Marine Biology (1997) 130: 1±10
Ó Springer-Verlag 1997
B. S. T. Helmuth á B. E. H. Timmerman á K. P. Sebens
Interplay of host morphology and symbiont microhabitat
in coral aggregations
Received: 12 June 1997 / Accepted: 24 June 1997
Abstract The Belizean reef coral Agaricia tenuifolia
Dana forms aggregations in which rows of thin, upright
blades line up behind each other. On average, the
spacing between blades increases with depth and hence
with decreasing ambient irradiance. We designed and
built a small, inexpensive light meter and used it to
quantify the e€ect of branch spacing on light levels
within colonies at varying distances from branch tips.
Concurrently, we measured photosynthetic pigment
concentrations and population densities of symbiotic
dino¯agellates (zooxanthellae) extracted from coral
branches of colonies with tight (£3 cm) vs wide (³6 cm)
branch spacing, collected at 15 to 17 m and from colonies with tight branch spacing collected at 1 to 2 m.
Light levels decreased signi®cantly with tighter branch
spacing and with distance from the branch tips. Total
cellular pigment concentrations (chlorophylls a, c2 and
peridinin) as well as chlorophyll a:c2 and chlorophyll a:
peridinin ratios all increased signi®cantly with distance
from the branch tip, indicating very localized di€erences
in photoacclimation within individual branches. Zooxanthellae from colonies with widely-spaced branches
displayed signi®cantly lower chlorophyll a:c2 and chlorophyll a:peridinin ratios, and were present at signi®cantly higher population densities than those from colonies with tightly-spaced branches collected at the same
depth (15 m). Tightly-spaced colonies collected from
shallow environments (1 to 2 m) displayed pigment ratios similar to those from widely-spaced colonies from
deeper water (15 m), but maintained zooxanthellae
populations at levels similar to those in tightly-branched
colonies from deeper water. Thus, variation in colony
morphology (branch spacing and distance from branch
tip) can a€ect symbiont physiology in a manner comparable to an increase of over 15 m of water depth.
These results show that a host's morphology can
strongly determine the microhabitat of its symbionts
over very small spatial scales, and that zooxanthellae can
in turn display steep gradients in concordance with these
altered physical conditions.
Introduction
Communicated by M.F. Strathmann, Friday Harbor
B.S.T. Helmuth (&)1
University of Washington, Department of Zoology,
Box 351800, Seattle, Washington 98195-1800, USA
B.E.H. Timmerman
University of Washington, Department of Botany,
Box 355325, Seattle, Washington 98195-5325, USA
K.P. Sebens
University of Maryland, Department of Zoology,
College Park, Maryland 20742, USA, and
Horn Point Environmental Laboratory,
University of Maryland Center for Environmental
and Estuarine Studies, Cambridge,
Maryland 21613, USA
Present address:
1
Hopkins Marine Station, Stanford University,
Department of Biological Sciences, Paci®c Grove,
California 93950-3094, USA
Hermatypic scleractinian corals and their associated
photosynthetic dino¯agellates (commonly termed ``zooxanthellae'') are among the best known examples of
symbiosis in the marine environment. The role of zooxanthellae in determining the distribution and productivity of corals has received considerable attention,
especially in response to changes in light (e.g. Jokiel
1989). For example, Dustan (1979) demonstrated that
corals living in deeper, lower-light environments maintained populations of zooxanthellae which were better
suited to shade conditions, but that when these corals
were transplanted to shallower water, their symbionts
showed some degree of plasticity in response to the altered light regime. Traditionally, the responses of zooxanthellae and their photosynthetic pigments to changes
in irradiance have been lumped under the term ``photo-
2
adaptation,'' which encompasses time scales of minutes
to years (reviewed in Falkowski et al. 1990). However,
with heightened awareness of the importance of genetic
and taxonomic variability among zooxanthellae inhabiting corals (e.g. Iglesias-Prieto and Trench 1994; Rowan
and Knowlton 1995), it is perhaps more appropriate to
distinguish true photoadaptation (a genetic change in the
population structure of a coral's symbionts due to differential growth and survival) from photoacclimation (a
physiologically plastic response of a clone of zooxanthellae to changing light conditions, e.g. BjoÈrkman 1981;
Iglesias-Prieto and Trench 1994). In general, studies of
zooxanthellar photoadaptation and photoacclimation
have tended to focus on the e€ects of large-scale environmental variability such as occurs over a depth gradient (e.g. Graus and Macintyre 1976; Fricke et al. 1987;
Battey and Porter 1989), and to quantify the response of
the entire coral±zooxanthellae symbiosis as a single unit.
Recent evidence suggests that this scale of approach may
not always be appropriate. As suggested by Goulet and
Co€roth (1997) for gorgonians, a coral host may be
considered as a spatially variable habitat for symbiotic
zooxanthellae, where local light and water ¯ow potentially vary within a single colony. For example, Jokiel
and Morrissey (1986) demonstrated changes in the photosynthetic characteristics of zooxanthellae with increasing canopy development in the coral Pocillopora
damicornis, and Muller-Parker (1987) found di€erences
in pigment concentration between symbionts inhabiting
the tentacles of anemones (Aiptasia pulchella) compared
with those living in the body column. Furthermore,
Rowan and Knowlton (1995) demonstrated that populations of zooxanthellae can display taxonomic zonation
within individual coral (Montastrea spp.) colonies.
