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Phylogenetic relationships among zooxanthellae (Symbiodinium) associated to
excavating sponges (Cliona spp.) reveal an unexpected lineage in the Caribbean
C. Granados a,1,2
, C. Camargo a,1
, S. Zea b
, J.A. Sánchez a,*
a
Laboratorio de Biología Molecular Marina—BIOMMAR, Departamento de Ciencias Biológicas-Facultad de Ciencias, Universidad de los Andes, P.O. Box 4976, Bogotá, Colombia
b
Departamento de Biología y Centro de Estudios en Ciencias del Mar—CECIMAR, Universidad Nacional de Colombia, INVEMAR, Cerro de Punta Betín, Santa Marta, Colombia
a r t i c l e i n f o
Article history:
Received 18 March 2008
Revised 19 July 2008
Accepted 30 July 2008
Available online 7 August 2008
Keywords:
Zooxanthellae
Clade G
Symbiodinium
Excavating sponges
Cliona
Caribbean
a b s t r a c t
Phylogenetic relationships of symbiotic dinoflagellate lineages, distributed in all tropical and subtropical
seas, suggest strategies for long distance dispersal but at the same time strong host specialization. Zoo-
xanthellae (Symbiodinium: Dinophyta), which are associated to diverse shallow-water cnidarians, also
engage in symbioses with some sponge species of the genus Cliona. In the Caribbean, zooxanthellae-bear-
ing Cliona has recently become abundant due to global warming, overfishing, and algae abundance. Using
molecular techniques, the symbionts from five excavating species (Cliona caribbaea, C. tenuis, C. varians,
C. aprica and C. laticavicola) from the southern and southwestern Caribbean were surveyed. Several
DNA sequence regions were used in order to confirm zooxanthellae identity; 18S rDNA, domain V of chlo-
roplast large subunit (cp23S), internal transcribed spacer 2 (ITS2), and ITS2 secondary structure.
Sequence analyses corroborated the presence of three zooxanthellae clades: A, B, and G. Presence of
clades A and B in common boring sponges of the Caribbean fit with the general pattern of the province.
The discovery of clade G for the first time in any organism of the Atlantic Ocean leads us to consider this
unusual finding as a phylogenetic relict through common ancestors of sponge clades or an invasion of the
sponge from the Indo-Pacific.
Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction
Zooxanthellae are symbiotic dinoflagellates (Symbiodinium:
Dinophyta) distributed in all tropical and subtropical seas includ-
ing coral reefs (Baker, 2003). Symbiodinium engage symbioses with
different organisms including octocorals (e.g. Goulet and Coffroth,
2004), scleractinians (e.g. Pochon et al., 2006; Santos et al., 2002),
and foraminiferans (Pochon et al.,2004, 2006, 2001). Some studies
have revealed that zooxanthellae provide their hosts metabolites
important in the ocean which is poor nutrient water (Santos and
Coffroth, 2005). Zooxanthellae have different strategies among
them long distance dispersal and strong host specialization (e.g.
Goulet, 2006; LaJeunesse, 2005). Moreover, Symbiodinium lineages
evolution should reflect a combination of ecological drift together
with symbiotic preferences. Using molecular techniques, eight ma-
jor lineages of zooxanthellae have been found (termed clades A–H)
symbiotic to shallow water cnidarians and a few other organisms
such as molluscs and foraminifera (e.g. Baker, 2001, 2003; Pochon
et al., 2001; Rowan, 1998). Only one study has focused on the
molecular identity of symbiotic dinoflagellates associated to the
boring sponge Cliona orientalis in the Indo-Pacific, finding a new
subclade within clade G (Schönberg and Loh, 2005) although other
studies have considered the sponge–zooxanthellae symbiosis (Car-
los et al., 1999). Clade G has also been found in Great Barrier Reef
octocorals (Goulet et al., 2008; van Oppen et al., 2005) and Pacific
foraminifera (Pochon et al., 2007, 2004, 2006, 2001). Caribbean
coral reefs have suffered a change in composition since Acropora
palmata died off due to disease and bleaching during the early
1980s (see review in López-Victoria and Zea, 2004). Sponges,
including Cliona spp., have become more abundant but, despite this
abundance, studies on their zooxanthellae symbionts have not yet
been undertaken.
