ArticlePDF Available

Abstract and Figures

With the continued and unprecedented decline of coral reefs worldwide, evaluating the factors that contribute to coral demise is of critical importance. As coral cover declines, macroalgae are becoming more common on tropical reefs. Interactions between these macroalgae and corals may alter the coral microbiome, which is thought to play an important role in colony health and survival. Together, such changes in benthic macroalgae and in the coral microbiome may result in a feedback mechanism that contributes to additional coral cover loss. To determine if macroalgae alter the coral microbiome, we conducted a field-based experiment in which the coral Porites astreoides was placed in competition with five species of macroalgae. Macroalgal contact increased variance in the coral-associated microbial community, and two algal species significantly altered microbial community composition. All macroalgae caused the disappearance of a γ-proteobacterium previously hypothesized to be an important mutualist of P. astreoides. Macroalgal contact also triggered: 1) increases or 2) decreases in microbial taxa already present in corals, 3) establishment of new taxa to the coral microbiome, and 4) vectoring and growth of microbial taxa from the macroalgae to the coral. Furthermore, macroalgal competition decreased coral growth rates by an average of 36.8%. Overall, this study found that competition between corals and certain species of macroalgae leads to an altered coral microbiome, providing a potential mechanism by which macroalgae-coral interactions reduce coral health and lead to coral loss on impacted reefs.
Content may be subject to copyright.
Macroalgae Decrease Growth and Alter Microbial
Community Structure of the Reef-Building Coral,
Rebecca Vega Thurber
*, Deron E. Burkepile
, Adrienne M. S. Correa
, Andrew R. Thurber
Andrew A. Shantz
, Rory Welsh
, Catharine Pritchard
, Stephanie Rosales
1 Florida International University, Deptartment of Biological Sciences, North Miami, Florida, United States of America, 2 Oregon State University, Deptartment of
Microbiology, Corvallis, Oregon, United States of America, 3 Oregon State University, College of Earth, Ocean and Atmospheric Sciences, Corvallis, Oregon, United States
of America, 4 Oregon Institute of Marine Biology, Charleston, Oregon, United States of America
With the continued and unprecedented decline of coral reefs worldwide, evaluating the factors that contribute to coral
demise is of critical importance. As coral cover declines, macroalgae are becoming more common on tropical reefs.
Interactions between these macroalgae and corals may alter the coral microbiome, which is thought to play an important
role in colony health and survival. Together, such changes in benthic macroalgae and in the coral microbiome may result in
a feedback mechanism that contributes to additional coral cover loss. To determine if macroalgae alter the coral
microbiome, we conducted a field-based experiment in which the coral Porites astreoides was placed in competition with
five species of macroalgae. Macroalgal contact increased variance in the coral-associated microbial community, and two
algal species significantly altered microbial community composition. All macroalgae caused the disappearance of a c-
proteobacterium previously hypothesized to be an important mutualist of P. astreoides. Macroalgal contact also triggered: 1)
increases or 2) decreases in microbial taxa already present in corals, 3) establishment of new taxa to the coral microbiome,
and 4) vectoring and growth of microbial taxa from the macroalgae to the coral. Furthermore, macroalgal competition
decreased coral growth rates by an average of 36.8%. Overall, this study found that competition between corals and certain
species of macroalgae leads to an altered coral microbiome, providing a potential mechanism by which macroalgae-coral
interactions reduce coral health and lead to coral loss on impacted reefs.
Citation: Vega Thurber R, Burkepile DE, Correa AMS, Thurber AR, Shantz AA, et al. (2012) Macroalgae Decrease Growth and Alter Microbial Community Structure
of the Reef-Building Coral, Porites astreoides. PLoS ONE 7(9): e44246. doi:10.1371/journal.pone.0044246
Editor: Christian R. Voolstra, King Abdullah University of Science and Technology, Saudi Arabia
Received April 16, 2012; Accepted July 31, 2012; Published September 5, 2012
Copyright: ß 2012 Vega-Thurber et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by the Florida International University College of Arts and Sciences and grant OCE #1130786 from the National Science
Foundation to DEB and RVT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail:
Corals typically host species-specific communities of bacteria [1]
that perform a wide variety of context-dependent roles on reefs
[2]. For example, some bacteria may ward off pathogenic
microbes by occupying available physical niches on coral colonies
and/or producing antibiotics [3,4,5]. Within coral skeletons,
cyanobacteria and other endolithic organisms may be important
nitrogen fixers [6,7,8] supplying 50–200% of their host’s nitrogen
requirement [9]. Cyanobacterial symbionts may be especially
important for providing nutrients to the host during stressful
conditions, such as bleaching events, when other symbionts (e.g.,
Symbiodinium dinoflagellates) are not performing adequately [10].
Common coral-associated bacteria also may play a variety of other
roles such as digesting recalcitrant carbon sources (e.g., chitin,
cellulose), scavenging micronutrients (e.g., iron), and impacting
elemental cycling [3,11,12]. Therefore, coral-associated microbes
are likely critical for the maintenance of coral colony health and
survival [4,13].
Anthropogenic impacts such as climate change, eutrophication,
and overfishing are important drivers of the loss of coral cover and
biodiversity [14,15]. These stressors appear particularly severe on
reefs in the Caribbean Sea where corals have declined ,80% in
recent decades [16,17,18]. Concomitantly, the combined forcing
of reduced herbivore abundance from overfishing, increased
nutrient input, and loss of coral cover due to bleaching and
disease has led to a significant increase in macroalgal cover on
many Caribbean reefs [19,20,21]. Abundant macroalgae may
reinforce a coral-depauperate state by facilitating the spread of
coral diseases [22,23], reducing the survival and growth of adult
corals [24,25,26,27], and/or preventing the recruitment of
juvenile corals [24,28,29].
Despite evidence showing that increased macroalgal abundance
has negative effects on corals, we understand little about the
mechanisms by which macroalgal competition may impact the
coral-microbial mutualism and how these impacts relate to overall
coral fitness. Increases in macroalgal abundance may alter the
normal microbial communities on corals and potentially trigger
episodes of microbial disease [22,23,30,31,32]. Although it is
unlikely that macroalgae mediate all coral disease outbreaks, shifts
in macroalgal diversity and abundance likely influence the
taxonomic and metabolic diversity of coral-associated bacteria.
PLOS ONE | 1 September 2012 | Volume 7 | Issue 9 | e44246
Shifts in the microbial community on corals may result in
a decreased abundance of beneficial bacteria, which could
potentially increase colony vulnerability to water-borne pathogens,
thermal bleaching, or other stressors. Alternatively, the presence of
macroalgal competitors may influence the growth of rare members
of the coral microbiome or even directly vector new, harmful
microbial taxa onto corals. Here, we test whether coral-macroalgal
interactions affect the microbial community on the scleractinian
coral, Porites astreoides, and whether such alterations impact coral
Materials and Methods
Study Site and Experimental Design
The Florida Keys Reef Tract consists of a large bank reef system
located approximately 8 km offshore of the Florida Keys, USA,
paralleling the island chain. Our study site, Pickles Reef (25u 009
050N, 80u 249 550W), is a 5–6 m deep relict spur and groove reef
system located off Key Largo, FL. Live coral cover ranges between
5–10% and macroalgal cover often varies between 20–30%
In July-September 2009, coral-algal competition experiments
were conducted over 10 weeks using the coral P. astreoides and
the macroalgal species: Dictyota menstrualis , Galaxuara obtusata,
Lobophora variegata, Halimeda tuna, and Sargassum polyceratium. Porites
astreoides is an encrusting/mounding coral and is now a spatial
dominant after recent declines of other corals such as Acropora
cervicornis and Montastraea faveolata [32]. Dictyota spp., H. tuna, and
L. variegata are often the most abundant components of the
macroalgal community on reefs in the Florida Keys and often
compete with corals [31]. All of the macroalgal species that
were used tend to increase when herbivory rates decline and
thus represent species that might compete with corals where
overfishing is common [26,27]. Porites astreoides colonies were
collected from a nearby shallow inshore reef at 3 m depth.
Experimental corals were fragmented, transported to our field
site, and then common garden acclimated at 5 m on site for 2
weeks prior to the experiment. Algae were collected either from
our field site or nearby Pickles Reef or Conch Reef.
Experiments were conducted using cinderblocks
(10620640 cm) as competition arenas. Porites astreoides fragments
(,15 cm
surface area as determine with the tin foil method
[33]) were attached to plastic mesh using marine epoxy. For
each replicate experiment, a single P. astreoides colony was split
to provide all the fragments that underwent manipulation. This
was done to allow comparison in growth rates of P. astreoides
among the different replicates without being biased by in-
traspecific growth rates. A total of six colonies were split
providing six independent replicates and a ‘blocked’ experimen-
tal design. The mesh attached to each fragment was used to
anchor the fragment to cinderblocks using cable ties with a single
coral fragment on either end of a single cinderblock (Figure
S1A). Approximately equal volumes (5 ml) of one of the five
algal species were attached to the mesh next to each fragment,
so that it was in direct contact with the coral (Figure S1A). One
replicate of the experiment included each of the five macroalgal
species plus a control randomly assigned to one of the three
cinderblocks (Figure S1B). Controls were a treatment where no
macroalgae were transplanted next to the coral. Each coral-algal
pairing (including control) was replicated 6 times. The
combination of macroalgal treatments on a specific cinderblock
was randomized within each block of the experiment. Algae and
cable ties were replaced every 1–2 weeks to minimize fouling by
other algae or invertebrates. An exclosure of plastic mesh
(2.5 cm diameter) was constructed around each cinderblock to
prevent corallivores from preying on corals and herbivores from
grazing on macroalgae.
