Content uploaded by Jose V Lopez
Author content
All content in this area was uploaded by Jose V Lopez on Nov 07, 2014
Content may be subject to copyright.
ORIGINAL RESEARCH ARTICLE
published: 04 November 2014
doi: 10.3389/fmicb.2014.00581
Two distinct microbial communities revealed in the sponge
Cinachyrella
Marie L. Cuvelier1*, Emily Blake2, Rebecca Mulheron2, Peter J. McCarthy3, Patricia Blackwelder2,4,
Rebecca L. Vega Thurber5and Jose V. Lopez2
1Biological Sciences Department, Florida International University, Miami, FL, USA
2Oceanographic Center, Nova Southeastern University, Dania Beach, FL, USA
3Marine Biomedical and Biotechnology Research, Harbor Branch Oceanographic Institute, Florida Atlantic University, Fort Pierce, FL, USA
4Marine Geosciences, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL, USA
5Department of Microbiology, Oregon State University, Corvallis, OR, USA
Edited by:
Torsten Thomas, The University of
New South Wales, Australia
Reviewed by:
Megan Jane Huggett, Edith Cowan
University, Australia
Heidi M. Luter, Charles Darwin
University, Australia
Robert W. Thacker, University of
Alabama at Birmingham, USA
*Correspondence:
Marie L. Cuvelier, Biological
Sciences Department, Florida
International University, 3000 NE
151st Street, Miami, FL, USA
e-mail: cuvelierml@gmail.com
Marine sponges are vital components of benthic and coral reef ecosystems, providing
shelter and nutrition for many organisms. In addition, sponges act as an essential carbon
and nutrient link between the pelagic and benthic environment by filtering large quantities
of seawater. Many sponge species harbor a diverse microbial community (including
Archaea, Bacteria and Eukaryotes), which can constitute up to 50% of the sponge
biomass. Sponges of the genus Cinachyrella are common in Caribbean and Floridian
reefs and their archaeal and bacterial microbiomes were explored here using 16S rRNA
gene tag pyrosequencing. Cinachyrella specimens and seawater samples were collected
from the same South Florida reef at two different times of year. In total, 639 OTUs
(12 archaeal and 627 bacterial) belonging to 2 archaeal and 21 bacterial phyla were
detected in the sponges. Based on their microbiomes, the six sponge samples formed
two distinct groups, namely sponge group 1 (SG1) with lower diversity (Shannon-Wiener
index: 3.73 ±0.22) and SG2 with higher diversity (Shannon-Wiener index: 5.95 ±
0.25). Hosts’ 28S rRNA gene sequences further confirmed that the sponge specimens
were composed of two taxa closely related to Cinachyrella kuekenthalli. Both sponge
groups were dominated by Proteobacteria, but Alphaproteobacteria were significantly
more abundant in SG1. SG2 harbored many bacterial phyla (>1% of sequences) present
in low abundance or below detection limits (<0.07%) in SG1 including: Acidobacteria,
Chloroflexi,Gemmatimonadetes,Nitrospirae, PAUC34f, Poribacteria,andVerrucomicrobia.
Furthermore, SG1 and SG2 only had 95 OTUs in common, representing 30.5 and 22.4%
of SG1 and SG2’s total OTUs, respectively. These results suggest that the sponge host
may exert a pivotal influence on the nature and structure of the microbial community and
may only be marginally affected by external environment parameters.
Keywords: marine sponge, symbionts, diversity, archaea, pyrosequencing, 16S rRNA, microbiome
INTRODUCTION
Sponges are one of the most primitive Metazoan life forms with
fossils dating from at least 580 million years ago (Li et al., 1998;
Ryan et al., 2013). Today, there are more than 8500 described
extant sponge species, most of which are marine (van Soest et al.,
2012). Marine sponges are ecologically important components
of the benthic community due to their wide diversity and high
biomass (Ilan et al., 2004; de Goeij et al., 2013). In addition,
they play a key functional role linking benthic and pelagic ecosys-
tems, as they efficiently remove particulate organic carbon from
theseawater(Díaz and Rützler, 2001; Ilan et al., 2004; Webster
et al., 2011). Indeed, these sessile invertebrates are able to filter
Abbreviations: AOA, ammonia-oxidizing archaea; AOB, ammonia-oxidizing bac-
teria; LMA, low microbial abundance; HMA, high microbial abundance; SG1,
sponge group 1; SG2, sponge group 2.
considerable amounts of seawater; a 1 kg sponge can filter up to
24000 L of water per day (Vogel, 1977). Because they are efficient
filter feeders, many sponges can live in nutrient-poor habitats
such as tropical reefs. However, because of their feeding mode,
they are also directly affected by water quality and are vulnera-
ble to marginal environmental conditions (Webster and Blackall,
2009).
Many sponge species consistently harbor dense and diverse
microbial communities including bacteria, archaea and eukary-
otes (Taylor et al., 2007b). Symbionts can contribute up to
50% of the sponge biomass (Wilkinson, 1978a,b,c; Hentschel
et al., 2006). Sponge-associated microorganisms include mem-
bers of two archaeal lineages and >30 different bacterial and
candidate phyla (Taylor et al., 2007b; Webster et al., 2008;
Zhu et al., 2008; Sipkema et al., 2009; Schmitt et al., 2012).
Many of these taxa form monophyletic sponge-specific clusters
www.frontiersin.org November 2014 | Volume 5 | Article 581 |1
Cuvelier et al. Microbes associated with Cinachyrella sponge
even though they are found in geographically and phylogeneti-
cally distinct sponge hosts (Taylor et al., 2007b; Simister et al.,
2012).
Although sequencing technology has revealed much about
the structural diversity of sponge associated microbiomes, rela-
tively little is known about the specific ecological relationships
and interactions among these sponge symbionts and their host
(Taylor et al., 2007a; Webster and Taylor, 2012). While sponges are
believed to provide a favorable environment to their symbionts,
the contribution of the symbionts to the host is less well under-
stood. However, phylogenetic inference suggests that associated
bacteria and archaea are capable of a range of metabolic pro-
cesses that can benefit their hosts such as ammonium-oxidation
(Steger et al., 2008), nitrite-oxidation (Hentschel et al., 2002),
nitrogen fixation (Wilkinson and Fay, 1979), sulfate reduction
(Hoffmann et al., 2005), and photosynthesis (Wilkinson and Fay,
1979; Bayer et al., 2008; Hoffmann et al., 2009; Mohamed et al.,
2010; Schläppy et al., 2010). However, it is possible that sponges
and some, or all, of their microbes coexist in a more commensal
or even parasitic style relationship with their hosts as opposed to
a truly mutualistic one.
Further, how sponges distinguish between symbionts, food
and pathogens is still unclear (Webster and Blackall, 2009). Recent
studies have compared sponge microbial communities from phy-
logenetically distant hosts in the same location and from closely
related sponges at different locations (Hentschel et al., 2002;
Webster et al., 2010; Schmitt et al., 2012; Jeong et al., 2013;
Montalvo et al., 2014; Kennedy et al., 2014). Thus, studies have
established a “core microbial community” that would be present
in many host taxa under various space and time conditions
(Schmitt et al., 2012).
Here, we compare the microbial communities of different
specimens of the sponge genus Cinachyrella collected from the
same South Florida location at two different times of year.
Cinachyrella (class Demospongiae), is common in coastal waters
of South Florida as well as the Caribbean, with three species
(C. kuekenthali,C. alloclada,andC. apion) present in these loca-
tions (Cárdenas et al., 2009). While C. apion is usually small and
lives mainly near the mangrove area in shallow waters, C. kueken-
thali and C. alloclada typically occur on reefs (Rützler and Smith,
1992; Cárdenas et al., 2009). However, these species are extremely
difficult to visually differentiate and require careful examination
of the spicules for identification at the species level (Cárdenas
et al., 2009, personal observation).
Much debate currently exists concerning the identification of
these species, with morphological diagnostic characters conflict-
ing with molecular phylogenies created from marker genes. For
example, using the 28S rRNA gene, cox1 gene and a combination
of the two former genes and 18S rRNA, Szitenberg et al. (2013)
showed that, Cinachyrella australiensis contains several cryptic
sympatric populations. Within the present study, we explore the
microbiome of Cinachyrella specimens collected from the same
natural environment. The purpose of the study was to describe
the baseline microbial community of Cinachyrella in order to
develop this sponge as a future experimental model. Interestingly,
we discovered that based on different microbial communities, our
samples formed two distinct groups of sponges, independent of
the time of collection, indicating that Cinachyrella can harbor
very distinct symbionts.
MATERIAL AND METHODS
SPONGE AND SEAWATER COLLECTION
Cinachyrella specimens were collected by SCUBA diving from the
Inner Reef (as defined by Walker, 2012), Broward County, Florida,
USA (N 26◦0301,W80
◦0618)atadepthof6.1m,onAug2,
2011, on Oct 24, 2011, and Feb 15, 2012, under a Florida Fish and
Wildlife Conservation Commission Fishing License and a Special
Activity License (-12-1372-372a). Sponges were identified as the
genus Cinachyrella (family Tetillidae, Sollas, 1886; van Soest et al.,
2014) given their characteristic orange to yellow color, subglob-
ular shape and hispid surface. Water temperatures reached 30.3,
23.9, and 22.8◦C in August, October and February, respectively.
A total of 64 individuals were collected in total. Here, we present
detailed results for six individuals consisting of three individu-
als on October and February (henceforth labeled as Sponge 1, 2,
3 (Sp1, Sp2, Sp3) Oct and Sponge 4, 5, 6 (Sp4, Sp5, Sp6) Feb.