What then is the appropriate scale for examining
zooxanthella populations and to what degree are these
symbionts a€ected by the morphology and spatial position of their host? These questions are pertinent to any
symbiotic relationship in which the host modi®es the
physical environment of its symbionts. The assumption
that colony morphology signi®cantly a€ects the amount
of light received by its zooxanthellae is largely accepted,
but has seldom been quanti®ed (but see Titlyanov 1991
and Stambler 1996). Here we address the following
questions: what is the role of a coral's morphology in
determining the microhabitat of its symbionts, and to
what extent are the photosynthetic characteristics of
zooxanthellae correlated with the altered physical conditions of their local habitat?
The coral Agaricia tenuifolia Dana is a dominant
scleractinian on the barrier reef of Belize, Central
America [RuÈtzler and Macintyre 1982; Chornesky 1991
± however, evidence suggests that as recently as a decade
ago this species was not the spatial dominant on many
parts of the reef (Aronson and Precht 1997)]. A. tenuifolia grows almost exclusively in aggregations ranging in
size from a few branches (= blades) to many square
meters (Chornesky 1991; Helmuth et al. 1997a). Especially in larger aggregations, plate-like branches in the
center of colonies are often aligned in rows, one behind
the other, with the ¯at surfaces perpendicular to the
dominant direction of ¯ow. Previously, we have shown
that the spacing between these plates increases with
depth on the reef and that branch spacing appears to be
more strongly (negatively) correlated with light than
with water ¯ow (Helmuth et al. 1997a). However,
whether variability in branch spacing is genetically determined or is a plastic response to environmental conditions remains to be studied. Despite the fact that
branch spacing appeared to be insensitive to the ¯ow
regime, experimental trials indicated that tight spacing
could potentially limit the delivery of mass [i.e. gases
(O2, CO2), nutrients and ions (e.g. ammonium, bicarbonate, nitrate)] to the coral's tissues, and that regions
within individual branches farther removed from branch
tips experienced signi®cantly reduced rates of mass ¯ux
(Helmuth et al. 1997a). This latter ®nding was supported
by the observation that tight branch spacing was correlated with a signi®cant decrease in the amount of live
tissue in these regions. Because photosynthesis is driven
both by light and mass ¯ux (e.g. Dennison and Barnes
1988; Patterson et al. 1991; Patterson 1992), lowered
mass ¯ux could result in reduced rates of net photosynthesis by the zooxanthellae and of host respiration,
potentially limiting productivity and growth. In this
paper we quantify the e€ect of branch spacing on light
availability within coral aggregations, and attempt to
measure the subsequent response of zooxanthella populations living within the coral's tissues to these altered
conditions of light and mass ¯ux.
Speci®cally, the goals of this study were as follows:
(1) to quantify the degree to which the physical environment changes (spatially) within a single branch (upright plate); (2) to assess the e€ects of branch spacing on
these patterns, and (3) to determine patterns in the response of zooxanthellae populations (pigment concentration, population density) to changes in their
microhabitat, as created by their host. Many factors are
likely to a€ect population growth and pigment characteristics of zooxanthellae; our aim here is not to examine
explicitly the speci®c environmental controls of zooxanthella populations, but rather to discern the potential for
a host to create ®ne-scale partitioning in the physical
conditions (light and mass ¯ux) experienced by its
symbionts.
Materials and methods
Light levels in aggregations
To quantify the e€ect of branch spacing on self-shading in Agaricia
tenuifolia Dana, we constructed small submersible light meters with
photosensors capable of measuring small-scale di€erences in total
light (400 to 700 nm, 'PAR) within colonies and at varying distances from the branch tips (Fig. 1). In 1995 and 1996, a small
digital multimeter (Micronta Model 22-169) was used to measure
the voltage output from a silicon solar cell (2 ´ 4 cm, Radio Shack
Model 276-124) encased between two thin pieces of bu€ed Plexiglas. A small piece of nonconductive tape was placed between one
3
of the multimeter's batteries and the adjacent battery post as a
means of breaking the circuit of the power source. The post was
wired to the battery through a magnetic reed switch so that the
assembly functioned only in the presence of a magnet (Fig. 1a). The
assembly was sealed in a Plexiglas case ®lled with mineral oil.
The leads of the meter were then wired through the case to the
photocell, and the connecting wires were encased in tubing. The
total probe size was 5 ´ 7 cm, and it integrated total visible light
over an area of '8 cm2. The voltage output of the solar cell was
calibrated (in air) against a Licor 2p (¯at) pyranometer under ¯uorescent lighting [the range of wavelengths produced by this type of
light is similar to that measured by a photocell, and does not include infrared components which are normally measured by a Licor
pyranometer (e.g. Bickford and Dunn 1972)]. The relationship
between irradiance measured by the pyranometer and voltage
output conformed best to an exponential curve (R2 ' 0.99). While
the nonlinearity of this response curve limited the accuracy of the
meter at high levels of irradiance (>2000 W m)2), levels in the ®eld
were generally under this limit and as such, the volt meter provided
accurate measures of light over the entire depth range tested
(Fig. 1a, inset). Similar methods were used in 1997, except that the
output from the solar cell was measured using a small panel meter
(Acculex Model DPM-2S) encased in a small waterproof housing
(Ikelite clear Subcase A; Fig. 1b).