To measure growth, corals were buoyant weighed at the
beginning and end of the experiment [34]. Growth rates were
calculated as g/cm
of colony area/day. Differences in growth
over the course of the experiment were assessed by comparing
growth of each coral fragment in the macroalgal competition
treatments to growth of the control within each experimental block
via paired t-tests. Since data from the control corals were used for
multiple statistical tests, we controlled for Type I errors using the
Bonferroni-Holm correction [35]. This approach is analogous to
applying a Dunnet’s post-hoc test to an ANOVA model, however,
it allows the maximum amount of power from the paired
experimental design as it takes into account the different intrinsic
growth rates for each replicate colony that was divided into the
treatment fragments. The Dunnet’s post-hoc test is based on
repetitive t-tests that have been corrected for Type I errors [36],
exactly as we have done here except we have used a paired t-tests.
During the experiment, a storm dislodged several cinderblocks
from the experiment and thus potentially compromised the health
of these corals. However, none of the control fragments were
damaged. In cases where a treatment was lost, the control from
that replicate was not included in the statistical analyses, which
lead to different replication for each treatment. Final replication
for each algal treatment was: (1) control: n = 6, (2) D. menstrualis:
n = 5, (3) L. variegata: n = 5, (4) H. tuna: n = 5, (5) G. obtusata:n=4,
and (6) S. polyceratium:n=5.
Isolation of Coral- and Macroalgal-associated Microbial
To characterize the microbial community on each macroalgal
species, small portions of each macroalgae (n = 5 of each
species) were sampled from the coral-algal competition arenas
prior to the experiment. A 1 cm
portion of each macroalgal
thallus was patted dry and swabbed with a sterile cotton swab
to collect surface-associated microbes and avoid seawater-
associated microbes. Swabs were placed in 15 ml conical tubes
containing 10 mls of 95% ethanol and stored at 4uC. For
microbial DNA extractions, swabs were placed in an o-ring
sealed, 2 ml centrifuge tube containing 1.5 ml of lysis buffer
(0.36 M NaCl, 45 mM EDTA, 1% SDS), vortexed for 15
minutes in a bead beater, and incubated at 65uC for 1.5 hours.
DNA was archived and later extracted according to previously
published methods [37].
At the conclusion of the experiment, corals were placed in sterile
Whirl-paks, brought to the surface, and placed on ice. Once on
shore, corals were rinsed in 0.2 mm filtered seawater to remove
seawater associated microbes, weighed, separated from their mesh
base, and placed in 50 ml conical tubes containing 30 ml of 95%
ethanol and stored at 4uC (n = 4 per treatment). For microbial
DNA isolation, coral tissue was removed using sterile razor blades,
placed in 2 ml centrifuge tubes, and extracted using same method
as described for the macroalgal swabs except that the coral
samples were not bead beaten [37].
Community Analyses of Coral- and Mac roalgal-associated
Relative microbial taxonomic diversity was measured using
terminal restriction fragment length polymorphisms (T-RFLP)
[38], using the primer sets, FAM-Univ 9F and Univ 1509R, to
amplify the 16S rRNA gene from each sample in two replicate
50 ml PCR reactions (10 mlof56 buffer, 2.4 mM of MgCl,
Macroalgal Effects on Coral Microbiome
PLOS ONE | 2 September 2012 | Volume 7 | Issue 9 | e44246
0.2 mM each primer, 2.5 U of Taq polymerase, and 0.2 mM of
each dNTP) using the following touchdown thermo-cycler pro-
gram: 95uC 2 min, 34 cycles of 95uC 1 min, 55.6uC 1 min
(20.3uC), and 72uC 1 min, and a final extension of 72uC 5 min
step [39]. Successful duplicate amplifications were combined and
cleaned using the Promega PCR Wizard kit (Madison, WI). Seven
samples (including both coral tissues and algae thalli specimens,
notably the G. obtusata coralline algae) could not be amplified and
were eliminated from the study. To normalize the amount of
sample analyzed, exactly 960 ng of DNA from each pooled
amplification was digested at 65uC for 4 h using the restriction
enzymes Rsa1 and Hha1 from Promega (Madison, WI) and
analyzed at Laragen, Inc (Culver City, CA) with a 0.5 ml
Liz1200 size standard and an ABI 3730 sequencer
(Applied Biosystems). Overall this large dataset includes 84
TRFLP profiles (42 individual samples, and digested with two
TRFs were determined using the Local Southern size-calling
algorithm of the Peak Scanner Software Version 1.0 (Applied
Biosystems). Sample versus TRFLP peak data matrices were
constructed using a conservative threshold of 50 units above
background. Peaks smaller than 50 base pairs (bp) and larger than
1200 bp (outside of the standard’s linear range) were removed in
silico. Peak area was linearly related to peak height (r
.0.95), therefore, relative microbial taxa abundance data was
obtained from peak heights following sample standardization
including rounding to the nearest integer and a two basepair bin of
fragment sizes [40]. Since the selected primers target a large
portion of the 16S rRNA gene, TRFs can represent one or more
bacterial and/or archaeal sequence fragments. Therefore, for the
purposes of this paper, we refer to the bacteria and archaea as
‘‘microbial communities.’’
One way analysis of variance (ANOVA) followed by Tukey
post-hoc tests were used to identify if: 1) relative microbial
abundance differed across macroalgae species and among the
coral-algal competition experiment, 2) there were differences
among specific microbial TRFs within each treatment, and 3)
there were differences in diversity of the microbial taxa as
a function of the treatment. The underlying assumptions of this
test were determined graphically (homogeneity of variance) and
using a Kolmogorov-Smirnov test (normality). A variety of
transformations were applied (see results) when necessary to meet
these assumptions. Diversity indices employed included species
richness (total number of TFRLP peaks) and CHAO1 predicted
relative taxonomic abundance [41].
Multidimensional analyses were used to identify whole micro-
bial community differences among the macroalgae and treat-
ments. These analyses, along with the later two measures of
diversity listed above, were performed in PRIMER v. 6 [42]. Bray-
Curtis similarity was used to compare log-transformed peak
heights of microbial abundance and distribution data (as measured
by TRFs). The similarity of these data was visualized using
multidimensional scaling (MDS) plots. Significant differences
between these microbial communities among the algal treatments
were evaluated using an analysis of similarity (ANOSIM), and the
identification of which taxa were most important at driving the
differences among the groups was conducted using similarity
percentage (SIMPER) analysis.
Permit # FKNMS-2009-047 was obtained for this study from
the Florida Keys National Marine Sanctuary.
Corals Exposed to Macroalgae have Reduced Growth
Macroalgal competition decreased coral growth rates by a mean
of 36.8% (0.1260.007 g cm
for control corals vs.
0.0860.01 cm
across all corals with algal competitors;
t = 3.4, P = 0.008). Yet, there were interspecific differences in how
macroalgae affected coral growth. Dictyota menstrualis did not
significantly suppress growth in P. astreoides (Figure 1). The other
four macroalgal species all lowered coral growth rates relative to
controls with no macroalgal competitors.
Microbial Community Com position Differs Among
Macroalgal Species and Among Control and Treated
Among the macroalgae, 429 individual TRFs were identified
while in the coral-algae experiments 232 TRFs were identified.
Using the relative abundance of each TRF, mean microbial
community profiles were generated for all the different
macroalgae and coral-algae experimental specimens. A majority
of these TRFs were singletons or comprised less than 3% of any
one community profile. However, forty-one TRFs were identi-
fied that compromised a mean of 3% or more of any
community profile (Figure S2). All samples demonstrated
variation in the combination of these TRFs with the macroalgae
samples only containing 5 TRFs (63, 421, 424, 826, 1048) in
common with any coral community. The number of major
TRFs in each sample varied from as low as 2 (G. obtusata thalli)
to as many as 20 (D. menstrualis exposed corals). Overall, while
there were more individual TRFs identified among macroalgal
samples, there were only ,9 major TRFs (mean 6 SE:
9.4062.0 TRFs) that comprised more than 3% of algae
microbial community. In contrast, coral samples contained
more than twice the number (mean 6 SE: 16.861.30 TRF) of
major TRFs.
Using all of the TRFs it was found that relative taxonomic
richness of the microbial community on macroalgae varied ,80
fold among species (Figure 2A). Sargassum polyceratium hosted the
highest species richness (mean of 103 TRFs observed, black bars).
CHAO 1 estimators (grey bars) also predicted that S. polyceratium
thalli statistically contained the most species-rich microbial
community (ANOVA F
= 62.85, p#0.001; post-hoc results
indicated on Figure 2A) compared to all other macroalgae (mean
6 SE: 822.069.26 taxa). Of the coral treatments, control corals
contained statistically fewer taxa compared to all other macroalgal
treatments, with an observed species richness of only 19 TRFs
resulting in a mean of 51 (SE 612.18) predicted microbial taxa or
groups of taxa (ANOVA F
= 88.0, p#0.01; see Figure 3B for
post-hoc results). Species evenness also was determined for all
algae species and coral-algae interaction experiments, but no
major differences among macroalgae or among coral treatments
were detected.