The other 58 individuals were subjected to various experimental
conditions in aquaculture, and we provide a preliminary analy-
sis of these samples (Supplementary Material). In-depth results
of the different experiments for these samples are not shown.
Individuals were cut at the base with a dive knife, placed in indi-
vidual Nasco Whirl Pak bags filled with ambient seawater and
brought to the surface. Samples were stored in the shade and
maintained at ambient seawater temperature until transported
back to the laboratory (within 2 h of collection). Surface seawa-
ter was also collected each time (one replicate in October and one
replicate in February) from the dive site in 50 L carboys. These
seawater samples were used to confirm that microbial commu-
nities associated with the sponge were specific to the sponges and
not amplified from seawater DNA. Upon return to the laboratory,
sponges were quartered with a sterile knife, frozen in liquid nitro-
gen, and placed at −80◦C for long-term storage. Seawater (0.5 L)
was filtered onto a 0.22 µm Supor filter (Pall Life Science, Ann
Arbor, MI) by vacuum filtration (<10 mm Hg), the filters were
frozen in liquid nitrogen, and stored at −80◦C.
DNA EXTRACTION
Approximately ¼ of a sponge was used for DNA extraction. In
a sterile petri dish, the sample was defrosted and the ectoderm
(darker outer layer) was immediately removed using a sterile
scalpel. The endoderm was transferred to a new petri dish and
5mlofbuffer(10mMTrispH=7.6, 100 mM EDTA, 20 mM
NaCl) was added. The sponge endoderm was minced, mixed
in buffer, and the cell suspension collected into 1.7 mL tubes.
These sponge suspensions were centrifuged for 15 min at 16,000
gat 4◦C. Supernatant was decanted and the pellets transferred
and extracted using the MO BIO PowerSoil DNA isolation kit
according to the manufacturer’s instructions (MO BIO, Carlsbad,
CA).
Seawater filters also were extracted with the MO BIO
PowerSoil kit to avoid yield discrepancy between DNA extraction
protocols. The filters were placed into bead tubes (provided by
the kit) and cut into fine pieces using sterile dissection scissors.
DNA was extracted according to the manufacturer’s instructions
Frontiers in Microbiology | Aquatic Microbiology November 2014 | Volume 5 | Article 581 |2
Cuvelier et al. Microbes associated with Cinachyrella sponge
using a 2 min bead-beating step (instead of 10 min vortexing
step).
SPONGE 28S rRNA GENE PCR AND ANALYSIS
For molecular systematics, our methods followed those pro-
scribed by the Porifera Tree of Life project (Thacker et al.,
2013). Specifically, the 28S rRNA gene was amplified using the
28F63mod (5- ACC CGC TGA AYT TAA GCA TAT HAN TMA
G- 3) and 28R2077sq (5- GAG CCA ATC CTT WTC CCG ARG
TT- 3)(Thacker et al., 2013). PCR consisted of one reaction of
50 µLwith:1µM each forward and reverse primer, 1 µLoftem-
plate DNA, 2.5 mM MgCl2, 0.2 mM dNTPs and 1.25 unit of Taq
(High Fidelity Taq, TaKARa Otsu, Shiga, Japan). Thermal cycling
was initiated with denaturation at 94◦C for 3 min, followed by
30 cycles of: 45 s at 94◦C, 60 s at 55◦C, and 72◦C for 6 min and
a final extension step for 10 min at 72◦C. PCR products were
visualized on a 1.5% agarose gel (containing Gel Red). PCR prod-
ucts were cloned and sequenced on an ABI 377 automated DNA
sequencer at the University of Alabama, Birmingham using the
primer: 28R1411 (5-GTT GTT ACA CACTCC TTA GCG G-3).
Two samples (Sp5 Feb and Sp6 Feb) had low quality sequences
and were removed from the study. The nearest relative for each
sequence was determined using the NCBI BLASTn tool against
the GenBank non redundant database.
16S rRNA GENE PCR AND ANALYSIS
Approximately 291bp of the 16S rRNA gene was amplified by
PCR using the universal bacterial and archaeal primers (target-
ing the V4 region of the gene): 515F (5- GTGCCAGCMGCCG
CGGTAA- 3) and 806R (5- GGACTACHVGGGTWTCTAAT- 3)
(Caporaso et al., 2011), which contained a unique barcode used
to tag each PCR product. This primer set was chosen because it
targets a broad range of bacterial and archaeal taxa with the excep-
tion of a few groups (Bates et al., 2011; Caporaso et al., 2011). PCR
consisted of two reactions of 30 µL with (for each reaction): 1 µM
each forward and reverse primer, 1µLoftemplateDNA,2.5mM
MgCl2, 0.2 mM dNTPs and 1.25 unit of Taq (High Fidelity Taq,
TaKARa Otsu, Shiga, Japan). Thermal cycling was initiated with
denaturation at 94◦C for 3 min, followed by 30 cycles of: 45 s at
94◦C, 60 s at 50 and 72◦C for 90 s and a final extension step for
10 min at 72◦C. PCR products were visualized on a 1.5% agarose
gel (containing Gel Red). Successful reactions (i.e., with a clear
band, two reactions of 25 µL) were pooled and purified with the
Agencourt AMPure kit (Beckman Coulter, Beverly, MA), using
1.8×vol.ofAMPurebeadslurryandelutedin10mMTrispH
7.5. Each sample was quantified using PicoGreen dsDNA reagent
(Invitrogen, Carlsbad, CA). Purified products were sequenced
on a 454 Life Science Genome Sequencer FLX (Roche) at
Advanced Genetic Technologies Center at the University of
Kentucky.
Sequences were analyzed using QIIME version 1.6 (Caporaso
et al., 2010b). Only sequences with a mean quality score >25
and of length >280 bp were included in the analysis. Sequences
were then assigned to each barcode and denoised using the
denoise_wrapper option (Reeder and Knight, 2010) in QIIME.
Operational Taxonomic Units (OTU) were picked using the
UCLUST method (Edgar, 2010) and sequences with ≥97%
identities were considered as one OTU. A representative sequence
was chosen for each OTU and the taxonomic identity of each
representative was assigned (in QIIME) using the RDP Classifier
(Wang et al., 2007) against the Greengene 12_10 database
(McDonald et al., 2012). Chimera sequences were removed using
the ChimeraSlayer option (Haas et al., 2011). Sequences were
aligned (using PyNAST with default paramaters set in QIIME,
Caporaso et al., 2010a) and screened with Lane mask to remove
gaps and hypervariable regions (Lane, 1991). A representative
phylogenetic tree was built using FastTree (Price et al., 2010)
and used for further analysis in QIIME (alpha, beta diversity
from weighted UniFrac, Lozupone and Knight, 2005 and prin-
cipal coordinate analysis generated from the UniFrac distances).
T-tests (Microsoft Excel) were used to compare the relative abun-
dance of each microbial phylum present in the samples of SG1 and
SG2. A P value less than 0.05 was considered statistically signifi-
cant. A principal coordinate analysis generated from the weighted
UniFrac distances and an analysis of similarity (ANOSIM, 999
permutations) were generated in QIIME for all the 64 sponge
individuals.
RESULTS
MOLECULAR PHYLOGENETICS CONFIRM SPONGES ARE
CINACHYRELLA
All the partial 28S rRNA gene sequences obtained were most
similar to the single C. kuekanthali 28S rRNA sequence present
in Genbank (KC869490.1). Two 28S rRNA gene sequences (Sp5
and Sp6) could not be included in this study because of poor
quality. Sp1 Oct and Sp4 Feb displayed 97% identity to C. kuekan-
thali and Sp2 Oct and Sp3 Oct had 99% identity to the same
sequence (C. kuekanthali). Results showed that Sp1 Oct and Sp4
Feb were most closely related to each other (99.3% identity com-
pared to ∼97% identity to the other two samples). Similarly,
Sp2 Oct and Sp3 Oct were 100% identical to each other respec-
tively, but only ∼97% identical to the other two samples (Ta b l e 1 ).
Based on the 28S rRNA gene sequences, the samples therefore
form two groups, one group including: Sp1 Oct and Sp4 Feb and
another group including: Sp2 Oct and Sp3 Oct. These are similar
to the two groups observed after analysis of the microbiomes (see
below).
CINACHYRELLA SPECIMENS HARBOR A DIVERSITY OF UNIQUE
BACTERIA AND ARCHAEA
After quality control and chloroplast sequence removal, a
total of 16,811 sequences were analyzed including 13,947 from
Table 1 | Percent identity between the 28S rRNA gene partial
sequences of Cinachyrella samples (Sp1- 4: sponge 1- 4) collected in
October 2011 (Oct) and February 2012 (Feb) from South Florida and
C. kuekenthali (C. kuek.; GenBank: KC869490.1; Panama).
Sp1 Oct Sp2 Oct Sp3 Oct Sp4 Feb C. kuek.
Sp1 Oct 100
Sp2 Oct 97.3 100
Sp3 Oct 97.3 100 100
Sp4 Feb 99.3 96.9 96.9 100
C. kuek 96.9 99.8 99.8 96.5 100
www.frontiersin.org November 2014 | Volume 5 | Article 581 |3
Cuvelier et al. Microbes associated with Cinachyrella sponge
sponges (ranging from 1185 to 3616 sequences/animal) and 2864
from seawater (ranging from 1340 to 1524 sequences/sample)
(Table 2 ). Results indicated that Cinachyrella specimens harbor a
diverse community of symbionts, including members of all three
Domains of life (Bacteria, Archaea and Eukaryotes). Here, the
analysis of the eukaryotic community is not presented. In total,
951 OTUs (measured at 97% identity) were identified among all
samples (including seawater), of which 19 were archaeal and 932
were bacterial. A total of 639 OTUs (12 archaeal and 627 bacte-
rial OTUs) were present in the sponge symbiont community, and
OTU richness in the sponges was lower than the seawater except
for one sample (Sp3 Oct, 341 OTUs). The seawater microbial
community contained a total of 450 OTUs (10 archaeal and 440
bacterial OTUs), and OTU richness was similar in both samples
(246 vs. 285 OTUs) across sampling times (Tab l e 2 ).