Sponges constitute one of the main groups of the animal king-
dom, being present and abundant in most marine ecosystems,
including coral reefs (Díaz and Rützler, 2001). Particularly, sponges
able to excavate tunnels and galleries into calcium carbonate play a
fundamental ecological role on coral reefs. Besides participating as
important bioeroders of the reef framework (e.g. Glynn, 1997),
those species able to encrust the excavated substratum often com-
pete for space with reef corals (López-Victoria et al., 2006; Rützler,
2002; Schönberg and Wilkinson, 2001; Vicente, 1978). In impacted
coral reefs, that is, reefs that had been affected by several stressors
such as sediment and overfishing (Bellwood et al., 2004), excavat-
ing sponges tend to augment in abundance, thus increasing bioero-
1055-7903/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2008.07.023
* Corresponding author. Fax: +57 1 3 394949 2817.
E-mail address: juansanc@uniandes.edu.co (J.A. Sánchez).
1
These two authors contributed equally to this paper.
2
Present address: Department of Biology, University of Louisiana at Lafayette,
Lafayette, LA, 70504, USA.
Molecular Phylogenetics and Evolution 49 (2008) 554–560
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev

sion and killing corals in the process (Holmes, 2000; López-Victoria
and Zea, 2005; Rose and Risk, 1985). For instance, any physical
damage to coral colonies may favor sponge recruitment and bor-
ing, which can then initiate a massive bioerosion process (Rützler,
2002). In addition, under elevated temperature and irradiance
stress due to global warming, these sponges tend to spread faster
into and over corals (Rützler, 2002) owing to physiological dys-
function and loss of their crucial symbionts (Walther et al.,
2002). Interestingly, some excavating sponges, especially those
that are also able to encrust the excavated substratum and com-
pete for space in the reef, engage in symbioses with zooxanthellae
(e.g. Rützler, 1990). Furthermore, some sponges, as the boring
sponge Cliona varians, are able to acquire symbionts from the envi-
ronment when they are subject to bleaching events (Hill and Wil-
cox, 1998). Although sponges are potentially threatened by the
same environmental factors affecting reef-building corals, such as
bleaching, they seem to be less affected (Schönberg, 2006; Vicente,
1990).
In spite of the increasing understanding in the molecular ecol-
ogy of the coral–zooxanthellae symbioses (e.g. Goulet, 2006),
there has been only one such study carried on one species of
Indo-Pacific Cliona, as mentioned above (Schönberg and Loh,
2005). Fifteen species of excavating sponges are found in the
Caribbean Sea, but not all of them have symbioses with photo-
synthetic dinoflagellates. Studies about these symbioses have
confirmed that sponges receive metabolic energy from the dino-
flagellates and that symbionts use animal waste as nutrient
source (Garson et al., 1998). These symbioses also enhance the
boring rate, reproductive output, and storage capability of the
sponge (Hill, 1996). In reef-building corals, different types of zoo-
xanthellae offer differential energetic outputs to their hosts
whereas others are more tolerant to certain levels of UVR or tem-
perature, which have been related to coral resistance and resil-
ience to thermal or irradiance stress (e.g. Berkelmans and van
Oppen, 2006; Goulet and Coffroth, 2004; Rowan, 2004). It is un-
known, however, whether the differential boring rates, tolerances
and strategies of Cliona spp. are related to particular types of zoo-
xanthellae or not, although recent research is being carried on
(Schönberg et al., 2008). In this study, we characterized the Sym-
biodinium lineages associated with five Caribbean symbiotic bor-
ing species (Cliona caribbaea, C. tenuis, C. varians, C. aprica, C.
laticavicola: Porifera, Hadromerida, Clionaidae: Fig. 1) using DNA
sequences and phylogenetic approaches.
2. Materials and methods
Tissue samples of boring sponges were collected using SCUBA at
different reefs in Colombia, which included southern (Santa Marta
and Cartagena) and southwestern (San Andrés island) Caribbean
reefs. S. Zea provided the San Andrés samples from previous sur-
veys. Samples from Santa Marta and Cartagena were collected be-
tween May and August 2005, taking a pinch with metal tweezers
<<1 cm3
. The samples were kept in sterile sealed bags (WhirlpalkTM
)
with seawater and were quickly fixed after collection in 95% etha-
nol or dimethyl sulfoxide (DMSO). In the laboratory, a portion of
the sponge sample was digested with sodium hypochlorite to re-
lease the siliceous spicules and slides were prepared for micro-
scopic identification with the aid of keys by Pang (1973) and Zea
and Weil (2003). DNA was extracted following the protocol of Cof-
froth et al. (1992), where the cells were lysed using CTAB, protein-
ase K and purifications steps with a mixture of phenol, chloroform,
and isoamyl alcohol (25:24:1), chloroform:isoamyl alcohol (24:1)
and ethanol precipitation. DNA was stored in TE buffer and diluted
with double distilled water (1:20).