We used the meters to measure light within colonies growing on
the barrier reef of Belize as a function of branch spacing and dis-
tance from branch tips. Measurements were collected over a range
of depths (1 to 17 m) in the fore reef and patch reef environments
near Carrie Bow Cay (16°48.0¢N; 88°05.0¢W) and the reef surrounding Manatee Cay (1 to 2 m only; 16°40.0¢N; 88°11.5¢W) in
March/April 1995, March 1996 and February/March 1997.
Fig. 1 a Small multimeter, used in 1995 and 1996, wired to photocell
encased in Plexiglas. Output was not linear with respect to light, but
usable range (inset) provided accurate measurements of light in the
®eld. b Light meter used in 1997 to measure light within aggregations
of Agaricia tenuifolia; note parallel orientation of branches within
coral colony. c Stylized sketch of methodology for measuring light
within aggregations; ambient light was measured directly above
colony with solar cell oriented parallel to face of the blade (Position 1
± in this example facing east); measurements were repeated with light
sensor oriented in same direction at distances of 0 to 4 cm (Position 2),
4 to 8 cm (Position 3) and 8 to 12 cm (Position 4) from branch tip;
during periods of directional sunlight (>1 h before or after solar
noon, shown here), measurements were repeated on face of opposing
blade (here facing west ± Positions 5 to 8) and values for the two
directions were averaged for each region (e.g. total ambient
light = average for Positions 1 and 5, total light at 0 to 4 cm
= average for Positions 2 and 6, etc.)
4
Transects of light were conducted only during periods of clear
skies, and involved time scales of only several minutes to reduce
any artifact of changes in ambient light. For each colony tested, the
distance between two blades within the colony was measured to the
nearest 0.5 cm. Light was quanti®ed immediately above (parallel to
the face of) one of these blades as a measure of total ambient light.
Light measurements were repeated in 4 cm increments at distances
of 0 to 4, 4 to 8 and 8 to 12 cm from the branch tip (Fig. 1c). The
total amount of light which a blade receives is the sum of the light
traveling through the surrounding water as well as that re¯ected by
the opposite blade it faces. In situations where light was highly
directional, we found that irradiance experienced by a blade facing
away from the sun can be higher than that directly above the blade,
due to light re¯ected by the opposing branch. During such conditions (>1 h before or after solar noon), corresponding measurements were taken above and along the length of the opposing blade
to eliminate the e€ect of the angle of solar incidence (Fig. 1c). Light
levels at each height were calculated as a percent of total available
irradiance, and the measurements from the two facing blades were
averaged. Light levels from a total of 56 branches and branch pairs
were collected in this manner. Results collected using the two light
meters (1995 and 1996 [n = 24] vs 1997 [n = 32]) were similar and
were therefore combined. While measurements were conducted
over a depth range, most measurements were conducted at the two
extremes of 1 to 2 and 15 to 17 m. Samples were fairly evenly
distributed over a range of branch spacings, although widelybranched colonies were sampled more commonly in deeper water,
and tightly-branched colonies more commonly in shallow water.
E€ects of branch spacing and distance from branch tips were tested
for homogeneity of slopes followed by a one-factor analysis of
covariance [ANCOVA; SuperANOVA V. 1.11 (Abacus Concepts,
Inc.)].
because data from 1995 suggested that intra-aggregation variation
within any given region was less than inter-aggregation variation,
only one branch was collected from each colony sampled. Each
blade was divided into two (in tightly-spaced branches) or three (in
widely-spaced branches) regions for measurements of chlorophyll.
In addition, pieces immediately adjacent to those sampled for
chlorophyll were removed for zooxanthella counts. Coral tissue was
removed with a ®ne high pressure stream of seawater from a WaterpikÒ (Teledyne Corporation). Samples were then centrifuged,
concentrated and resuspended, and at least four (usually 6 to 8)
subsamples were counted using a hemacytometer. Projected surface
areas of coral skeletal pieces used for chlorophyll (chl) extractions
and zooxanthella counts were measured by video image-analysis.
Surface areas and zooxanthella densities from each sample were
used to calculate levels of chl a, chl c2 and carotenoids (peridinin)
per zooxanthella cell (= cellular pigment concentration) and per
unit surface area of coral (= aerial pigment concentration). Total
cellular pigment concentration and pigment ratios (chl a:c2 and
chl a:peridinin) were used as measures of photoacclimation (e.g.