CINACHYRELLA CONTAIN DISTINCT AND CANALIZED MICROBIOMES
COMPARED TO SEAWATER
Rarefaction analysis demonstrated that for some samples
(Seawater Oct, Seawater Feb, Sp2 Oct and Sp3 Oct), the diversity
was high enough such that sequencing depth was likely not suf-
ficient to evaluate the rarer members of the community and that
further sequencing would be necessary to reveal the true diversity
(Supplementary Figure 1). Yet the rarefaction analysis here con-
firmed that most sponge samples’ microbiome was less diverse
than seawater (SupplementaryFigure1). Chao1 richness esti-
mates for sponges varied from 124 to 529 phylotypes and 440 and
510 OTUs for the seawater (t=−1.9, 0.05 <P<0.1). Similarly,
the Shannon-Wiener indices for the Cinachyrella samples were
lower on average (3.1–6.2), but not statistically different than for
the seawater (6.2 and 6.3; Tab l e 2 t=−1.9, 0.05 <P<0.1).
Comparatively, 21 bacterial and 2 archaeal phyla and candi-
date phyla were detected in the sponges vs. 27 bacterial and 2
Table 2 | Overview of the number of sequences, OTUs (97%
identities) and diversity indices for six sponges (Sp1- 6: sponge 1- 6)
and seawater (SW) samples collected in October 2011 (Oct) and
February 2012 (Feb).
Sample ID Total Total Chao1*Observed Shannon*
#reads OTUs#OTUs*
SEAWATER
SW Oct 1340 246 (0) 440 221 6.2
SW Feb 1524 285 (1) 510 239 6.3
GROUP “SG1”
Sp1 Oct 1185 90 (2) 176 86 3.7
Sp4 Feb 2386 179 (0) 267 119 4.2
Sp5 Feb 1755 105 (1) 191 79 3.3
Sp6 Feb 3616 115 (1) 124 61 3.1
GROUP “SG2”
Sp2 Oct 2254 220 (1) 289 156 5.7
Sp3 Oct 2751 341 (0) 529 203 6.2
#Number in parentheses denotes the number of unclassified OTUs included in
the total.
*1100 reads were subsampled to calculate diversity indices.
archaeal phyla and candidate phyla in the seawater. Here, we use
the term “candidate phylum” to define a phylum that can be iden-
tified from genetic sequences, but lacks cultured representatives
(Hugenholtz et al., 1998). Most bacterial sequences were clas-
sified, but a small portion (2.7 ±0.9% in sponges and 2.8 ±
0.003% in seawater samples) could not be assigned to any known
phylum.
MICROBIAL COMMUNITY COMPOSITION DEFINES TWO
CINACHYRELLA TAXA
Both sequence taxonomy (Tab l e 1 ) and PCoA analyses (Figure 1)
suggestthattheCinachyrella specimens in this study form two
distinct groups and may represent different taxa of sponge. We
defined here these groups as Sponge Group 1 (SG1) and Sponge
Group 2 (SG2; Figure 1). SG1 incorporates samples that spanned
both seasons (Sp1 Oct, Sp4 Feb, Sp5 Feb, and Sp6 Feb) while
SG2 is composed of just two samples from one season (Sp2 Oct
and Sp3 Oct). In addition, the PCoA analysis for all 64 sponges
samples (See Material and Methods) confirmed that Sp1-6 were
split among two groups of sponges defined by their microbial
communities (Supplementary Figure 2), even though 58 of these
samples were placed in aquaculture under various conditions
(results of experiments not shown). ANOSIM analysis (using
all 64 sponge samples) confirmed that these were statistically
different (R=0.9926, P=0.001).
The marked differences in these two groups are demonstrated
by comparisons of the diversity of microbial taxa in each. SG2
FIGURE 1 | PCoA analysis of weighted UniFrac distance. UniFrac
measures phylogenetic distances between OTUs sets within a
phylogenetic tree. Here, we used weighted UniFrac, which takes into
account relative abundances of OTUs (as opposed to presence/absence
only). Samples formed three groups: a seawater group (samples circled in
gray), SG1 (samples circled in red) and SG2 (samples circled in blue). Ovals
circling samples are for visual guidance and do not represent any statistical
grouping.
Frontiers in Microbiology | Aquatic Microbiology November 2014 | Volume 5 | Article 581 |4
Cuvelier et al. Microbes associated with Cinachyrella sponge
FIGURE 2 | Relative abundance of pyrosequencing reads at the phylum
(or classes in the case of Proteobacteria) level present in six sponges
(Sp1- 6: sponge 1- 6) and seawater (SW) samples collected in October
2011 (Oct) and February 2012 (Feb). Phyla comprised of <0.1% of
sequences per sample are not shown. Based on the microbial community
structure, samples were placed into two groups: sponge group 1 (SG1: Sp1
Oct, Sp4 Feb, Sp5 Feb and Sp6 Feb) and sponge group 2 (SG2: Sp2 Oct and
Sp3 Oct).
samples harbored a more diverse community of microbes as
measured by a mean Shannon-Wiener diversity index of 5.95
±0.25 (s.e.m.) compared to 3.73 ±0.22 in the SG1 commu-
nity (t=−6.8, P<0.01; Table 2). Further, SG2 contained taxa
from 21 different bacterial phyla and candidate phyla and 2
archaeal phyla; SG1 contained about half that with 12 bacterial
and candidate phyla, and 2 archaeal phyla.
Overall, both sponge groups were dominated by Proteobacteria
(SG1: 63.5 ±2.9%; SG2: 38.9 ±1.0%), but Alphaproteobacteria
were more abundant (t=5.23, P<0.01) in SG1 (38.3 ±3.8%)
than in SG2 (7.9±0.2%). Proteobacteria in SG2 were dom-
inated by the Gammaproteobacteria (22.1 ±1.1%, Figure 2).
Actinobacteria were also present in both sponge groups, but were
in significantly greater numbers (t=3.23, P<0.05) in SG1
(12.2 ±2.0%, Figure 2)thanSG2(2.6±0.6%, Figure 2). SG2
harbored the candidate phylum Poribacteria (6.4 ±2.9%) that
was first discovered from sponge tissues and can be widespread
in these invertebrates (Fieseler et al., 2004; Lafi et al., 2009).
In contrast Poribacteria was below the detection limit in SG1
(t=−3.67, P<0.05; Figure 2).
Only a few bacterial phyla or classes were not significantly
different in abundance between SG1 and SG2: Bacteroidetes
(t=−0.049, P>0.05), Chlamydiae (t=−2.08 P>0.1),
Firmicutes (t=−1.63, P>0.1), Beta- (t=1.22, P>0.1),
Delta- (t=0.08, P>0.1), Gammaproteobacteria (t=−2.23,
P>0.05), and SAR406 (t=−0.42, P>0.1, Figure 2). On
the contrary, many phyla were present in SG2 at >1% (mean),
but in very low abundance (<0.07% mean) or below detection
limits in SG1 and included: Acidobacteria (t=−4.03, P<0.02),
Chloroflexi (t=−22.09, P<0.001), Gemmatimonadetes
(t=−4.154, P<0.02), Nitrospirae (t=−18.01, P<0.001),
PAUC34f (t=−17.63, P<0.001) and Verrucomicrobia
(t=−11.99, P<0.001, Figure 2).
In SG1, a few OTUs noticeably dominated the commu-
nity and composed >10.0% of all the sequences. These
included one unclassified Alphaproteobacteria OTU (30.0 ±
4.4%), one OTU in the Cenarchaeaceae family (18.3 ±1.1%;
Supplementary Figure 3), and one unclassified Actinobacteria
OTU (11.9 ±2.0%). In SG2, none of the OTUs represented more
than 10% of all the community.
Another striking difference in the communities was the relative
abundance of archaeal sequences. Archaeal sequences represented
a large portion (18.5 ±1.1%) of all the sequences recovered from
SG1 samples, but only 6.9 ±0.7% for SG2 samples (t=9.23,
P<0.01; Figure 2). In SG1, one archaeal OTU in Cenarchaeaceae
family (mentioned above) was dominant (99.3 ±0.3%). In SG2,
68.2 ±15.0% of archaeal reads also fell into one Cenarchaeaceae
family OTU, but this OTU was different from the main one
in SG1. A small proportion (5.8 ±2.3%) of the SG2 archaeal
sequences were assigned to the phylum Thaumarchaeota,which
was almost absent (except for three sequences) from SG1 (t=
7.48, P<0.01). These data indicate that the sponges collected in
our study, while physically reminiscent, in the same genus, and
from the same environment harbor distinct enough microbial
communities to warrant a re-evaluation of their phylogenetic
relationship.
www.frontiersin.org November 2014 | Volume 5 | Article 581 |5
Cuvelier et al. Microbes associated with Cinachyrella sponge
SEAWATER ARCHAEAL AND BACTERIAL COMMUNITIES ARE DISTINCT
FROM SPONGES’
In the overlying seawater, Proteobacteria (45.0 ±0.9%)—
and particularly Alpha- (23.6 ±0.6%) and Gamma- (19.0 ±
2.1%)—were the most abundant taxa of bacteria. In addition,
Bacteroidetes (19.4 ±2.2%), Cyanobacteria (17.6 ±0.3%), and
Actinobacteria (7.0 ±3.7%) were the only other bacterial phyla
that comprised >2% of all the reads.