To determine if microbial communities differed among
macroalgal species and among corals in competition with
macroalgae, multi-dimensional scaling (MDS) and analysis of
similarity (ANOSIM) were performed. The microbial communi-
ties present on all macroalgae, except G. obtusata, were different
from the communities present on the coral samples (Table S1).
Further, distinct microbial communities were found associated
with different macroalgal species, with the exception for G. obtusata
(Figure 3A, circles) and H. tuna (diamonds), which were not
significantly different from one another (Table S1). In an MDS
plot containing just the macroalgal samples, S. polyceratium-
Macroalgal Effects on Coral Microbiome
PLOS ONE | 3 September 2012 | Volume 7 | Issue 9 | e44246
associated microbial communities were particularly distinct,
forming a tight cluster (squares; Figure 3A). The similarity of
microbial communities within macroalgal samples of a given
species ranged from a low of 32% for L. variegata to a high of 67%
for G. obtusata (Table S2). The microbial community associated
with S. polyceratium was highly (mean 92.2% 61.93 SE) dissimilar
from all other macroalgal species (Table S2). For example, TRF
895 was unique to S. polyceratium and contributed most to its
grouping, yet this TRF still only comprised ,7% of the
Community analysis also revealed that corals in competition
with certain macroalgae harbored microbial communities
significantly different from those on control corals (Figure 3B).
Specifically, exposure to S. polyceratium (Figure 3B, squares)
resulted in microbial communities that were different from those
on control corals (triangles), as well as from microbial
communities sampled on all other coral-macroalgal pairings
(Table S1). Exposure to G. obtusata (circles) also significantly
altered microbial communities relative to controls. Further,
treated corals exhibited higher within-group community varia-
tion (mean 33.56% 64.63 SE) than the control corals, which
had the most similar (,44%) microbial communities of all the
samples (Table S3). Microbial communities on corals exposed to
D. menstrualis showed the most variation within only 16%
similarity to each other. On average control corals were 75%
dissimilar to the corals exposed to macroalgae (mean 74.82%
61.78 SE).
Analysis of every TRF identified from the P. astreoides samples
showed that a total of 15 TRFs were significantly different across
the macroalgal treatments (Table S4). All macroalgal treatments
produced at least one significant change in the abundance of
a TRF as compared to TRFs associated with controls (Figure 4A).
Corals exposed to D. menstrualis had the fewest TRF changes (i.e.,
two), while corals challenged with H. tuna had the most altered
TRFs (i.e., 12). Observed TRF changes included significant
increases and/or decreases, depending on the treatment
(Figure 4B). For example, S. polyceratium exposure led to four
TRF increases and two decreases, while L. variegata led to only
a single TRF increase but three TRF decreases. Furthermore, the
relative amount that each TRF changed compared to the control
was different across treatments (Figure 4C). While all decreases in
TRF abundance were similar across the treatments (from 275 to
2146 units), the increase in TRF abundance in corals exposed to
S. polyceratium (.12,000 units) was an order of magnitude greater
than TRF increases in any of the other macroalgal treatments
(#2,000 units).
Changes in the abundances of individual TRFs were complex
(Figure 5); the abundance of some TRFs was altered by more than
one macroalgal treatment while other TRFs changed within
a single treatment only. For example, TRF 341 was reduced in
every macroalgal competition treatment, whereas TRF 126
increased in response to G. obtusata, H. tuna, and L. variegata.
Halimeda tuna-exposed corals had 6 unique TRF increases (TRFs
75, 227, 802, 858, 882, 899). TRFs 63, 564, and 879 were elevated
only in corals exposed to S. polyceratium; increases in these TRFs
were substantial relative to other observed TRF increases
(Figure 5).
Figure 1. Effects of treatments on coral growth. Comparisons of growth rates (means 6 SE) between corals competing with one of five
macroalgal species vs. control corals. Each algal treatment in the block design had its own paired controls that had no algae. The number of controls
were constant among treatments except when lost due to storm damage; in that instance the corresponding control was removed from the analysis.
Statistics are from paired t-tests. P-values are based on Bonferroni-Holm correction for multiple comparisons with the controls. * p,0.05.
Macroalgal Effects on Coral Microbiome
PLOS ONE | 4 September 2012 | Volume 7 | Issue 9 | e44246
Macroalgae Significantly Alter the Structure of Coral-
Associated Microbial Communities
Our findings suggest that direct competition with some
macroalgal species increases the taxonomic variability of microbes
on P. astreoides. Similar decreases in the specificity of the coral
microbiome have previously been observed in corals that differed
in their health state and/or proximity to sewage outflows [39].
These increases in microbial community variability may be
a general response to disturbance as has been seen in communities
of macro-organisms [43]. Although the overall microbial commu-
nity significantly changed only on corals in competition with two
macroalgal species (S. polyceratium and G. obtusata) (Figure 3B; Table
S1), some microbial taxa were significantly altered on all corals
challenged with macroalgal competitors (Figure 5). Four types of
alterations in the microbial TRFs associated with corals in
competition with macroalgae were identified. First, some taxa
present on control corals increased in abundance on macroalgae-
treated corals. This increase was the least frequently observed
change to microbial TRFs, occurring only with TRF 63. This
TRF was present on every coral and macroalgae tested, and drove
much of the clustering among microbial communities (Tables S2
& S3). Yet, the abundance of TRF 63 was significantly altered only
on S. polyceratium-exposed corals, where it increased 4-fold relative
to control corals (Figure 5).
Conversely, a bacterial taxon previously documented [3] to be
a member of the P. astreoides holobiont always declined on corals in
competition with macroalgae. TRF 341 was the most abundant
TRF comprising ,50% of the community in control corals, but its
presence was reduced in every macroalgal treatment to below the
detection level. Further, this TRF was not observed on any of the
macroalgal species in this study. Rohwer et al. [3] first discovered
TRF 341 on P. astreoides in Panama. TRF 341 was the most
commonly identified taxa [3] (comprising an average of 61% of
clones detected), and it was therefore named P. astreoides 1 (PA1).
Sequencing identified PA1 as a c-proteobacteria (GenBank
accession # AF365457) [3]; the TRF 341 observed in corals in
this study also represents the PA1 phylotype. PA1 has also
previously been identified in Diploria strigosa corals as TRF 342
[39], where it exhibited lower abundances in diseased colonies,
relative to apparently healthy conspecifics. Together, these
previous studies have indicated that TRF 341 (PA1) is a likely
important, but context dependent, mutualistic symbiont of corals.
With the observation that there is concomitant reduction in PA1
abundance and coral growth in this study, it is possible that the loss
of coral-associated microbial symbionts can potentially lead to
reduced coral health. Alternatively the loss of this symbiont may
Figure 2. Comparisons of relative bacterial diversity among macroalgal species and among coral-algal competition treatments. The
presence of individual TRFs were used to determine bacterial species richness (black bars) and CHAO 1 estimates (grey bars) were used to predict the
relative number of taxa (means 6 SE) in macroalgae (A) and on corals challenged with macroalgae (B). Letters represent significant differences
(p,0.05) in CHAO1 estimates among sample types; richness was not found to be significantly different among samples. Differences among
macroalgae are denoted by uppercase letters A-D while difference among coral-algae treatments and controls are denoted by lowercase letters w-z.
*Galaxaura obtusata macroalgae data were not included in statistical analysis due to low replication.
Macroalgal Effects on Coral Microbiome
PLOS ONE | 5 September 2012 | Volume 7 | Issue 9 | e44246
Macroalgal Effects on Coral Microbiome
PLOS ONE | 6 September 2012 | Volume 7 | Issue 9 | e44246
have arisen from altered coral physiology that resulted from
exposure to the algae. Both hypotheses should be tested in the
A third group of TRFs were present in control corals and
changed non-uniformly across the macroalgal treatments. For
example, TRF 423 increased in two of the competition treatments
(S. polyceratium and H. tuna) but decreased in another (L. variegata).
These data suggest that competition with macroalgae can have
idiosyncratic effects on coral-associated microbial communities
depending on the species of macroalgal competitor and, poten-
tially, the mechanisms that macroalgae use to compete with corals
(e.g. allelopathy, abrasion, smothering).
The last and most commonly identified alteration to microbial
communities was the detection of TRFs on macroalgal-treated
corals that were not observed on controls. In our study, this
included TRFs 75, 126, 227, 564, 802, 826, 828, 858, 879, and
899. We additionally searched for these TRFs within datasets
generated from nine other P. astreoides colonies collected from this
same reef in a long-term study (Burkepile & Vega Thurber,
unpublished data), and TRFs 227, 564, 828, 858, 879, and 899
were never detected in these unmanipulated colonies (data not
shown). This apparent relaxation of specificity in coral-microbial
associations may represent colonization of corals by opportunist
microbes during disturbance events.