Titlyanov et al. 1980; Masuda et al. 1993; Iglesias-Prieto and Trench
1994). In the absence of any data concerning the genetics of zooxanthellae within Agaricia tenuifolia, we tentatively consider any
di€erences in pigment characteristics to represent photoacclimation
rather than photoadaptation). In total (1995, 1996 and 1997 data
combined) 22 colonies were sampled from each branch-spacing
group (tightly-branched 15 m, widely-branched 15 m) for aerial
pigment concentrations and pigment ratios. Of these, 12 from each
group were sampled for cellular pigment concentrations and zooxanthella population density. In addition, polyp densities (polyp
cm)2) were counted from all samples collected in 1997 as a crude
measure of small-scale di€erences in colony morphology.
E€ects of aggregation morphology on zooxanthellae
In¯uence of habitat depth
Branches were collected in March/April 1995, March 1996 and
February/March 1997 from coral colonies growing on the fore reef
of Carrie Bow Cay. Branch spacing (distance to nearest branch) on
either side of the sample branch was measured to the nearest
0.5 cm. Aggregations were designated as either tightly (distance
from branches on both sides £3 cm)-or widely (distance ³6 cm)branched. Branches were brought to the surface in covered containers, maintained in running sea water and low light, and
sampled within 4 h. In 1995, we collected three branches from each
of ®ve tightly-branched and ®ve widely-branched aggregations.
Because smaller aggregations of presumed clonemates (based on
color) often join with other clones to form large aggregations
(Chornesky 1991), we collected only closely adjacent and similarlycolored branches from each aggregation. Each branch was divided
into three ''regions'' for analysis of chlorophylls: 0 to 2 cm, 4 to
6 cm and, in widely-spaced corals, 8 to 10 cm from the branch tip
(tissue does not often extend more than 6 cm from the branch tip in
tightly-spaced branches: Helmuth et al. 1997a). Pigments were extracted for 18 to 24 h in 100% acetone from small, fresh coral
pieces ('1 to 3 cm2 per side, both sides of coral piece) from each
region. Samples were kept cool and dark during extraction. Repeated measurements of pigment concentration from a series of
samples (n = 10) con®rmed that pigment concentrations were
highest after 18 h extraction and began to decline only after ³36 h.
Pigment absorption spectra were calculated using a Shimadzu UV1201 spectrophotometer and equations from Je€rey and Humphrey
(1975; for chlorophylls a and c2) and Parsons and Strickland (1963;
for peridinin). While the latter was based on carotenoid extractions
in 90% acetone, Je€rey and Haxo (1968) suggested that extinction
coecients from cells extracted in 100% acetone were similar to
those extracted in 90%. Projected surface areas of each coral piece
were measured from video using image-analysis (NIH Image 1.60
with an Apple Macintosh computer). Data from each region were
averaged for the three blades from each aggregation (= presumed
clone) to yield a single data point per region per colony.
Similar methodologies were used in 1996 and 1997, with the
addition of zooxanthella counts from some samples; however,
Blades were additionally collected from tightly-branched colonies
growing at 1 to 2 m depth in 1996 and 1997 (widely-branched
colonies are rare at this depth: Helmuth et al. 1997a) to assess the
e€ect of habitat depth (1 to 2 m vs 15 m) on pigments and population densities of zooxanthellae. Sample sizes for each assay were
identical to those for tightly-branched colonies collected from
deeper water (n = 22 for aerial pigment concentrations and pigment ratios, n = 12 for cellular pigment concentrations and zooxanthella densities).
Analysis
A series of general linear two-way analyses of variance (ANOVAs;
SuperANOVA V. 1.11) were used to test for the e€ects of branch
spacing and habitat depth (tight 1 m, tight 15 m, wide 15 m) and
intrabranch region (0 to 2, 4 to 6 and 8 to 10 cm from branch tip)
on total cellular pigment concentrations (chl a + chl c2 + peridinin) and zooxanthellae population densities (n = 12 blades per
sample group) as well as pigment density ratios (chl a:c2 and
chl a:peridinin; n = 22 blades per sample group). A ®fth ANOVA
tested for di€erences in polyp density from samples collected in
1997 (n = 10 blades per sample group). The acceptable level of
statistical signi®cance was adjusted for the number of tests (®ve
tests, signi®cant at p £ 0.01). Post-hoc pairwise comparisons between the three morphology/depth groupings were completed using
Fisher's protected least signi®cant di€erence (PLSD) tests.
Results
Light levels in aggregations of Agaricia tenuifolia
Light levels between branches was negatively correlated
with distance from branch tip (F = 13.58, p £ 0.0004)
5
Table 1 Agaricia tenuifolia. Cellular pigment concentrations
(lg 10)5 zooxanthellae) from symbionts extracted from tightlybranched colonies living at 15 to 17 m (tight 15 m) and 1 to 2 m
(tight 1 m) and from widely-branched aggregations living at 15 m
(wide 15 m). Values are presented as means ‹ SDs, and are divided by intrabranch location: 0 to 2, 4 to 6 and (for wide 15 m
only) 8 to 10 cm from branch tip. There was no signi®cant di€erence in total cellular pigment concentrations of the three morphology/depth groups, but concentrations increased signi®cantly
with distance from branch tip
Pigment
Fig. 2 Light levels (% of ambient immediately above Agaricia
tenuifolia colony) as a function of branch spacing and intrabranch
region (distance from branch tip). Light levels increased signi®cantly
with spacing, and decreased markedly with distance from branch tip.