The seawater-derived archaeal sequences represented 2.5 ±
0.2% of sequences and mostly belonged to the Thaumarchaeota,
in particular the Marine Group II or Marine Group III. Marine
Group II represented 89.3 ±4.4% of all seawater archaeal
sequences with a single OTU with pronounced dominance
(58.5 ±6.0%; Supplementary Figure 2).
CINACHYRELLA’S CORE AND VARIABLE MICROBIAL COMMUNITIES
To further examine the distinct microbial communities, core and
variable members of each group were compared. The numbers
of common OTUs between SG1 and SG2 was relatively low, with
only 95 shared OTUs representing 22.4% of the OTUs in SG2 and
30.5% in SG1. This was approximately equivalent to the numbers
of OTUs the seawater shared with SG1 (94 OTUs) and SG2 (103
OTUs, Figure 3A).
Within each sponge group, 136 common OTUs were found
in SG2 samples, as compared to 21 shared in the two SG1 sam-
ples (Figures 3B,C). In SG2, these common OTUs belonged to
2 archaeal and 14 bacterial phyla, with the most abundant being
(≥8 shared OTUs): Bacteroidetes,Chloroflexi,Cyanobacteria,and
Proteobacteria. Interestingly, the samples in SG2 also shared 12
unclassified bacterial OTUs. In SG1, the shared OTUs belonged
to the Crenarchaeota,Actinobacteria,Bacteroidetes,Cyanobacteria
and Proteobacteria. The diversity among sponge samples of the
same group was similar at the class level, but not shared at the
family or genus level. In most cases, many of the OTUs were
present in only one of the sponge samples. Out of all the sponge
samples, 83 OTUs were present in at least 50% of the samples and
23 OTUs in at least 70% of the sponge samples. Within SG1 and
SG2, 107 and 424 OTUs respectively were present in at least 50%
of the samples and 55 and 135 OTUs respectively were present in
at least 70% of the samples. The only 11 OTUs common to all the
sponge samples (i.e., the core community) were assigned to the
Proteobacteria (Alpha- and Gamma-)aswellasCyanobacteria,the
Bacteroidetes and the Actinobacteria.
MICROBIAL COMMUNITY FUNCTIONAL INSIGHTS
QIIME analysis of the 16S rRNA gene sequences revealed that
microbes with potential contribution to the nitrogen cycle were
present. SG2 samples contained OTUs belonging to the gen-
era Cenarchaeum (18.4 ±12.1% of the archaeal reads) and
Nitrosopumilus (6.9 ±5.9% of archaeal reads). These genera
are part of the ammonia-oxidizing archaea (AOA) that oxidize
ammonia to nitrite (Preston et al., 1996; Walker et al., 2010). In
SG1, AOA sequences belonging to the family Cenarchaeaceae were
also present. Bacteria involved in the second step of nitrification,
the oxidation of nitrite to nitrate were present in SG2 samples.
These belonged to two OTUS in the family Nitrospiracea (phylum
FIGURE 3 | Venn diagrams of specific and shared classified OTUs between. (A) Sponge group 1 (SG1)-SG2-seawater (SW); (B) SG1 samples; (C) SG2
samples.
Frontiers in Microbiology | Aquatic Microbiology November 2014 | Volume 5 | Article 581 |6
Cuvelier et al. Microbes associated with Cinachyrella sponge
Nitrospirae, 2.3 ±0.2%; Supplementary Figure 3) with 98.3 ±
0.02% of these reads affiliated to one OTU. This OTU had 99%
identity to sponge-derived sequences
In the Chloroflexi (which was almost absent from SG1), two
classes were abundant in SG2: Anaerolinae (7.4 ±1.6%) and
SAR202 (6.4 ±0.6%), with most OTUs in the latter class
belonging to sponge-specific clusters.
The most abundant Cyanobacteria OTU in SG1 (1.6% ±
0.6%) and SG2 (4.7% ±0.1%) was 100% identical to
Synechoccocus strain WH8109. This also was the second most
abundant OTU in seawater. One noteworthy finding related
to Cyanobacteria involved the numerically dominant OTU in
seawater, which was 100% identical to a UCYN-A clone,
Candidatus Atelocyanobacterium thalassa (Thompson et al.,
2012), a widespread cyanobacterium and likely a significant con-
tributor to N2-fixation in marine waters (Zehr et al., 2001;
Moisander et al., 2010).
In the phylum Proteobacteria, many OTUs were obtained that
could not be further classified, but had little overlap between
SG1 and SG2. In SG1, the unclassified sequences in each of the
Proteobacteria class had one clear dominant OTU. In the Alpha-,
Beta-,Delta- and Gammaproteobacteria sequences, this OTU
encompassed 77.4 ±4.2%, 92.1 ±3.2%, >95.5 ±1.4%, 55.4 ±
5.9% of the reads in each class, respectively. In contrast, in SG2,
none of the unclassified OTU at the class level included more
than 37.1 ±0.2% of the sequences and a few abundant OTUs
were usually present. In all sponge samples, many unclassified
OTUs (at the class level) were closely related to uncultured bac-
teria derived from sponge tissues. In particular, the most abun-
dant unclassified Alpha- and Gammaproteobacteria OTUs were 99
and 100% identical, respectively, to a sequence from Cinachyra
sp. from India. Within the classified Alphaproteobacteria,the
families Rhodobacteraceae and Rhodospirillaceae were common
and diverse in both sponge groups and seawater. As expected
Pelagibacteraceae were the most abundant Alphaproteobacteria in
the seawater.
DISCUSSION
Since our field collections were confined to a relatively small
portion of the reef, we did not intend or expect to collect two
apparently divergent Cinachyrella taxa. The sponges in this study
were collected as part of a broader study involving greater num-
ber of specimens used for aquaculture. Upon analysis of the all the
samples, it became clear that sponges formed two groups based on
their microbial communities. The sponges in aquaculture (data
not shown) were subject to different conditions. We therefore
decided to present here only the data from sponges collected
from the reef and never kept in aquaculture. In the present study,
although we have confirmed that these specimens belong to the
genus Cinachyrella, their exact taxonomic and phylogenetic iden-
tification goes beyond the scope of this paper, as the taxonomy
of this genus and family (Tetill i d a e ) is still under much debate
(see introduction and Szitenberg et al., 2013). However, our find-
ings are consistent, but not totally sufficient (due to low sample
number and the low 28S rRNA sequence quality of two of our
six samples) to prove the idea presented by Cárdenas et al. (2012)
that microbiome signatures may be useful traits to delineate some
sponge taxa. Thus, additional samples and a more comprehen-
sive histology and electron microscopy analyses of the spicules
would be needed to confirm the species identity of these sponge
individuals. However, given the clear differences in the micro-
biomes of these sponge taxa, a simple PCR diagnostic of one or
more variable members of the sponges’ microbiota could also
be used.
Overall, our results are similar to those of Chambers et al.
(2013). There, the authors showed that two sponge morphs ini-
tially assigned to the genus Paratetilla (Demospongiae, Tetillidae)
had different microbial communities, sharing less than 43% simi-
larity. Within each morph group, microbial community similarity
varied between 65 and 94% between individuals. Using COI gene,
the authors confirmed that one of the sponge morphs actually
belonged to the genus Cinachyrella,“challengingthevalueof
the morphological characters used in the classification of these
genera” (Chambers et al., 2013). Similar to our results, the bac-
terial communities were different for the two groups, even for
specimens collected from the same location.
DIVERSE MICROBES ARE PRESENT IN CINACHYRELLA
Multiple studies have shown that marine sponges can harbor
a large diversity of microbes and the microbial taxa richness
present in our Cinachyrella tissue samples (90–341 OTUs) was
within the range of other sponge species. An extensive study
targeting 32 species from eight different locations worldwide
revealed each sponge carried between 225 and 364 OTUs (at 97%
identity) with sequence coverage similar to our study (Schmitt
et al., 2012). As expected, when sequencing depth was much
greater, OTU richness was higher, reaching numbers between
1099 and 2996 OTUs (95% identity) in three Pacific sponge
species (Webster et al., 2010). Total taxon richness (at a higher
sequencing depth) was also greater in C. australiensis sampled
from the coast of Indonesia, in which 800 phylotypes were present
(Cleary et al., 2013). In subtropical waters of Key Largo, FL, USA
(close to our study site), the barrel sponge Xestospongia muta
had Shannon diversity indices comparable to the lower range of
our Cinachyrella samples (Montalvo and Hill, 2011). However,
Cinachyrella contained fewer OTUs than Axinella corrugata (at
least 1000 OTUs per specimen) collected less than a few miles
away from our study site (White et al., 2012). Compared to the
coral Orbicella faveolata (formerly Montastraea faveolata;Kimes
et al., 2013), our sponge samples showed similar diversity, for
which 943 bacterial clones contained 178 OTUs (97% similar-
ity threshold), with Chao1 estimates of 307 ribotypes (Sunagawa
et al., 2009). Similarly, the coral O. annularis sampled from var-
ious sites at Curaçao Island harbored 163–323 bacterial OTUs
(Barott et al., 2011).
CINACHYRELLA HARBOR FUNCTIONALLY DIVERSE MICROBES
A small percentage of the bacterial 16S rRNA gene fragments
could not be further classified indicating that some of the bacte-
rial diversity remains unexplored. This number was much lower
than those reported for A. corrugata collected nearby, in which
36% of the reads obtained by amplification of the 16S rRNA gene
V1-V3 regions were not assigned to any bacterial phylum (White
et al., 2012). In their pyrosequencing study of C. australiensis and
www.frontiersin.org November 2014 | Volume 5 | Article 581 |7
Cuvelier et al. Microbes associated with Cinachyrella sponge
Suberites diversicolor microbiomes, Cleary et al. (2013) also found
34% of bacterial OTUs unclassified at the phylum level. There, the
primers used targeted the V3-V4 regions while the V4 region was
used for this Cinachyrella study.