The most significant taxon introduction to macroalgae-treated
corals was TRF 879. This TRF was not detected from any control
corals (n = 3) or local unmanipulated corals (n = 9) but represented
up to ,53% (mean 42.6% 66.11 SE) of the microbial community
on corals in competition with S. polyceratium. TRF 879 was not
detected from thalli of S. polyceratium, and therefore does not
appear to be vectored directly to the coral by the algae. In
contrast, TRF 564 also was not observed on controls or local
corals but was abundant on corals exposed to S. polyceratium (mean
,26% of the coral-associated microbial community). Importantly,
however, TRF 564 was detected from S. polyceratium thalli, albeit at
lower relative abundances (,1.5% of the algae-associated
microbial community) than on treated corals, and TRF 564 was
never detected on any other type of macroalgae. Taken together,
these data indicate that TRF 564 was vectored from S. polyceratium
to P. astreoides colonies and induced to proliferate. Although we
currently do not know the identity of this TRF 564 microbial
taxon, to our knowledge, this is first example of a macroalga
vectoring a microbe to a coral. An alternative hypothesis is that
TRF 564 also is present in the overlying water column and thus
the surrounding seawater could have contributed to the increase in
the relative abundance of this TRF on corals after exposure to the
S. polyceratium algae. However, if that were the case, then TRF 564
should have been present in all of the samples (like TRF 63), yet, it
was only ever detected on thalli of the S. polyceritium algae and
corals exposed to that same algae. Therefore it is more
parsimonious to suggest that TRF 564 is vectored from the algae
to the corals.
While these data clearly demonstrate alterations in microbial
diversity on corals, a caveat of TRFLP analysis is that it is not as
sensitive at detecting rare members of the community, compared
to other methods such as pyrosequencing [44]. It cannot be ruled
out that rare members of the coral and macroalgal microbiomes
were not detected using this technique. Therefore, it is possible
that some microbial taxa, which appeared to be present on
macroalgae-treated corals but not on controls, were in fact also
present on control corals but below the detection threshold of our
TRFLP analysis. For example, TRF 564 could normally be a rare
member of the coral microbiome, whose growth is highly
stimulated by the presence of S. polyceratium. Nevertheless, such
dramatically large shifts in any one member of the microbiome in
response to algal competition (e.g., from undetectable to 30% of
the community) are likely to affect the metabolism of the coral
holobiont [45,46] with potentially adverse consequences for the
Yet, our analysis did indicate that minor members of the coral
microbiome were affected by interactions with macroalgae. While
we found that the major ($3% of any one community) microbial
Figure 3. Microbial communities on algal thalli. Multidimensional ordination of Bray-Curtis similarity of microbial communities found on
macroalgae (A) and on corals in competition with different macroalgal species (B).
Figure 4. Significant Changes in Individual TRFs. Individual TRFs were compared between control corals and corals exposed to different
macroalgal species. The relative total number of TRFs changed (A), the number of relative increases and decreases in individual TRFs (B), and the
combined mean change in relative TRF abundance (C) compared to control TRFs.
Macroalgal Effects on Coral Microbiome
PLOS ONE | 7 September 2012 | Volume 7 | Issue 9 | e44246
members of coral and algae were different among species and
coral treatments, only 60% of the TRFs that were significantly
altered on corals exposed to algae were from this majority. Rare
taxa were also significantly altered, and contributed to 40% of all
the significant individual TRF changes. These rare TRFs that
were altered included: 51, 54, 199, 227, 802, 803, 839, 858, 882,
883, and 889. Together these data suggest that members of both
the coral’s common and rare biosphere are impacted by algal
Mechanisms Driving Macroalgal-Induced Changes to the
Coral Microbiome
Several mechanisms could drive shifts in microbial abundance
and community structure on corals competing with macroalgae.
Exudates and surface bound compounds, including organic
carbon and allelopathic chemicals may provide the mechanism
that resulted in reduced coral growth and microbial community
shifts in corals in contact with macroalgae. One perturbation that
shifts microbial communities is an increase in the available food for
heterotrophic bacteria, including those that feed upon dissolved
organic carbon (DOC) [47,48]. Some macroalgae exude DOC
into the surrounding water column and may stimulate microbial
growth at the expense of coral health [22,49]. These mechanisms
may be species-specific to different macroalgae or could differ
according to morphology or growth rates. Patterns of DOC release
vary widely among algal species and are influenced by algal
growth form and morphology [50,51]. Therefore, the differential
DOC released by each of these macroalgal taxa may drive the
species-specific shifts of the microbial community as well as the
changes in coral growth that we observed. In one recent study,
Sargassum dentifolium had the highest DOC exudation rates among
nine species of benthic macroalgae [51]. If this is a common trend
to the Sargassum genera, then DOC release may explain why corals
exposed to S. polyceratium had the greatest shift in microbial
community compared to all other treatments (Table S1).
In addition to DOC exudates, some macroalgae produce
allelopathic surface compounds that directly alter the growth of
surface bacteria on corals [52,53]. Morrow et al. [53] found that of
eight macroalgal species, L. variegata showed the most inhibitory
effects against coral-associated microbial growth. Our data
corroborate this effect, in that the competition between L. variegata
and P. astreoides had the greatest inhibition of microbial taxa and
also strongly depressed coral growth. In contrast, Morrow et al.
[53] reported that D. menstrualis extracts had significant effects on
coral-associated microbial dynamics, but in our study, this
macroalga had the least impact on coral growth and coral-
associated microbial communities. Likewise, H. tuna extracts had
minimal effects (stimulatory or inhibitory) on coral-associated
bacteria [53], yet in this study competition with H. tuna resulted in
the most significant number of individual microbial taxa changes
on corals (Figure 4). It could be that the changes in microbial
growth documented by Morrow et al. [53] in short laboratory
growth assays of several days were ephemeral and that these shifts
Figure 5. Contribution of Individual TRFs to Changes in Relative TRF Abundance. Changes in mean individual peak heights for specific
TRFs for macroalgal competition treatments relative to control corals. Positive/negative values indicate an increase/decrease of a bacterial TRF on
coral fragments in competition with a given macroalgal species, compared to TRFs on control corals.
Macroalgal Effects on Coral Microbiome
PLOS ONE | 8 September 2012 | Volume 7 | Issue 9 | e44246
do not persist under the more realistic field conditions of our
experiment. Regardless, allelopathic interactions may be re-
sponsible for some shifts in microbial abundance in response to
algal competition, but the role of allelopathy likely differs
significantly among macroalgal species.
Hypoxia, vectored bacteria, and physical abrasion may also be
important mechanisms that drive changes in the microbial
communities on corals. When in contact with colonies, some
macroalgae may create areas of persistent hypoxia, which facilitate
changes in the coral microbial community [22,54,55]. Hypoxia is
generated more commonly by filamentous turf algae or macro-
algae that grow prostrate along a coral surface (e.g., Dictyota or
Lobophora), rather than by upright macroalgae (e.g., Sargassum or
Halimeda) [54]. Finally, macroalgae may be important vectors of
bacteria that are not otherwise found on coral surfaces. As we
show here, S. polyceratium introduced a microbe (i.e., TRF 564) to
its coral competitor that was not observed in any other coral
treatments or macroalgal species in this study. The microbial
community on S. polyceratium appeared markedly different from
other algal species in terms of taxonomic composition (Figure 3B).
To our knowledge S. polyceratium -associated microbial communities
have not previously been examined, yet, this macroalga is of
particular interest as Sargassum spp. often increase on reefs when
herbivores are removed [24,26,27], and the increase of Sargassum
spp. on these reefs has led to lower coral recruitment, growth, and
survivorship [24,56].
One drawback of our experimental design is that we did not
include an algal mimic treatment: (e.g., a piece of plastic placed in
physical contact with coral fragments). Such a treatment would
have indicated if the effect of live macroalgae on microbial
communities and coral growth was a result of multiple factors or
solely physical abrasion or shading. However, previous studies of
coral-algal competition have shown that algal mimics have
minimal effects on coral growth and health [52,56]. Further, the
fact that different algal species had different effects on coral
growth, as well as coral-associated microbial communities, suggests
that macroalgae impact corals in species-specific ways, rather than
via general physical contact or shading.
Interactions with macroalgae could also lead to direct down-
stream changes in coral physiology (e.g., differential mucus layer
polysaccharide composition and output); these physiological shifts
could ultimately result in alterations to the coral microbiome. We
suggest, however, that a direct or combined effect of macroalgae
on the microbial community and coral host is most likely, given
that past work has demonstrated that: (1) applications of DOC and
alleopathic compounds to corals alter microbial growth rates in situ
[48,57], and (2) the application of antibiotics alleviates many of the
negative effects of algae competition on corals [22].
This study is among the first to empirically and quantitatively
analyze shifts in coral-associated microbial communities resulting
from competition with macroalgae. We show here that the
presence of certain macroalgal species reduces the growth rate of
the coral P. astreoides. These reduced coral growth rates occurred
concomitantly with changes in their microbial community
composition. Furthermore, contact with macroalgae can relax
coral-microbial specificity, allowing microbial taxa that are not
normally associated or exceedingly rare with a given coral host to
become established. Given the increasing abundance of macro-
algae in tropical coastal environments, interactions among
macroalgae, corals, and microbes are likely to play a role in
shaping the ecology of future reefs.
Supporting Information
Figure S1 Schematic and Picture of Coral-algal Competition
Experiment. (A). The combination of macroalgal treatments on
a specific cinderblock (e.g., D. menstrualis and H. tuna on
Cinderblock 1 below) was randomized within each block of the
experiment. (B). The figure represents one complete block
containing one replicate of each of five algal species treatments
and the no-algae control. Fragments of Porites astreoides within
a block of the experiment were all generated from the same
original colony in order to minimize intraspecific differences in
coral growth patterns.
Figure S2 Pie Charts of Major TRFs in Each Coral Treatment
of Algae Thalli as Measured by Mean Relative TRF abundance.
TRF peak heights were averaged and percent contribution to the
community measured. Any TRF that represented $3% of the
community was plotted in the pie charts.