Although analysis was conducted using actual branch-spacing data,
results are here divided into following spacing groups: tight (£3 cm),
intermediate (3 to 6 cm) and wide (6 to 9 cm). Light levels were
generally not obtainable in regions >8 cm from branch tips of tightlybranched aggregations due to blockage by debris and epibionts
and positively correlated with spacing between branches
(F = 34.58, p £ 0.001; Fig. 2). Measured values (% of
ambient irradiance) ranged from <20% at locations 8
to 12 cm from branch tips in colonies with intermediate
branch spacing (e.g. 5 cm ± we were unable to measure
light in this region of more tightly-spaced colonies due to
blockage by dead coral and epibionts) to ³75% at
branch tips (Fig. 2). While light decreased with spacing
at branch tips, di€erences in light reduction as a function
of branch spacing were more apparent in regions further
removed from branch tips (Fig. 2).
0 to 2 cm
4 to 6 cm
8 to 10 cm
Chlorophyll a
tight 1 m
tight 15 m
wide 15 m
0.92 ‹ 0.44
1.05 ‹ 0.39
0.9 ‹ 0.49
1.98 ‹ 1.08
1.73 ‹ 0.66
1.08 ‹ 0.46
±
±
1.20 ‹ 0.48
Chlorophyll c2
tight 1 m
tight 15 m
wide 15 m
0.43 ‹ 0.17
0.40 ‹ 0.16
0.42 ‹ 0.15
0.59 ‹ 0.26
0.52 ‹ 0.23
0.46 ‹ 0.22
±
±
0.48 ‹ 0.25
Peridinin
tight 1 m
tight 15 m
wide 15 m
1.97 ‹ 0.74
1.97 ‹ 0.70
1.91 ‹ 0.82
3.45 ‹ 1.69
2.86 ‹ 1.14
2.12 ‹ 0.81
±
±
2.23 ‹ 0.93
Total pigments
tight 1 m
tight 15 m
wide 15 m
3.31 ‹ 1.31
3.42 ‹ 1.23
3.28 ‹ 1.44
6.12 ‹ 2.98
5.10 ‹ 2.00
3.66 ‹ 1.46
±
±
3.91 ‹ 1.63
Table 2 Agaricia tenuifolia. Aerial pigment concentrations (lg
cm)2 projected coral surface area) and polyp densities from each
intrabranch region (0 to 2, 4 to 6 and 8 to 10 cm from branch tip)
for three morphology/depth groups (means ‹ SDs). Because of
higher population densities of zooxanthellae in widely-branched
colonies (Fig. 5) total aerial pigment concentrations were highest in
these colonies, despite lower cellular concentrations. Polyp densities
increased signi®cantly with distance from branch tip, and were
signi®cantly higher in widely-branched colonies than in tightlybranched colonies from either 1 or 15 m
Pigment
Photoacclimation and population densities
of zooxanthellae
Measures of photoacclimation within aggregations indicated highly signi®cant e€ects of both morphology/
depth group (tight 1 m, tight 15 m, wide 15 m) and intrabranch region (Tables 1 and 2; Figs. 3 to 5). Speci®cally, there were signi®cant e€ects of branch spacing/
habitat depth and distance from branch tip on chl a:c2
(group: F = 8.91, p £ 0.0002, region: F = 11.41,
p £ 0.0001; interaction = NS; Fig. 3) and chl a:peridinin
(group: F = 17.45, p £ 0.0001; region: F = 53.86,
p £ 0.0001; interaction: F = 9.97, p £ 0.0001; Fig. 4)
ratios. For both these metrics of photoacclimation,
zooxanthellae extracted from tightly-branched colonies
in 15 m water displayed markedly higher chl a:c2 and chl
a:peridinin ratios than zooxanthellae taken from either
tightly-branched colonies collected from 1 to 2 m (chl
a:c2 p £ 0.0009; chl a:peridinin: p £ 0.0002; Figs. 3 and
4), or from widely-branched colonies inhabiting the same
Distance from branch tip
Distance from branch tip
0 to 2 cm
4 to 6 cm
8 to 10 cm
Chlorophyll a
tight 1 m
tight 15 m
wide 15 m
4.65 ‹ 1.87
4.49 ‹ 1.67
4.40 ‹ 2.09
11.88 ‹ 2.42
9.28 ‹ 1.67
9.18 ‹ 2.59
±
±
10.01 ‹ 2.09
Chlorophyll c2
tight 1 m
tight 15 m
wide 15 m
2.35 ‹ 0.86
1.81 ‹ 0.82
2.02 ‹ 0.80
4.41 ‹ 1.20
2.99 ‹ 0.88
3.80 ‹ 1.17
±
±
3.94 ‹ 1.28
Peridinin
tight 1 m
tight 15 m
wide 15 m
10.30 ‹ 3.71
8.84 ‹ 3.34
9.13 ‹ 3.86
20.90 ‹ 4.22
15.93 ‹ 2.95
17.98 ‹ 4.44
±
±
18.77 ‹ 3.91
Total pigments
tight 1 m
tight 15 m
wide 15 m
17.30 ‹ 6.29
15.14 ‹ 5.72
15.55 ‹ 6.64
37.19 ‹ 7.60
28.20 ‹ 5.19
30.96 ‹ 7.92
±
±
32.72 ‹ 6.96
6.44 ‹ 1.69
5.72 ‹ 1.37
7.09 ‹ 1.03
8.36 ‹ 1.97
7.42 ‹ 0.69
8.82 ‹ 1.23
±
±
8.25 ‹ 1.76
Polyp density
tight 1 m
tight 15 m
wide 15 m
6
Fig. 3 Agaricia tenuifolia. Ratios (means ‹ SDs) of chl a:c2, a
measure of photoacclimation, as a function of branch spacing (tight vs
wide), habitat depth (comparison within tightly-spaced colonies only)
and distance from branch tips
depth (Fisher's PLSD; chl a:c2: p £ 0.001; chl a:peridinin
= p £ 0.0001). Symbionts from tightly-branched colonies at 1 to 2 m displayed pigment ratios statistically
identical to those from widely-branched aggregations at
15 m. While there was no signi®cant e€ect of branch