In sponges, the dominant microbial phyla can vary with taxon-
omy and across geographical location or habitat. High microbial
abundance (HMA) sponges usually harbor many bacterial taxa
while low microbial abundance (LMA) sponges typically have one
or few numerically dominant taxa and a few less abundant ones
(Hentschel et al., 2003; Giles et al., 2013). In this study, SG1 sam-
ples contained few taxa with pronounced dominance, resembling
LMA sponges in terms of microbial equitability, but also encom-
passed many other phylotypes, atypical of LMA sponges. SG2
samples clearly harbored a more diverse microbial community,
similar to HMA sponges. It is important to note that the similar-
ity of these sponge groups to HMA and LMA was inferred solely
based on the structure of the microbiomes and an in-depth his-
tological study was not performed on these samples to confirm
microbial abundance.
SG2 samples contained the candidate phylum Poribacter ia,
but this taxon was below detection limits in both SG1 and
seawater. This is notable because Por ibacteria are typical mem-
bers of sponge microbiomes, but have mostly been detected
in HMA sponges (Hochmuth et al., 2010). This taxon can be
diverse, as shown by Schmitt et al. (2012) who detected a
total of 437 Poribacteria OTUs in the 32 sponges species stud-
ied, with up to 79 different Poribacteria OTUs (97% identity)
per species. In our Cinachyrella,Poribacter ia were only classi-
fied as two OTUs. This lower diversity related to Poribacteria
might be distinctive of Cinachyrella because only four OTUs were
present in C. autraliensis specimens from open ocean habitats in
Indonesia and similar to our SG1, Poribacteria were undetected
in specimens collected from nearby marine lakes (Cleary et al.,
2013).
Chloroflexi also was below detection limits in SG1. This again
might be typical of LMA sponges as the Chloroflexi were absent in
LMA sponges from the Red Sea, the Caribbean Sea and the South
Pacific Ocean and present in low numbers in other LMA sponges
(Schmitt et al., 2011; Giles et al., 2013). In SG2, Chloroflexi
sequences were grouped into 12 OTUs, close to the range (14–
21 OTUs) Schmitt et al. (2011) reported for HMA sponges, but
lower than the 502 OTUs (97% identity) retrieved from another
32 sponge species (Schmitt et al., 2012).
Giles et al. (2013) studied the microbiomes in six species
of LMA sponges using clone libraries and found that the
phyla Acidobacteria, Chloroflexi and Gemmatimonadetes were not
detected. Here, SG1 samples also were missing these phyla (with
the exception of three sequences of SAR202-Chloroflexi and two
sequences in the Gemmatimonadetes). These three bacterial phyla
were also missing in eight of the 13 species analyzed by Jeong et al.
(2013). The other five species contained a high microbial diversity
with a large proportion of Chloroflexi (this group was called the
CF group because of the Chloroflexi).
We also found a large portion of unclassified Proteobacteria
in the sponge, but not in the seawater suggesting that it was
not a consequence of the analysis. In the sponges Raspailia
ramosa and Stelligera stuposa, 32 and 17% of the Proteobacteria
sequences, respectively, were unclassified as opposed to only 1%
in the seawater (Jackson et al., 2012). Further exploration suggests
that many of our unclassified Proteobacteria OTUs are sponge-
specific and the presence of large clusters of sponge-specific and
sponge- and coral-specific bacteria in the invertebrates have been
described (Simister et al., 2012). Interestingly, our results related
to Proteobacteria were similar to Cleary et al. (2013).Intheir
study, Alphaproteobacteria were more abundant in C. australiensis
from marine lakes than open ocean habitats. In our Cinachyrella
samples, Alphaproteobacteria were significantly more abundant in
the SG1 than SG2. These might again be typical of some LMA
sponges as Kamke et al. (2010) also recovered a large portion of
Alphaproteobacteria clones from LMA sponges.
Cinachyrella symbionts also belonged to the Archaea (6.9–
18.5%), in proportions within the wide range recorded for four
deep water (4–65%) and three shallow water sponges from the
Red Sea (4–28%) (Lee et al., 2011; Kennedy et al., 2014). All
of the archaeal sequences in Cinachyrella fell within two phyla:
Thaumarchaeota and Euryarchaeota, with most of the archaea
belonging to the Thaumarchaeota, which is widespread in sponges
(Webster et al., 2001; Margot et al., 2002; Lee et al., 2011; Kennedy
et al., 2014; Polónia et al., 2014). Archaeal reads grouped into
a low number of OTUs, with a few numerically dominant ones,
similar to the four species sampled by Kennedy et al. (2014),
which had 70% of the Thaumarchaeota sequences separated in
three OTUs. The phylum Thaumarchaeota includes AOA per-
forming the first step of nitrification using ammonium excreted
by sponges as a metabolic waste product (Jiménez and Ribes,
2007; Bayer et al., 2008; Hoffmann et al., 2009). Ammonia oxi-
dation by archaea is believed to be widespread in marine envi-
ronments (Francis et al., 2005; Könneke et al., 2005; Schleper
et al., 2005) and was detected both the LMA and HMA sponges
(Schläppy et al., 2010). In addition to the AOA, nitrite-oxidizing
bacteria catalyzing the second step of nitrification were found
in SG2. Hentschel et al. (2002) detected early on clones affili-
ated with nitrite-oxidizing phylum Nitrospirae in sponges. The
proportion of this phylum varies greatly between host species,
ranging from 0.6% in X. testudinaria from the Red Sea (Lee et al.,
2011) to 24% in Stelligera stuposa from Irish waters (Jackson et al.,
2012). Overall, in the present study, it appears that only one group
of Cinachyrella (SG2) harbors the microbes required for both
steps of nitrification.
THE TWO SPONGE GROUPS ONLY SHARE A SMALL CORE MICROBIOME
Symbionts in SG1 and SG2 were very different at the OTU level
with both groups only sharing a small core microbial commu-
nity as seen in many sponges. For example, C. australiensis from
open ocean habitat and marine lakes only shared 9.4% of their
OTUs (Cleary et al., 2013), lower than the percentage shared
between SG1 and SG2. In contrast, the sponge genus Xestospongia
often showed exceptionally high overlap in OTUs. For exam-
ple, X. muta (collected from Florida) and X. testudinaria (from
Indonesia) shared 85% of the reads (=245 OTUs) between the
two species (Montalvo et al., 2014). However, after surveying
32 sponge species, Schmitt et al. (2012) concluded that phy-
logeny of the host (i.e., how closely related sponges were) did not
correlate with the bacterial composition. Similarly, host sponge
Frontiers in Microbiology | Aquatic Microbiology November 2014 | Volume 5 | Article 581 |8
Cuvelier et al. Microbes associated with Cinachyrella sponge
phylogeny—except for the genus Xestospongia—did not affect
the similarity of the symbionts communities in sponges from
Orpheus Island (Webster et al., 2013). Nevertheless, when trip-
licate individuals of the same species (including Cinachyra sp.)
were analyzed, conserved (>65% similarity) microbial commu-
nities were observed (Webster et al., 2013). This is consistent with
the pyrosequencing characterization of A. corrugata symbiont
communities in S. Florida (White et al., 2012), which showed rel-
atively high similarities among multiple individuals and across
hundreds of km. In Cinachyrella, the numbers of shared OTUs
between SG1 samples (12–24%) and SG2 samples (39–62%) was
low. Giles et al. (2013) and Schmitt et al. (2012) suggest envi-
ronmental factors such as temperature, salinity or nutrient levels
might impact symbionts population structures. In their study,
species from tropical waters had more similar bacterial commu-
nities. This did not hold true at a smaller scale as we observed
distinct communities in the two sponge groups from the same
environment, independent of spatial or temporal scales.
Considering many sponges (including Cinachyrella)havea
reduced core and large variable microbial community, it would
be reasonable to assume that different OTUs perform distinct
functions within the sponge. However, using a metagenomic
approach, a recent study showed that taxonomically divergent
sponges can harbor phylogenetically diverse symbionts with func-
tional equivalence (Fan et al., 2012). The authors were able
to show that six sponge species possess similar functional pro-
files distinct from the ones obtained for the seawater microbial
communities (Fan et al., 2012). These findings suggest that key
functions in marine sponges might be performed by different
microbial taxa and a phylogenetically similar “core microbial
community” may therefore not be essential to meet the sponge
requirements. Moreover, perhaps the concept of a “core” micro-
biome, for Porifera at least, may have to be redefined altogether
to emphasize function over symbiont identity. This view may not
be so far fetched when considering that bacteria can often dras-
tically change their metabolic activities through horizontal gene
transfers (Costa et al., 2009).
Together with recent and ongoing molecular microbiome
analyses of adjacent coastal waters and reef invertebrate hosts
(unpublished), this study contributes to a growing spatio-
temporal profile of microbiome dynamics in subtropical South
Florida (Negandhi et al., 2010; White et al., 2012). These results
also help provide a baseline characterization for Cinachyrella,
which may be developed for further experimental studies, due
to its hardiness in aquaculture, relative ease of collection and
maintenance.
AUTHOR CONTRIBUTIONS
Marie L. Cuvelier, Emily Blake, Rebecca L. Vega Thurber, Peter J.
McCarthy, and Jose V. Lopez designed research; Marie L. Cuvelier,
Emily Blake, and Jose V. Lopez performed sampling; Marie L.