Table S1 ANOSIM Results of Macroalgal-associated and
Coral-associated Microbial Communities. Global R is 0.772 and
significance level of sample statistic is 0.001. Bold text indicates
a significant difference.
Table S2 SIMPER Analysis of Macroalgae-associated Microbial
Communities. Bold indicated total percent similarity. The most
similar (Sim) or dissimilar (Diss) TRFs are followed by their
average contribution to similarity or dissimilarity between two the
macroalgae taxa.
Table S3 SIMPER Analysis of Coral-associated Communities
After Prolonged Contact with Macroalgae. Bold indicated total
similarity. The most similar (Sim) or dissimilar (Diss) TRFs are
followed by their percent contribution to the total similarity or
Table S4 TRF ANOVA Data for Coral-Algal Competition
Experiments Statistical values for one way ANOVA on TRF
abundance data. C = control corals, D = D. menstrualis exposed
corals, G = G. obtusata exposed corals, H = H. tuna exposed corals,
L= L. variegata exposed corals, and S = S. polyceratium exposed
We thank the Florida Keys National Marine Sanctuary for Permit #
FKNMS-2009-047 to conduct this work. We are grateful to Drs. James S.
Klaus and Alan Piggot for helpful advice regarding T-RFLP and the use of
their T-RFLP protocol.
Author Contributions
Conceived and designed the experiments: RVT DEB AAS AMSC.
Performed the experiments: RVT DEB AMSC AAS CP ART SR RMW.
Analyzed the data: RVT DEB AMSC ART. Contributed reagents/
materials/analysis tools: RVT DEB AMSC ART SR. Wrote the paper:
Macroalgal Effects on Coral Microbiome
PLOS ONE | 9 September 2012 | Volume 7 | Issue 9 | e44246
1. Knowlton N, Rohwer F (2003) Multispecies microbial mutualisms on coral reefs:
The host as a habitat. American Naturalist 162: S51–S62.
2. Ainsworth TD, Vega Thurber R, Gates RD (2010) The future of coral reefs:
a microbial perspective. Trends in Ecology & Evolution 25: 233–240.
3. Rohwer F, Seguritan V, Azam F, Knowlton N (2002) Diversity and distribution
of coral-associated bacteria. Marine Ecology Progress Series 243: 1–10.
4. Reshef L, Koren O, Loya Y, Zilber-Rosenberg I, Rosenberg E (2006) The Coral
Probiotic Hypothesis. Environmental Microbiology 8: 2068–2073.
5. Ritchie KB (2006) Regulation of microbial populations by coral surface mucus
and mucus-associated bacteria. Marine Ecology Progress Series 322: 1–14.
6. Williams WM, Viner AB, Broughton WJ (1987) Nitrogen-fixation (acetylene-
reduction) associated with the living coral Acropora variabilis. Marine Biology 94:
7. Lesser MP, Mazel CH, Gorbunov MY, Falkowski PG (2004) Discovery of
symbiotic nitrogen-fixing cyanobacteria in corals. Science 305: 997–1000.
8. Shashar N, Cohen Y, Loya Y, Sar N (1994) Nitrogen fixation (acetylene
reduction) in stony corals - evidence for coral-bacteria interactions. Marine
Ecology Progress Series 111: 259–264.
9. Ferrer LM, Szmant AM (1988) Nutrient regeneration by the endolithic
community in coral skeletons. Proceedings of the 6th International Coral Reef
Symposium 2: 1–4.
10. Fine M, Loya Y (2002) Endolithic algae: an alternative source of photo-
assimilates during coral bleaching. Proceedings of the Royal Society of London
Series B 269: 1205–1210.
11. Raina JB, Tapiolas D, Willis BL, Bourne DG (2009) Coral-associated bacteria
and their role in the biogeochemical cycling of sulfur. Applied and
Environmental Microbiology 75: 3492–3501.
12. Wegley L, Edwards R, Beltran R-B, Hong L, Forest R (2007) Metagenomic
analysis of the microbial community associated with the coral Porites astreoides.
Environmental Microbiology 9: 2707–2719.
13. Rosenberg E, Koren O, Reshef L, Efrony R, Zilber-Rosenberg I (2007) The role
of microorganisms in coral health, disease and evolution. Nature Reviews
Microbiology 5: 355–362.
14. Hughes TP, Baird AH, Bellwood DR, Card M, Connolly SR, et al. (2003)
Climate change, human impacts, and the resilience of coral reefs. Science 301:
15. Jackson JBC (2008) Ecological extinction and evolution in the brave new ocean.
Proceedings of the National Academy of Sciences of the United States of
America 105: 11458–11465.
16. Gardner TA, Cote IM, Gill JA, Grant A, Watkinson AR (2003) Long-term
region-wide declines in Caribbean corals. Science 301: 958–960.
17. Cote IM, Gill JA, Gardner TA, Watkinson AR (2005) Measuring coral reef
decline through meta-analyses. Philosophical Transactions of the Royal Society
B-Biological Sciences 360: 385–395.
18. Schutte VGW, Selig ER, Bruno JF (2010) Regional spatio-temporal trends in
Caribbean coral reef benthic communities. Marine Ecology Progress Series 402:
19. Aronson RB, Precht WF (2006) Conservation, precaution, and Caribbean reefs.
Coral Reefs 25: 441–450.
20. Mumby PJ (2009) Herbivory versus corallivory: are parrotfish good or bad for
Caribbean coral reefs? Coral Reefs 28: 683–690.
21. Burkepile DE, Hay ME (2006) Herbivore vs. nutrient control of marine primary
producers: Context-dependent effects. Ecology 87: 3128–3139.
22. Smith JE, Shaw M, Edwards RA, Obura D, Pantos O, et al. (2006) Indirect
effects of algae on coral: algae-mediated, microbe-induced coral mortality.
Ecology Letters 9: 835–845.
23. Nugues MM, Smith GW, Hooidonk RJ, Seabra MI, Bak RPM (2004) Algal
contact as a trigger for coral disease. Ecology Letters 7: 919–923.
24. Hughes TP, Rodrigues MJ, Bellwood DR, Ceccarelli D, Hoegh-Guldberg O,
etal. (2007) Phase shifts, herbivory, and the resilience of coral reefs to climate
change. Current Biology 17: 360–365.
25. Nugues MM, Bak RPM (2006) Differential competitive abilities between
Caribbean coral species and a brown alga: a year of experiments and a long-
term perspective. Marine Ecology Progress Series 315: 75–86.
26. Burkepile DE, Hay ME (2008) Herbivore species richness and feeding
complementarity affect community structure and function on a coral reef.
Proceedings of the National Academy of Sciences of the United States of
America 105: 16201–16206.
27. Lewis SM (1986) The role of herbivorous fishes in the organization of
a Caribbean reef community. Ecological Monographs 56: 183–200.
28. Kuffner IB, Walters LJ, Beccero MA, Paul VJ, Ritson-Williams R, et al. (2006)
Inhibition of coral recruitment by macroalgae and cyanobacteria. Marine
Ecology Progress Series 323: 107–117.
29. McCook LJ, Jompa J, Diaz-Pulido G (2001) Competition between corals and
algae on coral reefs: a review of evidence and mechanisms. Coral Reefs 19: 400–
30. Paddack MJ, Cowen RK, Sponaugle S (2006) Grazing pressure of herbivorous
coral reef fishes on low coral-cover reefs. Coral Reefs 25: 461–472.
31. Burkepile DE, Hay ME (2011) Feeding complementarity versus redundancy
among herbivorous fishes on a Caribbean reef. Coral Reefs 30: 351–362.
32. Green DH, Edmunds PJ, Carpenter RC (2008) Increasing relative abundance of
Porites astreoides on Caribbean reefs mediated by an overall decline in coral cover.
Marine Ecology Progress Series 359: 1–10.
33. Marsh JA (1970) Primary productivity of reef-building calcareous red algae.
Ecology 51: 255–263.
34. Davies PS (1989) Short-term growth measurements of corals using an accurate
buoyant weighing technique. Marine Biology 101: 389–395.
35. Holm S (1979) A simple sequential rejective multiple test procedure.
Scandinavian Journal of Statistics 6: 65–70.
36. Underwood AJ (1997) Experiments in ecology: their logical design and
interpretation using analysis of variance. Cambridge, UK: Cambridge
University Press. 524 p.
37. Baker AC, Rowan R, Knowlton N (1997) Symbiosis ecology of two Caribbean
acroporid corals. Proceedings of the International Coral Reef Symposium, 8th,
Panama 2: 1295–1300.
38. Liu WT, Marsh TL, Cheng H, Forney LJ (1997) Characterization of microbial
diversity by determining terminal restriction fragment length polymorphisms of
genes encoding 16S rRNA. Applied and Environmental Microbiology 63: 4516–
39. Klaus JS, Frias-Lopez J, Bonheyo GT, Heikoop JM, Fouke BW (2005) Bacterial
communities inhabiting the healthy tissues of two Caribbean reef corals:
interspecific and spatial variation. Coral Reefs 24: 129–137.
40. Blackwood CB, Marsh T, Kim S-H, Paul EA (2003) Terminal Restriction
Fragment Length Polymorphism data analysis for quantitative comparison of
microbial communities. Applied and Environmental Microbiology 69: 926–932.
41. Hughes JB, Hellmann JJ, Ricketts TH, Bohannan BJM (2001) Counting the
uncountable: Statistical approaches to estimating microbial diversity. Applied
and Environmental Microbiology 67: 4399–4406.
