenko et al., 2007).
Consequently, understanding how this group of organ-
isms responds to longer term climatic change, and the
combination of factor(s) that drives ‘success’ alongside
elevated pCO2, will be an essential component of
confidently predicting the future form and function of
benthic ecosystems.
modifying whether
Acknowledgements
We wish to thank Marco Milazzo (University of Palermo) for
essential academic and logistical support throughout. We are
indebted to Simon Davy for critically reviewing and improving
the final version of the manuscript. This work contributes to the
EU FP7 project on ‘Mediterranean Sea Acidification under a
changing climate’ (MedSeA grant agreement no. 265103) and to
the UK Ocean Acidification Research Programme (NERC); also
we gratefully acknowledge additional funding support by
NERC (grant NE/G020116/1 to DJS and TL), Save Our Seas
Foundation (JHS), The National Science Foundation (grant
1040940 to MEW) and The Earls Colne and Halstead Educational
Charity (TGB).
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