spacing/depth on total cellular pigment density (Table 1), levels did increase signi®cantly with distance from
the branch tip (region: F = 7.17, p £ 0.0015). Thus,
levels of photoacclimation correlated well with absolute
light levels in the interior of coral aggregations, since
widely-branched colonies in deeper water (low light),
while signi®cantly di€erent from tightly-branched aggregations at the same depth, displayed similar charac-
Fig. 5 Agaricia tenuifolia. Zooxanthella densities (means ‹ SDs) in
colonies with tight (£3 cm) branch spacing collected at 1 to 2 m and
15 m and in aggregations with wide (³6 cm) branch spacing from
15 m
teristics to tightly-spaced colonies in shallow water (high
light).
Population densities of zooxanthellae, in contrast,
appeared to be correlated more strongly with morphology than with absolute light levels. There were signi®cant e€ects of morphology/depth group (F = 8.22,
p £ 0.006) and intrabranch region (F = 8.39, p £ 0.005;
interaction = NS) on zooxanthellae densities (Fig. 5).
In all groups, density increased with increasing distance
from branch tip. Post-hoc analysis indicated that zooxanthellar densities in widely-branched colonies from
15 m were signi®cantly di€erent from those in tightlybranched colonies at both 15 m ( p £ 0.001) and 1 to 2 m
( p £ 0.004). However, densities in tightly-spaced colonies were statistically independent of depth (Fig. 5).
Polyp densities also increased with distance from branch
tips (F = 12.67, p £ 0.001), although the e€ect of the
morphology/depth group was only marginally signi®cant (F = 4.39, p £ 0.016; Table 2).
Discussion and conclusions
Fig. 4 Agaricia tenuifolia. Ratios (means ‹ SDs) of chl a to
carotenoid (peridinin), an additional metric of physiological change
in zooxanthellar pigments to altered light conditions
The results of this study on Agaricia tenuifolia strongly
suggest that zooxanthellae populations respond to local
physical conditions, which are in turn determined to a
large extent by the morphology of the coral host. We
have previously shown that the ¯ux of gases (e.g. O2,
CO2), nutrients and ions (e.g. bicarbonate, ammonium)
to a coral's tissues can be markedly reduced by tight
branch spacing, and decreases with increasing distance
from the branch tip (Helmuth et al. 1997a). The results
presented here indicate similar e€ects of branch spacing
on light levels within the colony. In some cases selfshading is severe. For example, in regions >8 cm from
the branch tip, light levels can be as low as 15 to 20% of
7
the ambient available light, especially between more
tightly-spaced branches (Fig. 2). This reduction in light
is comparable in terms of absolute light reduction (but
not spectral composition) to an increase in water depth
of >15 m (vs subsurface: Helmuth et al. 1997a). Titlyanov (1991) has previously shown even greater reductions in light levels within the branches of Pocillopora
verrucosa. Di€erences in mass (gas, nutrient and ion)
¯ux have been reported both within branching colonies
(Lesser et al. 1994; Helmuth et al. 1997a) and over the
surface of mounding forms (e.g. Patterson and Price
1992; Helmuth et al. 1997b). Because both light and
water motion/gas ¯ux have been shown to drive rates of
photosynthesis and respiration (e.g. Dennison and
Barnes 1988; Patterson et al. 1991; Lesser et al. 1994),
colony morphology is likely to have a dual e€ect on
zooxanthellae physiology through its interaction with
these two parameters.
Our results indicate strong and very localized levels
of photoacclimation correlated with limited light and
lowered mass ¯ux conditions within colonies. Titlyanov
et al. (1980) found that corals in low-irradiance environments showed signi®cant increases in chlorophyll a,
while levels of chl c2 remained constant. Our results
similarly indicate an increase in chl a:c2 ratios, not only
in response to tighter branch spacing but also over
scales of several centimeters within a given branch.
Similar to the results of Titlyanov et al., this change in
ratio appears to result from an increase in chl a (Table 1). Thus, despite any potentially integrating e€ects
of translocation within coral branches (e.g. Gladfelter
1983), zooxanthella populations show distinct patterns
in photosynthetic characteristics within the host environment.