Cuvelier performed DNA extractions and 16S rRNA amplicon
preparation; Emily Blake performed sponge taxonomy analysis;
Rebecca Mulheron performed 28S rRNA PCR; Marie L. Cuvelier,
Rebecca L. Vega Thurber, and Jose V. Lopez analyzed data; Marie
L. Cuvelier, Emily Blake, Peter J. McCarthy, Patricia Blackwelder,
RebeccaL.VegaThurber,andJoseV.Lopezwrotethepaper.
Funding was awarded to Jose V. Lopez, Rebecca L. Vega Thurber,
Peter J. McCarthy, and Patricia Blackwelder.
ACKNOWLEDGMENTS
This work was supported by a Year 1 BP Gulf of Mexico Research
Initiative grant to the Florida Institute of Oceanography.
Therefore, data has been uploaded to the Gulf of Mexico Research
Initiative data portal—https://data.gulfresearchinitiative.org.
Molecular phylogeny of sponges was supported by the National
Science Foundation’s “Assembling the Porifera Tree of Life”
(PorToL.org) grant DEB-0820791 to JVL. Any opinions, findings,
and conclusions or recommendations expressed in this material
are those of the authors and do not necessarily reflect the views of
the National Science Foundation. We thank Alexandra Campbell,
Ari Halperin, Dawn Formica, Ian Rodericks, Katy Brown,
Keri O’Neal, Megan Zappe, Peter Grasso, Rory Welsh, Captain
Lance Robinson and Assistant Harbor Master Brian Buskirk for
assistance with the SCUBA diving and sample collections and
processing. We thank Dr. Robert Thacker, Dr. Paco Cárdenas,
Dr. Cristina Díaz and other PorToL members for assistance with
the Cinachyrella taxonomy. We also thank Dr. Jesse Zaneveld, Dr.
Dana Wilson for constructive discussion on the data analysis.
This is Harbor Branch Oceanographic Institute contribution
number 1940. 28S rRNA sequences have been deposited in
GenBank under accession no. KM588360 through 588363.
16S rRNA sequences have been deposited in NCBI SRA under
accession no. SRP047337.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: http://www.frontiersin.org/journal/10.3389/fmicb.2014.
00581/abstract
Supplementary Figure 1 | Rarefaction curves (note: SW Feb is under the
Sp6 Feb line).
Supplementary Figure 2 | PCoA analysis of weighted UniFrac distance.
UniFrac measure phylogenetic distances between OTUs sets within a
phylogenetic tree. Here, we used weighted UniFrac, which takes into
account relative abundances of OTUs (as opposed to presence/absence
only). 64 sponge individuals were used in total and Sp1 through Sp6 are
labeled.
Supplementary Figure 3 | Number of OTUs, abundance and classification
of all the sequences present in the seawater (Oct and Feb), Sponge Group
1 (Sp1 Oct, Sp4 Feb, Sp5 Feb, Sp6 Feb) and Sponge Group 2 (Sp2 Oct and
Sp3 Oct). Each OTU is classified at the lowest ranking.
REFERENCES
Barott, K. L., Rodriguez-Brito, B., Janouškovec, J., Marhaver, K. L., Smith, J. E.,
Keeling, P., et al. (2011). Microbial diversity associated with four functional
groups of benthic reef algae and the reef-building coral Montastraea annularis.
Environ. Microbiol. 13, 1192–1204. doi: 10.1111/j.1462-2920.2010.02419.x
Bates, S. T., Berg-Lyons, D., Caporaso, J. G., Walters, W. A., Knight, R., and Fierer,
N. (2011). Examining the global distribution of dominant archaeal populations
in soil. ISME J. 5, 908–917. doi: 10.1038/ismej.2010.171
Bayer, K., Schmitt, S., and Hentschel, U. (2008). Physiology, phylogeny and
in situ evidence for bacterial and archaeal nitrifiers in the marine sponge
Aplysina aerophoba.Environ. Microbiol. 10, 2942–2955. doi: 10.1111/j.1462-
2920.2008.01582.x
Caporaso, J. G., Bittinger, K., Bushman, F. D., DeSantis, T. Z., Andersen, G. L.,
and Knight, R. (2010a). PyNAST: a flexible tool for aligning sequences to a
www.frontiersin.org November 2014 | Volume 5 | Article 581 |9
Cuvelier et al. Microbes associated with Cinachyrella sponge
template alignment. Bioinformatics 26, 266–267. doi: 10.1093/bioinformatics/
btp636
Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger,K., Bushman, F. D., Costello,
E. K., et al. (2010b). QIIME allows analysis of high-throughput community
sequencing data. Nat. Methods 7, 335–336. doi: 10.1038/nmeth.f.303
Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Lozupone, C. A.,
Turnbaugh, P. J., et al. (2011). Global patterns of 16S rRNA diversity at a depth
of millions of sequences per sample. Proc. Natl. Acad. Sci. U.S.A. 108(Suppl. 1),
4516–4522. doi: 10.1073/pnas.1000080107
Cárdenas, P., Menegola, C., Rapp, H. T., and Díaz, M. C. (2009). Morphological
description and DNA barcodes of shallow-water Tetractinellida (Porifer a:
Demospongiae) from Bocas del Toro, Panama, with description of a new species.
Zootaxa 2276, 1–39. Available online at: http://www.mapress.com/zootaxa/list/
2009/zt02276.html
Cárdenas, P., Pérez, T., and Boury-Esnault, N. (2012). “Chapter two—sponge sys-
tematics facing new challenges,” in Advances in Marine Biology Advances in
Sponge Science: Phylogeny, Systematics, Ecology, eds M. A. Becerro, M. J. Uriz,
M. Maldonado, and X. Turon (London, UK: Academic Press), 79–209.
Chambers, K., Padovan, A., Alvarez, B., and Gibb, K. (2013). Microbial signatures
can help distinguish moon sponges (family Tetillidae) from Darwin Harbour,
Aust ral ia. Mar. Freshwater Res. 64, 716–725. doi: 10.1071/MF12226
Cleary, D. F. R., Becking, L. E., de Voogd, N. J., Pires, A. C. C., Polónia, A. R.
M., Egas, C., et al. (2013). Habitat- and host-related variation in sponge bac-
terial symbiont communities in Indonesian waters. FEMS Microbiol. Ecol. 85,
465–482. doi: 10.1111/1574-6941.12135
Costa, R., Van Aarle, I. M., Mendes, R., and Van Elsas, J. D. (2009). Genomics
of pyrrolnitrin biosynthetic loci: evidence for conservation and whole-operon
mobility within Gram-negative bacteria. Environ. Microbiol. 11, 159–175. doi:
10.1111/j.1462-2920.2008.01750.x
de Goeij, J. M., van Oevelen, D., Vermeij, M. J. A., Osinga, R., Middelburg, J.
J., Goeij, A. F. P. M., et al. (2013). Surviving in a marine desert: the sponge
loop retains resources within coral reefs. Science 342, 108–110. doi: 10.1126/sci-
ence.1241981
Díaz, M. C., and Rützler, K. (2001). Sponges: an essential component of Caribbean
coral reefs. Bull.Mar.Sci.69, 535–546. Available online at: http://www.ingenta-
connect.com/content/umrsmas/bullmar/2001/00000069/00000002/art00026
Edgar, R. C. (2010). Search and clustering orders of magnitude faster than BLAST.
Bioinformatics 26, 2460–2461. doi: 10.1093/bioinformatics/btq461
Fan, L., Reynolds, D., Liu, M., Stark, M., Kjelleberg, S., Webster, N. S., et al. (2012).
Functional equivalence and evolutionary convergence in complex communities
of microbial sponge symbionts. Proc. Natl. Acad. Sci. U.S.A. 109, E1878–E1887.
doi: 10.1073/pnas.1203287109
Fieseler, L., Horn, M., Wagner, M., and Hentschel,U. (2004). Discovery of the novel
candidate phylum “Poribacteria” in marine sponges. Appl. Environ. Microbiol.
70, 3724–3732. doi: 10.1128/AEM.70.6.3724-3732.2004
Francis, C. A., Roberts, K. J., Beman, J. M., Santoro, A. E., and Oakley, B. B. (2005).
Ubiquity and diversity of ammonia-oxidizing archaea in water columns and
sediments of the ocean. Proc. Natl. Acad. Sci. U.S.A. 102, 14683–14688. doi:
10.1073/pnas.0506625102
Giles, E. C., Kamke, J., Moitinho-Silva, L., Taylor, M. W., Hentschel, U., Ravasi,
T., et al. (2013). Bacterial community profiles in low microbial abundance
sponges. FEMS Microbiol. Ecol. 83, 232–241. doi: 10.1111/j.1574-6941.2012.
01467.x
Haas, B. J., Gevers, D., Earl, A. M., Feldgarden, M., Ward, D. V., Giannoukos,
G., et al. (2011). Chimeric 16S rRNA sequence formation and detection in
Sanger and 454-pyrosequenced PCR amplicons. Genome Res. 21, 494–504. doi:
10.1101/gr.112730.110
Hentschel, U., Fieseler, L., Wehrl, M., Gernert, C., Steinert, M., Hacker, J., et al.