42. Clarke KR (1993) Non-parametric multivariate analyses of changes in
community structure. Australian Journal of Ecology 18: 117–143.
43. Warwick RM, Clarke KR (1993) Increased variability as a symptom of stress in
marine communities. Journal of Experimental Marine Biology And Ecology 172:
44. Barott KL, Rodriguez-Brito B, Janousˇkovec J, Marhaver KL, Smith JE, et al.
(2011) Microbial diversity associated with four functional groups of benthic reef
algae and the reef-building coral Montastraea annu laris. Environmental Microbi-
ology: 1–13.
45. Littman RA, Willis BL, Bourne DG (2011) Metagenomic analysis of the coral
holobiont during a natural bleaching event on the Great Barrier Reef.
Environmental Microbiology Reports 3: 651–660.
46. Vega Thurber R, Willner-Hall D, Rodriguez-Mueller B, Desnues C, Edwards
RA, et al. (2009) Metagenomic analysis of stressed coral holobionts.
Environmental Microbiology 11: 2148–2163.
47. Nelson CE, Alldredge AL, McCliment EA, Amaral-Zettler LA, Carlson CA
(2011) Depleted dissolved organic carbon and distinct bacterial communities in
the water column of a rapid-flushing coral reef ecosystem. ISME Journal 5:
48. Haas AF, Nelson CE, Wegley Kelly L, Carlson CA, Rohwer F, et al. (2011)
Effects of coral reef benthic primary producers on dissolved organic carbon and
microbial activity. PLoS ONE 6: e27973.
49. Kline DI, Kuntz NM, Breitbart M, Knowlton N, Rohwer F (2006) Role of
elevated organic carbon levels and microbial activity in coral mortality. Marine
Ecology Progress Series 314: 119–125.
50. Brylinsky M (1977) Release of dissolved organic matter by some marine
macrophytes. Marine Biology 39: 213–230.
51. Haas AF, Jantzen C, Naumann MS, Iglesias-Prieto R, Wild C (2010) Organic
matter release by the dominant primary producers in a Caribbean reef lagoon:
implication for in situ O(2) availability. Marine Ecology Progress Series 409: 27–
52. Rasher DB, Hay ME (2010) Chemically rich seaweeds poison corals when not
controlled by herbivores. Proceedings of the National Academy of Sciences of
the United States of America 107: 9683–9688.
53. Morrow KM, Paul VJ, Liles MR, Chadwick NE (2011) Allelochemicals
produced by Caribbean macroalgae and cyanobacteria have species-specific
effects on reef coral microorganisms. Coral Reefs 30: 309–320.
54. Barott KL, Rodriguez-Mueller B, Youle M, Marhaver KL, Vermeij MJA, et al.
(2011) Microbial to reef scale interactions between the reef-building coral
Montastraea annularis and benthic algae. Proceedings of the Royal Society B:
Biological Sciences.
55. Barott KL, Smith J, Dinsdale E, Hatay M, Sandin S, et al. (2009) Hyperspectral
and physiological analyses of coral-algal interactions. PLoS ONE 4: e8043.
56. River GF, Edmunds PJ (2001) Mechanisms of interaction between macroalgae
and scleractinians on a coral reef in Jamaica. Journal of Experimental Marine
Biology and Ecology 261: 159–172.
57. Kuntz NM, Kline DI, Sandin SA, Rohwer F (2005) Pathologies and mortality
rates caused by organic carbon and nutrient stressors in three Caribbean coral
species. Marine Ecology Progress Series 294: 173–180.
Macroalgal Effects on Coral Microbiome
PLOS ONE | 10 September 2012 | Volume 7 | Issue 9 | e44246
... Environmental factors, such as geographic location, water depth, nutrient concentration, and temperature, also have significant effects (Apprill et al. 2009;Vega Thurber et al. 2009;Littman et al. 2011). Therefore, the relationship between bacterial communities and their hosts or the environment have been described in terms of bacterial diversity associated with corals (Rohwer et al. 2002), host specificity (Ceh et al. 2011), changes in bacterial communities in response to competition between corals and macroalgae (Vega Thurber et al. 2012), and coral disease (Rohwer et al. 2002;Ceh et al. 2011;Vega Thurber et al. 2012;Wilson et al. 2012). However, changes in the temporal and spatial diversity of bacterial communities in response to environmental changes have not been widely studied. ...
... Environmental factors, such as geographic location, water depth, nutrient concentration, and temperature, also have significant effects (Apprill et al. 2009;Vega Thurber et al. 2009;Littman et al. 2011). Therefore, the relationship between bacterial communities and their hosts or the environment have been described in terms of bacterial diversity associated with corals (Rohwer et al. 2002), host specificity (Ceh et al. 2011), changes in bacterial communities in response to competition between corals and macroalgae (Vega Thurber et al. 2012), and coral disease (Rohwer et al. 2002;Ceh et al. 2011;Vega Thurber et al. 2012;Wilson et al. 2012). However, changes in the temporal and spatial diversity of bacterial communities in response to environmental changes have not been widely studied. ...
Full-text available
Microbes in the coral holobiont play important roles in nitrogen fixation, carbon supply, antibiotic production , mucus recycling, and food supply to maintain homeostasis in corals. However, microbes can also induce coral diseases in response to environmental changes under non-optimal conditions. Therefore, studies of microbial communities are needed to understand the health statuses of corals in response to environmental changes. In this study, we performed 16S rDNA metabarcoding to investigate the bacterial communities in two healthy alcy-onacean soft coral species (Sinularia sp. and Litophyton sp.) inhabiting the coast of Weno Island (Chuuk, Micronesia) and in ambient seawater. We identified 18 bacterial phyla, 24 classes, 54 orders, 109 families, and 222 genera associated with the two corals and seawater. The bacterial communities differed in the corals and seawater. The bacterial community in Sinularia sp. was dominated by the genus Spirochaeta in Spirochaetaceae (63.9% relative abundance), followed by Endozoicomonas (10%). In Litophyton sp., the bacterial community also contained Spirochaeta (19.5%) and Endozoicomonas (4.7%), although Cellvibrionaceae (23.7%) was dominant and other groups such as Rhizobiales (11.5%) and Rhodospirillales (8.7%) were evenly distributed. In ambient seawater, the predominant bacteria were Pelagibacter (29.2%), Rhodobacteraceae (15.5%), Prochlorococcus (11.3%), and Vibrio (5.8%), which are distinct from the species in the two coral species. The microbial communities between the two alcyonacean soft corals and seawater were different, and the microbial community differences were coral species specific .
... Conversely, some algae produce chemical defenses that alter or inhibit bacterial colonization [26,27]. Thus, at coral-algal interfaces, it is conceivable that the presence of algal allelochemicals and the potential for a resultant coral immune response, combined with the interactions among members of these hosts' microbiomes, may create a unique environment that gives rise to production of novel chemicals [28][29][30][31][32][33]. ...
Full-text available
Microbes produce natural products that mediate interactions with each other and with their environments, representing a potential source of antibiotics for human use. The biosynthesis of some antibiotics whose constitutive production otherwise remains low has been shown to be induced by competing microbes. Competition among macroorganism hosts may further influence the metabolic outputs of members of their microbiomes, especially near host surfaces where hosts and microbial symbionts come into close contact. At multiple field sites in Fiji, we collected matched samples of corals and algae that were freestanding or in physical contact with each other, cultivated bacteria from their surfaces, and explored growth-inhibitory activities of these bacteria against marine and human pathogens. In the course of the investigation, an interaction was discovered between two coral-associated actinomycetes in which an Agrococcus sp. interfered with the antibiotic output of a Streptomyces sp. Several diketopiperazines identified from the antibiotic-producing bacterium could not, on their own, account for the antibiotic activity indicating that other, as yet unidentified molecule(s) or molecular blends, possibly including diketopiperazines, are likely involved. This observation highlights the complex molecular dynamics at play among microbiome constituents. The mechanisms through which microbial interactions impact the biological activities of specialized metabolites deserve further attention considering the ecological and commercial importance of bacterial natural products.
... After the human impact of overharvesting and disappearance of the large vertebrates, D. antillarum and small fishes functioned as the primary grazers (Jackson, 2001;Pandolfi et al. 2003;Steneck 2020). Hence, the disappearance of this keystone echinoid herbivore from disease resulted in immediate algal growth with broad repercussions on reef ecology that disrupted the trophic cascade with a swift and sustained phase shift that altered the reef biome dynamics from coral dominance to cover by macroalgae with associated reduction in species diversity (Liddell and Ohlhorst 1986;Hughes et al. 1987;Carpenter 1988Carpenter , 1990Hughes 1994;Ferrari Legorreta 2012;Vega Thurber et al. 2012;Burkepile et al. 2013;Menge et al. 2016;Schultz et al. 2016;Steneck 2020). This exact phase shift was predicted prior to the die-off event by exclusion experiments in which D. antillarum was removed from patches of reefs in Jamaica and demonstrated significant growth of soft algae in those patches that could out compete many coral species for space on the substrate (Sammarco 1980). ...
... Commonly called seaweeds, macroalgae are multicellular marine organisms that float as free-living forms or are affixed to hard substrates such as rocks. "Macroalgae forests" have important roles in coastal ecosystems: they provide habitat for many species, shape coralmicrobial ecology, protect organisms from storms, reduce deoxygenation and acidification, maintain biogeochemical cycling and storage, and contribute to fishery yields (Vega Thurber et al., 2012;Filbee-Dexter and Wernberg, 2018;Duffy et al., 2019). In addition, as a biomass resource, macroalgae can help achieve the United Nations' sustainable development goals (SDGs), mitigating CO 2 emissions but not competing with staple food crops. ...