Whether the observed distribution of zooxanthellar
pigments within Agaricia tenuifolia colonies represents
photoadaptation (genetic variability in symbiont populations), photoacclimation (a plastic response to an altered physical environment) or a combination of both
awaits further study. Rowan and Knowlton (1995)
found multiple clades of symbionts living within single
heads of the coral Montastrea annularis. Similarly,
Timmerman and Muller-Parker (in preparation) have
shown that the relative densities of zooxanthellae and
zoochlorellae (unicellular green algal photosymbionts)
within the temperate anemone Anthopleura elegantissima
appear to vary consistently between regions of the
anemone's body, as well as with ambient light and
temperature. Goulet and Co€roth (1997) hypothesized
that microhabitat variability within colonies of the gorgonian Plexaura kuna would determine the zonation of
zooxanthellar clones, but discovered that while several
clones can occur within a single colony, the distribution
of genotypes was uniform throughout the host. The
taxonomic status of symbionts within Agaricia tenuifolia
has not yet been reported, but T. Wilcox (Department of
Biology, University of Houston, personal communication) has found that zooxanthellae within three other
species of Agaricia (A. agaricites, A. fragilis and A. la-
marcki) show little or no intraspeci®c variation in zooxanthellar clade. These results tentatively suggest that
spatial patterns in pigment concentrations within the
symbionts of A. tenuifolia observed in the present study
are due to photoacclimation rather than to di€erential
survival of zooxanthellar clones. If this assumption
holds true, then, in contrast to prior comparisons between the symbionts of corals and of mobile hosts such
as scyphozoans (e.g. Iglesias-Prieto and Trench 1994),
clones of zooxanthellae inhabiting sessile hosts do not
necessarily experience a uniform physical environment.
Instead, as shown here, the physical environment experienced by zooxanthellae can vary over very small spatial
scales. Consequently, the physiological plasticity demanded of symbionts in corals may in many instances be
much greater than would be predicted by previous views
of coral aggregations as uniform habitats.
While increases in measures of photoacclimation are
clearly correlated with decreases in light, the proximal
causes of observed di€erences in zooxanthella population densities are more enigmatic. While Falkowski et al.
(1993) suggested that zooxanthellar densities remain
relatively una€ected by ambient light, Masuda et al.
(1993) found signi®cant decreases in areal zooxanthellae
density in the coral Fungia spp. with depth, and Lasker
(1981) found that nocturnal morphs of Montastrea
cavernosa contained fewer zooxanthellae per unit surface
area than did diurnal morphs. In contrast, Muller-Parker (1987) reported higher densities in shaded anemones
than in those living in full sunlight. The ¯ux of ammonium and nitrate ions to the symbionts has also been
shown to signi®cantly increase areal zooxanthella densities (e.g. Muller-Parker et al. 1991, Falkowski et al.
1993; Marubini and Davies 1996). For example, Marubini and Davies showed that the addition of nitrate to
the corals Porites porites and M. annularis increased not
only zooxanthellae densities but also cellular concentrations of chl a and chl c2. It is thus likely that patterns
of pigment concentrations and zooxanthellae density are
not strictly a function of light but also of mass (e.g.
ammonium, nitrate, O2, CO2) ¯ux, and the ®nding of
Marubini and Davies that nitrate ¯ux increased levels of
chlorophyll could at least partially explain why, in our
study, widely-branched colonies did not display total
cellular pigment concentrations lower than those in
tightly-branched colonies, despite signi®cant di€erences
in pigment ratios.
Within a given environment (i.e. identical ambient
nutrient concentrations, water ¯ow velocities and ambient light levels), zooxanthellae inhabiting widelybranched Agaricia tenuifolia colonies are predicted to
experience higher levels of both light and mass ¯ux than
those within tightly-branched aggregations. The observation that zooxanthella populations are signi®cantly
higher in widely-spaced aggregations could thus be explained as a positive e€ect of light and/or mass ¯ux.
However, two observations confound this hypothesis.
First, zooxanthella densities were signi®cantly higher in
regions distant from branch tips; these regions receive
8
not only lower irradiance (this study) but also lowered
rates of mass ¯ux (Helmuth et al. 1997a). This pattern is
consistent with results reported by Gladfelter et al.