(2003). Microbial diversity of marine sponges. Prog. Mol. Subcell. Biol. 37,
59–88. doi: 10.1007/978-3-642-55519-0_3
Hentschel, U., Hopke, J., Horn, M., Friedrich, A. B., Wagner, M., Hacker, J.,
et al. (2002). Molecular evidence for a uniform microbial community in
sponges from different oceans. Appl. Environ. Microbiol. 68, 4431–4440. doi:
10.1128/AEM.68.9.4431-4440.2002
Hentschel, U., Usher, K. M., and Taylor, M. W. (2006). Marine sponges as
microbial fermenters. FEMS Microbiol. Ecol. 55, 167–177. doi: 10.1111/j.1574-
6941.2005.00046.x
Hochmuth, T., Niederkrüger, H., Gernert, C., Siegl, A., Taudien, S., Platzer, M.,
et al. (2010). Linking chemical and microbial diversity in marine sponges:
possible role for Pori bacteria as producers of methyl-branched fatty acids.
Chembiochem 11, 2572–2578. doi: 10.1002/cbic.201000510
Hoffmann, F., Larsen, O., Thiel, V., Rapp, H. T., Pape, T., Michaelis, W.,
et al. (2005). An anaerobic world in sponges. Geomicrobiol. J. 22, 1–10. doi:
10.1080/01490450590922505
Hoffmann, F., Radax, R., Woebken, D., Holtappels, M., Lavik, G., Rapp, H. T.,
et al. (2009). Complex nitrogen cycling in the sponge Geodia barretti.Environ.
Microbiol. 11, 2228–2243. doi: 10.1111/j.1462-2920.2009.01944.x
Hugenholtz, P., Goebel, B. M., and Pace, N. R. (1998). Impact of culture-
independent studies on the emerging phylogenetic view of bacterial diversity.
J. Bacteriol. 180, 6793–6793.
Ilan, M., Gugel, J., and van Soest, R. (2004). Taxonomy, reproduction and
ecology of new and known Red Sea sponges. Sarsia 89, 388–410. doi:
10.1080/00364820410002659
Jackson, S. A., Kennedy, J., Morrissey, J. P., O’Gara, F., and Dobson, A. D. W. (2012).
Pyrosequencing reveals diverse and distinct sponge-specific microbial commu-
nities in sponges from a single geographical location in Irish waters. Microb.
Ecol. 64, 105–116. doi: 10.1007/s00248-011-0002-x
Jeong, I.-H., Kim, K.-H., and Park, J.-S. (2013). Analysis of bacterial diversity
in sponges collected off Chujado, an Island in Korea, using barcoded 454
pyrosequencing: analysis of a distinctive sponge group containing Chloroflexi.
J. Microbiol. 51, 570–577. doi: 10.1007/s12275-013-3426-9
Jiménez, E., and Ribes, M. (2007). Sponges as a source of dissolved inorganic
nitrogen: Nitrification mediated by temperate sponges. Limnol. Oceanogr. 52,
948–958. doi: 10.4319/lo.2007.52.3.0948
Kamke, J., Taylor, M. W., and Schmitt, S. (2010). Activity profiles for marine
sponge-associated bacteria obtained by 16S rRNA vs 16S rRNA gene compar-
isons. ISME J. 4, 498–508. doi: 10.1038/ismej.2009.143
Kennedy, J., Flemer, B., Jackson, S. A., Morrissey, J. P., O’Gara, F., and
Dobson, A. D. W. (2014). Evidence of a putative deep sea specific micro-
biome in marine sponges. PLoS ONE 9:e91092. doi: 10.1371/journal.pone.00
91092
Kimes, N. E., Johnson, W. R., Torralba, M., Nelson, K. E., Weil, E., and Morris,
P. J. (2013). The Montastraea faveolata microbiome: ecological and temporal
influences on a Caribbean reef-building coral in decline. Environ. Microbiol. 15,
2082–2094. doi: 10.1111/1462-2920.12130
Könneke, M., Bernhard, A. E., de la Torre, J. R., Walker, C. B., Waterbury, J. B.,
and Stahl, D. A. (2005). Isolation of an autotrophic ammonia-oxidizing marine
archaeon. Nature 437, 543–546. doi: 10.1038/nature03911
Lafi, F. F., Fuerst, J. A., Fieseler, L., Engels, C., Goh, W. W. L., and Hentschel, U.
(2009). Widespread distribution of Poribact eria in Demospongiae.Appl. Environ.
Microbiol. 75, 5695–5699. doi: 10.1128/AEM.00035-09
Lane, D. (1991). “16S/23S rRNAsequencing ,” in Nucleic AcidTechniques in Bacterial
Systematics, eds E. Stackebrandt and M. Goodfellow (West Sussex: John Wiley
and Sons), 115–175.
Lee, O. O., Wang, Y., Yang, J., Lafi, F. F., Al-Suwailem, A., and Qian, P.-Y.
(2011). Pyrosequencing reveals highly diverse and species-specific micro-
bial communities in sponges from the Red Sea. ISME J. 5, 650–664. doi:
10.1038/ismej.2010.165
Li, C.-W., Chen, J.-Y., and Hua, T.-E. (1998). Precambrian sponges with
cellular structures. Science 279, 879–882. doi: 10.1126/science.279.53
52.879
Lozupone, C., and Knight, R. (2005). UniFrac: a new phylogenetic method for com-
paring microbial communities. Appl. Environ. Microbiol. 71, 8228–8235. doi:
10.1128/AEM.71.12.8228-8235.2005
Margot, H., Acebal, C., Toril, E., Amils, R., and Puentes, J. F. (2002). Consistent
association of crenarchaeal Archaea with sponges of the genus Axinella.Mar.
Biol. 140, 739–745. doi: 10.1007/s00227-001-0740-2
McDonald, D., Price, M. N., Goodrich, J., Nawrocki, E. P., DeSantis, T. Z., Probst,
A., et al. (2012). An improved Greengenes taxonomy with explicit ranks for eco-
logical and evolutionary analyses of bacteria and archaea. ISME J. 6, 610–618.
doi: 10.1038/ismej.2011.139
Mohamed, N. M., Saito, K., Tal, Y., and Hill, R. T. (2010). Diversity of aerobic and
anaerobic ammonia-oxidizing bacteria in marine sponges. ISME J. 4, 38–48. doi:
10.1038/ismej.2009.84
Moisander, P. H., Beinart, R. A., Hewson, I., White, A. E., Johnson, K. S.,
Carlson, C. A., et al. (2010). Unicellular cyanobacterial distributions broaden
the oceanic N2 fixation domain. Science 327, 1512–1514. doi: 10.1126/science.
1185468
Frontiers in Microbiology | Aquatic Microbiology November 2014 | Volume 5 | Article 581 |10
Cuvelier et al. Microbes associated with Cinachyrella sponge
Montalvo, N. F., Davis, J., Vicente, J., Pittiglio, R., Ravel, J., and Hill, R. T.
(2014). Integration of culture-based and molecular analysis of a complex
sponge-associated bacterial community. PLoSONE 9:e90517. doi: 10.1371/jour-
nal.pone.0090517
Montalvo, N. F., and Hill, R. T. (2011). Sponge-associated bacteria are strictly
maintained in two closely related but geographically distant sponge
hosts. Appl. Environ. Microbiol. 77, 7207–7216. doi: 10.1128/AEM.05
285-11
Negandhi, K., Blackwelder, P. L., Ereskovsky, A. V., and Lopez, J. V. (2010). Florida
reef sponges harbor coral disease-associated microbes. Symbiosis 51, 117–129.
doi: 10.1007/s13199-010-0059-1
Polónia, A. R. M., Cleary, D. F. R., Duarte, L. N., de Voogd, N. J., and Gomes,
N. C. M. (2014). Composition of Archaea in seawater, sediment, and sponges
in the kepulauan seribu reef system, Indonesia. Microb. Ecol. 67, 553–567. doi:
10.1007/s00248-013-0365-2
Preston, C. M., Wu, K. Y., Molinski, T. F., and DeLong, E. F. (1996). A psychrophilic
crenarchaeon inhabits a marine sponge: Cenarchaeum symbiosum gen. nov.,
sp. nov. Proc. Natl. Acad. Sci. U.S.A. 93, 6241–6246. doi: 10.1073/pnas.93.
13.6241
Price, M. N., Dehal, P. S., and Arkin, A. P. (2010). FastTree 2 – approxi-
mately maximum-likelihood trees for large alignments. PLoS ONE 5:e9490. doi:
10.1371/journal.pone.0009490
Reeder, J., and Knight, R. (2010). Rapidly denoising pyrosequencing amplicon
reads by exploiting rank-abundance distributions. Nat. Methods 7, 668–669. doi:
10.1038/nmeth0910-668b
Rützler, K., and Smith, K. (1992). Guide to western Atlantic species of cinachyrella
(Porifera: Tetillidae). Proc. Biol. Soc. Wash. 105, 148–164.
Ryan, J. F., Pang, K., Schnitzler, C. E., Nguyen, A.-D., Moreland, R. T., Simmons,
D. K., et al. (2013). The genome of the Ctenophore Mnemiopsis leidyi and
its implications for cell type evolution. Science 342:1242592. doi: 10.1126/sci-
ence.1242592
Schläppy, M.-L., Schöttner, S. I., Lavik, G., Kuypers, M. M. M., de Beer, D.,
and Hoffmann, F. (2010). Evidence of nitrification and denitrification in
high and low microbial abundance sponges. Mar. Biol. 157, 593–602. doi:
10.1007/s00227-009-1344-5
Schleper, C., Jurgens, G., and Jonuscheit, M. (2005). Genomic studies
of uncultivated archaea. Nat. Rev. Microbiol. 3, 479–488. doi: 10.1038/
nrmicro1159
Schmitt, S., Deines, P., Behnam, F., Wagner, M., and Taylor, M. W. (2011).
Chloroflexi bacteria are more diverse, abundant, and similar in high than in
low microbial abundance sponges. FEMS Microbiol. Ecol. 78, 497–510. doi:
10.1111/j.1574-6941.2011.01179.x
Schmitt, S., Tsai, P., Bell, J., Fromont, J., Ilan, M., Lindquist, N., et al.