Full-text available
Marine macroalgae have huge potential as feedstocks for production of a wide spectrum of chemicals used in biofuels, biomaterials, and bioactive compounds. Harnessing macroalgae in these ways could promote wellbeing for people while mitigating climate change and environmental destruction linked to use of fossil fuels. Microorganisms play pivotal roles in converting macroalgae into valuable products, and metabolic engineering technologies have been developed to extend their native capabilities. This review showcases current achievements in engineering the metabolisms of various microbial chassis to convert red, green, and brown macroalgae into bioproducts. Unique features of macroalgae, such as seasonal variation in carbohydrate content and salinity, provide the next challenges to advancing macroalgae-based biorefineries. Three emerging engineering strategies are discussed here: (1) designing dynamic control of metabolic pathways, (2) engineering strains of halophilic (salt-tolerant) microbes, and (3) developing microbial consortia for conversion. This review illuminates opportunities for future research communities by elucidating current approaches to engineering microbes so they can become cell factories for the utilization of macroalgae feedstocks.
... They can help to mediate the competition between corals and macroalgae and enhancing the resilience of coral reef ecosystems following anthropogenic or natural disturbance (Adam et al. 2015). By exerting top-down control on algal communities in a cropped state can provide more space resources for corals and promote the attachment and recruitment of coral larvae, whcih is a vital ecological process (Bellwood et al. 2012;Thurber et al. 2012;Adam et al. 2015;Roos et al. 2016). Compared to other herbivorous sh, parrot sh have specialized feeding morphology that can remove the calcareous surface layers of the reef as they graze and get nutritional resources that are largely unavailable to other shes. ...
Full-text available
Based on the key ecological processes of parrotfish in coral reefs, we compiled species presence-absence data across 51 sites in the South China Sea to identify the distribution and composition of parrotfish and explore the relationship between species distribution and environmental factors, and 50 species (the Pacific: 57 species) of parrotfish were record. Nansha islands had the highest abundance with 41 parrotfish species. Nestedness analysis indicated parrotfish community had statistically significant nested patterns in the South China Sea and Nansha islands was the topmost site of nested matrix rank. Scleractinian coral species richness and Log(reef area) both had a significant effect on sites nested matrix rank ( P < 0.05), which supports habitat nestedness hypothesis in the South China Sea. Scrapers were the most important functional group composition while the browser had a greater contribution on species nested matrix rank. Linear regression model showed parrotfish species richness increased with increasing longitude, scleractinian coral species richness and reef area. Variations in the parrotfish species richness in longitude was related to distance from the biodiversity hotspot in the Indo-Australian Archipelago. Parrotfish was mainly distributed in the range of 26-29℃, which was almost the same as the optimum temperature for coral growth. Nansha islands should be as biodiversity conservation priority areas, which could provide important reference significance for conservation efforts of parrotfish in degraded coral reefs habitats, especially in the context of increasing natural variability and anthropogenic disturbance.
... The microbial communities of coral can change rapidly in response to host illness and stress (Reshef et al. 2006, Vega-Thurber et al. 2012, Kimes et al. 2013. Coral microbial communities have been shown to shift in as little as 2 days after being exposed to stressful environmental situations, such as spring tides (Sweet et al. 2017). ...
Full-text available
Coral reefs are in decline globally as anthropogenic induced climate change effects ravage our oceans. It is estimated that at least 50% of coral reefs have disappeared over the last 40 years and are declining at an alarming rate. While much scientific research focuses on healthy coral reef ecosystems, data suggests that degraded watersheds with high levels of selective pressure may harbor coral species that are well adapted to stress. A thriving population of corals exists in Honolulu Harbor, a highly degraded ocean habitat exposed to multiple anthropogenic stressors. Following the massive molasses spill in 2013, two species of corals have shown remarkable resilience to multiple stressors. Both species were observed to be brooders, with Leptastrea purpurea demonstrating a larval peak in the late summer. L. purpurea planula larvae are induced by a settlement cue originating from other coral colonies. When a coral scent is present, settlement rates are as high as 90-100% on biofilm and other substrates, including plain untreated glass. Field surveys reveal that L. purpurea colonies are found on average 18mm in distance from their nearest neighbor, and modeling suggests a non-random distribution of colonies at our survey sites. A second species, Harbor Porites, is genetically distinct from Porites lobata, though genetics show a similarity in origin. Harbor Porites larvae will settle in the presence of a biofilm cue, and both larvae and recruits show remarkable resilience to multiple chemical and physical stressors, as well as the ability to undergo reversible metamorphosis. Both coral species are in high abundance inside Honolulu harbor, and coral surveys reveal that the two species are found within an average 16mm of each other. To elucidate the high level of survival in these two species, a thermal tolerance exposure was performed to induce a bleaching response in both species. Molecular biomarkers were used to quantify relative stress levels. Molecular expression analyses could give us insights into how these corals are responding to stress, and if the basis for their resilience is tied to up-regulated molecular processes. While corals continue to face stress as a result of climate change, these two harbor coral species serve as excellent models for studying the resilience of corals to stress. Their persistence in a stressful environment makes them candidate species for coral reef restoration.
Full-text available
Effective coral restoration must include comprehensive investigations of the targeted coral community that consider all aspects of the coral holobiont—the coral host, symbiotic algae, and microbiome. For example, the richness and composition of microorganisms associated with corals may be indicative of the corals’ health status and thus help guide restoration activities. Potential differences in microbiomes of restoration corals due to differences in host genetics, environmental condition, or geographic location, may then influence outplant success. The objective of the present study was to characterize and compare the microbiomes of apparently healthy Acropora cervicornis genotypes that were originally collected from environmentally distinct regions of Florida’s Coral Reef and sampled after residing within Mote Marine Laboratory’s in situ nursery near Looe Key, FL (USA) for multiple years. By using 16S rRNA high-throughput sequencing, we described the microbial communities of 74 A. cervicornis genotypes originating from the Lower Florida Keys ( n = 40 genotypes), the Middle Florida Keys ( n = 15 genotypes), and the Upper Florida Keys ( n = 19 genotypes). Our findings demonstrated that the bacterial communities of A. cervicornis originating from the Lower Keys were significantly different from the bacterial communities of those originating from the Upper and Middle Keys even after these corals were held within the same common garden nursery for an average of 3.4 years. However, the bacterial communities of corals originating in the Upper Keys were not significantly different from those in the Middle Keys. The majority of the genotypes, regardless of collection region, were dominated by Alphaproteobacteria, namely an obligate intracellular parasite of the genus Ca. Aquarickettsia . Genotypes from the Upper and Middle Keys also had high relative abundances of Spirochaeta bacteria. Several genotypes originating from both the Lower and Upper Keys had lower abundances of Aquarickettsia , resulting in significantly higher species richness and diversity. Low abundance of Aquarickettsia has been previously identified as a signature of disease resistance. While the low- Aquarickettsia corals from both the Upper and Lower Keys had high abundances of an unclassified Proteobacteria, the genotypes in the Upper Keys were also dominated by Spirochaeta . The results of this study suggest that the abundance of Aquarickettsia and Spirochaeta may play an important role in distinguishing bacterial communities among A. cervicornis populations and compositional differences of these bacterial communities may be driven by regional processes that are influenced by both the environmental history and genetic relatedness of the host. Additionally, the high microbial diversity of low- Aquarickettsia genotypes may provide resilience to their hosts, and these genotypes may be a potential resource for restoration practices and management.
Nutrient pollution is linked to coral disease susceptibility and severity, but the mechanism behind this effect remains underexplored. A recently-identified bacterial species, ‘Ca. Aquarickettsia rohweri,’ is hypothesized to parasitize the Caribbean staghorn coral, Acropora cervicornis, leading to reduced coral growth and increased disease susceptibility. Aquarickettsia rohweri is hypothesized to assimilate host metabolites and ATP and was previously demonstrated to be highly nutrient-responsive. As nutrient enrichment is a pervasive issue in the Caribbean, this study examined the effects of common nutrient pollutants (nitrate, ammonium, and phosphate) on a disease-susceptible genotype of Acropora cervicornis. Microbial diversity was found to decline over the course of the experiment in phosphate-, nitrate-, and combined-treated samples, and quantitative PCR indicated that Aquarickettsia abundance increased significantly across all treatments. Only treatments amended with phosphate, however, exhibited a significant shift in Aquarickettsia abundance relative to other taxa. Furthermore, corals exposed to phosphate had significantly lower linear extension than untreated or nitrate-treated corals after 3 weeks of nutrient exposure. Together these data suggest that while experimental tank conditions, with an elevated nutrient regime associated with coastal waters, increased total bacterial abundance, only the addition of phosphate significantly altered the ratios of Aquarickettsia compared to other members of the microbiome.
Full-text available
Pollution is the major problem in the present era that has adversely affected the structure and functioning of all ecosystems on the earth. Coral reefs are no exception to the pollution that directly or indirectly possess health threat to coral and associated animals, which in turn adversely impact the functioning and productivity of the reef ecosystem. The present chapter aims to collate and assesses the available information on the pollutants in the coral environment and their effects on the coral and associated organisms. The present available tools and techniques for evaluation of pollution stress on coral reefs at organismal or community levels were discussed with the strategies to monitor, understand, and mitigate the effect of pollutants on the reef ecosystem. Overall, the present chapter improves the understanding of pollutants and their mitigation strategies to conserve the reef ecosystem.