(1989) for the coral Acropora palmata, and could re¯ect
the fact that our ``0 to 2 cm'' region included the
growing tip of the coral. Second, tightly-branched colonies from shallow water displayed statistically similar
densities to those from colonies with similar branch
spacing collected at 15 m, but di€ered from widelybranched colonies at depth. Mass ¯ux is determined by
the ambient concentration of the mass item of interest,
the morphology of the colony, and the ambient ¯ow
speed. Water velocities in the shallow fore reef of Carrie
Bow Cay are upwards of 2 to 4 times higher than those
at 15 m (Helmuth et al. 1997a). Thus, despite a reduction in mass ¯ux due to tight branch spacing, colonies
in shallow water are predicted to experience higher
nutrient ¯uxes than widely-branched colonies in deeper
water (assuming similar ambient concentrations). On
the basis of mass delivery alone, therefore, tightlybranched colonies at 1 to 2 m would have been expected to display zooxanthella densities more similar to
colonies with wide branch spacing at 15 m that to
tightly-branched colonies at this depth. However,
measures of polyp densities indicated an unexpected
potential complication of di€erences in ®ne-scale colony
morphology. While the results of this study are purely
correlative, increased zooxanthellae densities do appear
to be consistent with higher polyp densities, which may
harbor symbionts more e€ectively than the adjacent
tissue. While no obvious patterns were apparent, differences in true surface area (as opposed to the projected surface area measured here) could have a similar
e€ect. Alternatively, di€erences in ambient nutrient
concentration between 1 to 2 and 15 to 17 m could
potentially play a role in determining symbiont densities. Thus, while the relationship between colony morphology, local light levels and photoacclimation seems
clear, at present the proximal cause of the observed
pattern in zooxanthella population density must remain
speculative.
In summary, our results suggest a marked e€ect of
colony morphology on ambient light levels, and a corresponding photoacclimatory response of the symbionts.
The reduction in ambient light within a single aggregation of Agaricia tenuifolia can exceed that encountered
with depth increases of ³15 m. This interplay between
colony morphology and symbiont microhabitat is not
necessarily restricted to aggregations. Helmuth et al.
(1997b) have predicted di€erences in mass ¯ux within
single coral heads, and Shashar et al. (1993) have documented di€erences in di€usional boundary layers on
the level of individual polyps. Patterson and Price (1992)
have suggested that such di€erences in mass ¯ux could
determine intracolony variation in rates of bleaching.
Similarly, A. Szmant (unpublished data, cited in Goulet
and Co€roth 1997) measured light on the sides of coral
heads and found marked reductions compared to colony
tops. Thus, while depth e€ects are undoubtedly important, so are small-scale microhabitat di€erences within
individual corals and aggregations.
Why tight branch spacing?
Tight branch spacing clearly limits not only the delivery
of gases and other solutes to coral tissues and the
symbionts within (Helmuth et al. 1997a), but also severely decreases the amount of light impinging upon the
surface of the coral. From the host's perspective, however, optimal branch spacing is likely to result from
myriad selective pressures, several of which do not act
directly upon a coral's photosymbionts. For example,
water-¯ow patterns, and hence particle capture, have
been shown to be signi®cantly a€ected by coral morphology (Sebens and Johnson 1991; Helmuth and Sebens 1993) and branch spacing (Sebens et al. 1997). The
risk of breakage and dislodgement may be a€ected by
ramet spacing (Karlson et al. 1996), and has been suggested as a possible selective pressure a€ecting aggregation structure in Agaricia tenuifolia (Chornesky 1991).
Colony morphology and aggregation form may also be
driven by selective pressures resulting from competition
for space in reef environments (e.g. Jackson 1979;
Karlson et al. 1996), and, as we have previously suggested (Helmuth et al. 1997a), tight branch spacing may
maximize the amount of coral tissue per unit substrate.
Furthermore, tight branch spacing may limit the ability
of other sessile reef organisms, and in particular photosynthetic organisms such as other coral species, from
invading the aggregation of A. tenuifolia. Especially in
shallow environments, the coral Porites spp. frequently
grows among aggregations of A. tenuifolia, as do species
of algae (personal observations of present authors).
Thus, within the limits set by zooxanthellar acclimation
to reduced light and mass ¯ux, tight branch spacing may
a€ord some protection from incursion by spatial competitors. To some extent all of these selective pressures,
even predominantly biotic factors such as spatial competition, are a€ected by the interaction of the host with
the ambient environment. In turn, the microhabitats of
a coral's symbionts are a€ected by this interplay, which
in turn a€ects the growth of the host. Thus, in their
complex interactions with environmental parameters
such as light and water ¯ow, symbiotic associations such
as corals and their zooxanthellae o€er an added dimension in understanding the role of the physical environment in structuring benthic assemblages.
Acknowledgements We gratefully acknowledge the continuing
support of K. RuÈtzler, I. Macintyre, M. Carpenter and the
Smithsonian Institution's Caribbean Coral Reef Ecosystems
(CCRE) program, as well as the outstanding assistance of the
station managers and sta€ of the Smithsonian's research station on
Carrie Bow Cay. B. Timmerman was supported in part from a
Grant-in-Aid of Research from Sigma Xi, and supplemental
funding was provided to B. Helmuth from the Department of
Zoology, University of Washington. T. Shyka, D. Danaher,
9
B. Pfei€er and M. Phillips assisted with data collection in the ®eld,
and S. Edwards graciously supplied centrifuge tubes. T. Daniel,
J. Lee, S. Moore, A. Trimble and an anonymous reviewer provided
advice on constructing the light meters. Samples were collected and
exported with the permission of the Belizean government, and we
thank N. Jacobs for his assistance in obtaining permits. The
manuscript was greatly improved by comments from M. Strathmann, E. VanVolkenburgh and three anonymous reviewers. This is
Contribution No. 498 of the CCRE program, Smithsonian Institution.
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