(2012). Assessing the complex sponge microbiota: core, variable and species-
specific bacterial communities in marine sponges. ISME J. 6, 564–576. doi:
10.1038/ismej.2011.116
Simister, R. L., Deines, P., Botté, E. S., Webster, N. S., and Taylor, M.
W. (2012). Sponge-specific clusters revisited: a comprehensive phylogeny
of sponge-associated microorganisms. Environ. Microbiol. 14, 517–524. doi:
10.1111/j.1462-2920.2011.02664.x
Sipkema, D., Holmes, B., Nichols, S. A., and Blanch, H. W. (2009).
Biological characterisation of Haliclona (?gellius) sp.: sponge and associ-
ated microorganisms. Microb. Ecol. 58, 903–920. doi: 10.1007/s00248-009-
9534-8
Sollas, W. (1886). Preliminary account of the Tetractinellid sponges Dredged by
H.M.S. “Challenger” 1872-76. Part I. The Choristida. Sci. Proc. R. Dublin Soc. 5,
177–199.
Steger, D., Ettinger-Epstein, P., Whalan, S., Hentschel, U., De Nys, R., Wagner, M.,
et al. (2008). Diversity and mode of transmission of ammonia-oxidizing archaea
in marine sponges. Environ. Microbiol. 10, 1087–1094. doi: 10.1111/j.1462-
2920.2007.01515.x
Sunagawa, S., DeSantis, T. Z., Piceno, Y. M., Brodie, E. L., DeSalvo, M. K., Voolstra,
C. R., et al. (2009). Bacterial diversity and White Plague Disease-associated
community changes in the Caribbean coral Montastraea faveolata.ISME J. 3,
512–521. doi: 10.1038/ismej.2008.131
Szitenberg, A., Becking, L. E., Vargas, S., Fernandez, J. C. C., Santodomingo, N.,
Wörheide, G., et al. (2013). Phylogeny of Tetillidae (Porife ra,Demospongiae,
Spirophorida) based on three molecular markers. Mol. Phylogenet. Evol. 67,
509–519. doi: 10.1016/j.ympev.2013.02.018
Taylor, M. W., Hill, R. T., Piel, J., Thacker, R. W., and Hentschel, U. (2007a). Soaking
it up: the complex lives of marine sponges and their microbial associates. ISME
J. 1, 187–190. doi: 10.1038/ismej.2007.32
Taylor, M. W., Radax, R., Steger, D., and Wagner, M. (2007b). Sponge-associated
microorganisms: evolution, ecology, and biotechnological potential. Microbiol.
Mol. Biol. Rev. 71, 295–347. doi: 10.1128/MMBR.00040-06
Thacker, R. W., Hill, A. L., Hill, M. S., Redmond, N. E., Collins, A. G., Morrow,
C. C., et al. (2013). Nearly complete 28S rRNA gene sequences confirm
new hypotheses of sponge evolution. Integr. Comp. Biol. 53, 373–387. doi:
10.1093/icb/ict071
Thompson, A. W.,Foster, R. A., Krupke, A., Carter, B. J., Musat, N., Vaulot, D., et al.
(2012). Unicellular cyanobacterium symbiotic with a single-celled eukaryotic
alga. Science 337, 1546–1550. doi: 10.1126/science.1222700
van Soest, R. W. M., Boury-Esnault, N., Hooper, J. N. A., Rützler, K., De Voogd, N.
J., Alvarez de Glasby, B., et al. (2014). World Porifera Database. Available online
at: http://www.marinespecies.org/porifera (Accessed May 09, 2014).
van Soest, R. W. M., Boury-Esnault, N., Vacelet, J., Dohrmann, M., Erpenbeck, D.,
De Voogd, N. J., et al. (2012). Global diversity of sponges (Porifera). PLoS ONE
7:e35105. doi: 10.1371/journal.pone.0035105
Vogel, S. (1977). Current-induced flow through living sponges in nature. Proc. Natl.
Acad. Sci. U.S.A. 74, 2069–2071. doi: 10.1073/pnas.74.5.2069
Walker, B. K. (2012). Spatial analyses of benthic habitats to define coral reef
ecosystem regions and potential biogeographic boundaries along a latitudinal
gradient. PLoS ONE 7:e30466. doi: 10.1371/journal.pone.0030466
Walker, C. B., de la Torre, J. R., Klotz, M. G., Urakawa, H., Pinel, N., Arp,
D. J., et al. (2010). Nitrosopumilus maritimus genome reveals unique mecha-
nisms for nitrification and autotrophy in globally distributed marine crenar-
chaea. Proc. Natl. Acad. Sci. U.S.A. 107, 8818–8823. doi: 10.1073/pnas.0913
533107
Wang, Q., Garrity, G. M., Tiedje, J. M., and Cole, J. R. (2007). Naïve bayesian
classifier for rapid assignment of rRNA sequences into the new bacterial
taxonomy. Appl. Environ. Microbiol. 73, 5261–5267. doi: 10.1128/AEM.00
062-07
Webster, N. S., and Blackall, L. L. (2009). What do we really know about sponge-
microbial symbioses? ISME J. 3, 1–3. doi: 10.1038/ismej.2008.102
Webster, N. S., Cobb, R. E., and Negri, A. P. (2008). Temperature thresholds for
bacterial symbiosis with a sponge. ISME J. 2, 830–842. doi: 10.1038/ismej.
2008.42
Webster, N. S., Cobb, R. E., Soo, R., Anthony, S. L., Battershill, C. N., Whalan, S.,
et al. (2011). Bacterial community dynamics in the marine sponge Rhopaloeides
odorabile under in situ and ex situ cultivation. Mar. Biotechnol. 13, 296–304. doi:
10.1007/s10126-010-9300-4
Webster, N. S., Luter, H. M., Soo, R. M., Botté, E. S., Simister, R. L., Abdo, D.,
et al. (2013). Same, same but different: symbiotic bacterial associations in GBR
sponges. Front. Microbiol. 3:444. doi: 10.3389/fmicb.2012.00444
Webster, N. S., and Taylor, M. W. (2012). Marine sponges and their microbial
symbionts: love and other relationships. Environ. Microbiol. 14, 335–346. doi:
10.1111/j.1462-2920.2011.02460.x
Webster, N. S., Taylor, M. W., Behnam, F., Lücker, S., Rattei, T., Whalan, S., et al.
(2010). Deep sequencing reveals exceptional diversity and modes of transmis-
sion for bacterial sponge symbionts. Environ. Microbiol. 12, 2070–2082. doi:
10.1111/j.1462-2920.2009.02065.x
Webster, N. S., Watts, J. E. M., and Hill, R. T. (2001). Detection and phylogenetic
analysis of novel crenarchaeote and euryarchaeote 16S ribosomal RNA gene
sequences from a great barrier reef sponge. Mar. Biotechnol. 3, 600–608. doi:
10.1007/s10126-001-0065-7
White, J. R., Patel, J., Ottesen, A., Arce, G., Blackwelder, P., and Lopez, J. V. (2012).
Pyrosequencing of bacterial symbionts within Axinella corrugata sponges:
diversity and seasonal variability. PLoS ONE 7:e38204. doi: 10.1371/jour-
nal.pone.0038204
Wilkinson, C. R. (1978a). Microbial associations in sponges. I. Ecology, physiology
and microbial populations of coral reef sponges. Mar. Biol. 49, 161–167. doi:
10.1007/BF00387115
Wilkinson, C. R. (1978b). Microbial associations in sponges. II. Numerical anal-
ysis of sponge and water bacterial populations. Mar. Biol. 49, 169–176. doi:
10.1007/BF00387116
Wilkinson, C. R. (1978c). Microbial associations in sponges. III. Ultrastructure
of the in situ associations in coral reef sponges. Mar. Biol. 49, 177–185. doi:
10.1007/BF00387117
www.frontiersin.org November 2014 | Volume 5 | Article 581 |11
Cuvelier et al. Microbes associated with Cinachyrella sponge
Wilkinson, C. R., and Fay, P. (1979). Nitrogen fixation in coral reef
sponges with symbiotic cyanobacteria. Nature 279, 527–529. doi: 10.1038/27
9527a0
Zehr,J.P.,Waterbury,J.B.,Turner,P.J.,Montoya,J.P.,Omoregie,E.,
Steward, G. F., et al. (2001). Unicellular cyanobacteria fix N2in the
subtropical North Pacific Ocean. Nature 412, 635–638. doi: 10.1038/350
88063
Zhu, P., Li, Q., and Wang, G. (2008). Unique microbial signatures of the Alien
Hawaiian marine sponge Suberites zeteki.Microb. Ecol. 55, 406–414. doi:
10.1007/s00248-007-9285-3
Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Received: 30 June 2014; accepted: 15 October 2014; published online: 04 November
2014.
Citation: Cuvelier ML, Blake E, Mulheron R, McCarthy PJ, Blackwelder P, Vega
Thurber RL and Lopez JV (2014) Two distinct microbial communities revealed in the
sponge Cinachyrella. Front. Microbiol. 5:581. doi: 10.3389/fmicb.2014.00581
This article was submitted to Aquatic Microbiology, a section of the journal Frontiers
in Microbiology.
Copyright © 2014 Cuvelier, Blake, Mulheron, McCarthy, Blackwelder, Vega Thurber
and Lopez. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, distribut ion or reproduction in other
forums is permitted, provided the original author(s) or licensor are credited and that
the original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply with
these terms.
Frontiers in Microbiology | Aquatic Microbiology November 2014 | Volume 5 | Article 581 |12