Full-text available
Macroalgae play an intricate role in microbial-mediated coral reef degradation processes due to the release of dissolved nutrients. However, temporal variabilities of macroalgal surface biofilms and their implication on the wider reef system remain poorly characterized. Here, we study the microbial biofilm of the dominant reef macroalgae Sargassum over a period of one year at an inshore Great Barrier Reef site (Magnetic Island, Australia). Monthly sampling of the Sargassum biofilm links the temporal taxonomic and putative functional metabolic microbiome changes, examined using 16S rRNA gene amplicon and metagenomic sequencing, to the pronounced growth-reproduction-senescence cycle of the host. Overall, the macroalgal biofilm was dominated by the heterotrophic phyla Firmicutes (35% ± 5.9% SD) and Bacteroidetes (12% ± 0.6% SD); their relative abundance ratio shifted significantly along the annual growth-reproduction-senescence cycle of Sargassum. For example, Firmicutes were 1.7 to 3.9 times more abundant during host growth and reproduction cycles than Bacteroidetes. Both phyla varied in their carbohydrate degradation capabilities; hence, temporal fluctuations in the carbohydrate availability are potentially linked to the observed shift. Dominant heterotrophic macroalgal biofilm members, such as Firmicutes and Bacteroidetes, are implicated in exacerbating or ameliorating the release of dissolved nutrients into the ambient environment, though their contribution to microbial-mediated reef degradation processes remains to be determined.
Full-text available
Baker AC, Rowan R, Knowlton N (1997) Symbiosis ecology of two Caribbean acroporid corals
Full-text available
Coral cover has declined on reefs worldwide with particularly acute losses in the Caribbean. Despite our awareness of the broad-scale patterns and timing of Caribbean coral loss, there is little published information on: (1) finer-scale, subregional patterns over the last 35 yr, (2) regional-scale trends since 2001, and (3) macroalgal cover changes. We analyzed the spatiotemporal trends of benthic coral reef communities in the Caribbean using quantitative data from 3777 coral cover surveys of 1962 reefs from 1971 to 2006 and 2247 macroalgal cover surveys of 875 reefs from 1977 to 2006. A subset of 376 reefs was surveyed more than once (monitored). The largest 1 yr decline in coral cover occurred from 1980 to 1981, corresponding with the beginning of the Caribbean-wide Acropora spp. white band disease outbreak. Our results suggest that, regionally, coral cover has been relatively stable since this event (i.e. from 1982 to 2006). The largest increase in macroalgal cover was in 1986, 3 yr after the regional die-off of the urchin grazer Diadema antillarum began. Subsequently, macroalgal cover declined in 1987 and has been stable since then. Regional mean (±1 SE) macroalgal cover from 2001 to 2005 was 15.3 ± 0.4% (n = 1821 surveys). Caribbeanwide mean (±1 SE) coral cover was 16.0 ± 0.4% (n = 1547) for this same time period. Both macroalgal and coral cover varied significantly among subregions from 2001 to 2005, with the lowest coral cover in the Florida Keys and the highest coral cover in the Gulf of Mexico. Spatio-temporal patterns from the subset of monitored reefs are concordant with the conclusions drawn from the entire database. Our results suggest that coral and macroalgal cover on Caribbean reef benthic communities has changed relatively little since the mid-1980s.
Full-text available
The competitive replacement of corals by benthic algae is considered key to reef degradation. Such replacement could originate from direct competitive overgrowth of corals by algae or death of corals from other disturbances, followed by an increase in algal abundance. To assess the relative importance of these processes, this study experimentally tested the competitiveness of 6 Caribbean coral species against the brown alga Lobophora variegata on a fringing reef in Curaçao, Netherlands Antilles. This alga has a widespread distribution and is considered particularly aggressive towards corals due to its creeping growth form. We compared the growth of transplanted algae over living and dead coral, as well as coral tissue mortality in the presence and absence of transplanted algae over a 1 yr period. Competitive trends were also related to changes in species abundance from 1973 to 2002 on the same reef. The results indicated that only 1 species, Agaricia agaricites, was competitively inferior to L. variegata and suffered more tissue mortality when exposed to the algae. Surveys of naturally occurring interactions showed that less competitive species were generally more overgrown by L. variegata, further reinforcing our results. Importantly, A. agaricites experienced the greatest decline in percent cover from 1973 to 2002 among the studied species. A large proportion of this decline occurred following the die-off of Diadema antillarum in 1983, which generally marks the onset of increased algal abundance on Caribbean reefs. We concluded that Caribbean corals have different competitive abilities against algae, highlighting the complexity and species-specific nature of coral-algal interactions. Although our data supports that prior death of corals may be generally required for algae to become established, competition with algae could play a significant role in structuring coral communities by reducing the abundance of poor competitive species. We suggest that a species-by-species approach is needed to understand the factors influencing transitions from coral to algal dominance on Caribbean reefs.
Full-text available
Nitrogen fixation, as measured by acetylene reduct~on, has been detected to be associated with various hermatypic corals. Experiments were carried out on the massive coral Favia f a v u s both in situ and in the laboratory. Nitrogen fixation activity was found to be light dependent and fully inhibited by 5 X 10-6 M DCMU [3-(3,4-dichloropheny1)-1,l-dimethylurea Addition of glucose restored nitrogen fixation activity both in the dark and in the presence of DCMU. Removal of the coral tissue prevented acetylene reduction, while addition of glucose to the coral skeleton restored this activity. Bacteria isolated from the coral skeleton were found by dot blotting to contain the nif H gene. These results suggest that nitrogen-fixing bacteria found in the skeleton of corals benefit from organic carbon excreted by the coral tissue. The interaction between the nitrogen-fixing organisms and the coral may be of major lrnportance for the nitrogen budget of the corals.
Full-text available
Experimental manipulations of grazing intensity were used to examine the role of herbivorous fishes in the families Acanthuridae (surgeonfishes) and Scaridae (parrotfishes) in determining distributions and abundances of benthic species within and among shallow tropical reef habitats. A back reef habitat along the Belizean barrier reef was characterized by a diverse benthic assemblage of algal turfs, coralline algae, and the coral Porites astreoides, but by extremely low macroalgal abundance. In contrast, several nearby shallow habitats were dominated by dense stands of several macroalgal species. Experimental reduction of herbivorous fish grazing in the back reef (achieved by constructing exclosures) rapidly and dramatically altered existing patterns of benthic species composition and species abundances. After 10 wk of reduced herbivory, total macroalgal abundance increased significantly in herbivore exclusion areas relative to unmanipulated controls, and was correlated with decreased percent cover of available space, several algal turf species, crustose coralline algae, and Porites. Some macroalgal species were able to directly overgrow and kill portions of Porites colonies within herbivore exclusion treatments. Successful recruitment and growth of several algal species under experimentally reduced herbivory indicated that macroalgal species distributions may be limited by herbivory rather than by lack of spore availability or unsuitable physical conditions. Algal turfs characteristic of many reef habitats appear to represent herbivore-tolerant assemblages, persisting under high grazing intensity but responding rapidly to reduced herbivory with increased abundances, morphological changes, and altered reproductive status. These results suggest that herbivorous fish grazing profoundly influences benthic species distributions and abundances within some tropical reef habitats. Spatial variation in herbivory appears to be of fundamental importance in determining regional patterns of benthic community structure on tropical reefs. The spatial mosaic of benthic community composition among shallow reef habitats was associated with patterns of grazing intensity by herbivorous fishes. Several reef habitats supporting dense macroalgal stands represented spatial refuges from herbivory, with low herbivorous fish densities and reduced grazing intensities. Transplant experiments revealed that algal species characteristic of these low-herbivory habitats were highly susceptible to grazing by herbivorous fishes. Spatial heterogeneity in grazing intensity may contribute to high regional diversity among tropical reef habitats by maintaining different benthic species assemblages under fundamentally distinct selective regimes.
Full-text available
Coral reefs are the most biodiverse of all marine ecosystems; however, very little is known about prokaryotic diversity in these systems. To address this issue, we sequenced over 1000 bacterial 16S rDNAs from 3 massive coral species (Montastraea franksi, Diploria strigosa, and Porites astreoides) in Panama and Bermuda. Analysis of only 14 coral samples yielded 430 distinct bacterial ribotypes. Statistical analyses suggest that additional sequencing would have resulted in a total of 6000 bacterial ribotypes. Half of the sequences shared
Primary productivity of reef-building algae was studied by putting samples from the reef in a closed system and measuring oxygen exchange in the light and in the dark. Gross productivity determined for 32 samples in full sunlight had a mean value of 0.048 mg O"2 cm^@o^2 hr^@o^1. Photosynthesis was found to increase with the logarithm of light intensity up to 1,000 ft-c and was constant between 1,000 and 8,000 ft-c. Rates of gas exchange in flowing water showed no correlation with water velocity but were greater than rates in still water. Daily patterns of photosynthesis were calculated for populations of calcareous algae living on the submarine faces of the windward sides of atolls. During most of the daylight hours light is probably not a limiting factor for photosynthesis in these populations. Calculated productivity of various calcareous algal zones indicates that these do not contribute significantly to overall reef production on atolls of the northern Marshall Islands. Island reefs are less productive than previously studied inter-island reefs.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.