For the molecular determination of zooxanthellae, initially, to
find major groups or clades, the 18S rDNA was amplified using
the primers ‘‘SS3Z” (50
-AGCACTGCGGCAGTCCGAATAATTCA-30
)
and ‘‘SS5” (50
-GGTTGATCCTGCCAGTAGTCATATGC-30
) (Rowan and
Powers, 1992). Conditions for polymerase chain reaction (PCR)
were: an initial period of 2 min at 94 °C followed by 30 cycles of
30 s at 94 °C, 1 min at 61.6 °C and 1 min at 72 °C, and ended with
a final extension during 4 min at 72 °C (Taq Promega kit). The
18S rDNA products were digested with the restriction enzyme
Taq1 (New England Biolabs) for 2 h. The products were separated
in acrylamide gels 7% of 5 ml during 50 min, 150 V with a ladder
of 100 bp (Invitrogen). Taq1 RFLP patterns were compared with
those summarized in Santos et al. (2002).
The V domain of the chloroplast was amplified using primers
‘‘23SHYPERUP” (50
-TCAGTACAAATAATATGCTG-30
) and ‘‘23SHY-
PERDNM13” (50
-GATAACAATTTCACACAGGTTATCGCCCCAATTAAAC
AGT-30
) (Santos et al., 2003). Reactions were carried out under the
following conditions: one cycle at 94 °C for 4 min, 30 cycles
consisting of 94 °C for 1 min, 54.2 °C for 30 s, and 72 °C for 30 s,
and an extension period at 72 °C for 5 min. PCR products were
visualized in bromide stained agarose gels (1.0%).
For finer resolution of zooxanthellae types, the Internal Tran-
scribed Spacer 2 (ITS2) from nuclear ribosomal DNA was amplified
Fig. 1. Photographs of five Caribbean symbiotic boring species of sponges (Cliona caribbaea, C. tenuis, C. varians, C. aprica, C. laticavicola: Porifera, Hadromerida, Clionaidae) at
Barú Island, Colombian Caribbean.
C. Granados et al. / Molecular Phylogenetics and Evolution 49 (2008) 554–560 555

using the primers ‘‘ITSintfor2” (50
-GAATTGCAGAACTCCGTG-30
) and
‘‘ITS2” (50
-GGGATCCATA TGCTTAAG TT CAGCGGGT-30
) (Coleman
et al., 1994), with the following PCR thermocycling conditions:
an initial period of 3.0 min at 94 °C followed by 35 cycles of 30 s
at 94 °C, 40 s at 52 °C and 30 s at 72 °C, a final extension period
of 5 min at 72 °C. The ITS2 products were ran in a Denaturing Gra-
dient Gel Electrophoresis (DGGE) with a gradient gel of 3.15 M
urea/18% deionized formamide for 9 h at 150 V and a temperature
of 60 °C. It is important to note that we did not use a GC-clamp in
any of the primers. PCR amplifications were standardized to obtain
the best resolution of the bands.
PCR amplifications were purified for direct sequencing using the
QuickStep 2 PCR purification kit (Edge Biosystems) or using ExoSap
(Exonuclease I and Shrimp Alkaline Phosphatase, Fermentas). The
sequence was determined from both ends of the purified PCR prod-
uct using BigDye Terminators using capillary electrophoresis in an
ABI 310 automated sequencer (both Applied Biosystems). ITS2 and
cp23 S sequences were deposited in GenBank (Accession Nos.:
EF134611–EF134623 and EU006517–EU006529, respectively).
Initially for sequence analysis, a BLAST search in GenBank
(NCBI) was made with the partial sequences obtained as search
queries. Different sequences from various studies were used for
subsequent alignments and analyses (Table 1). Both maximum
parsimony and maximum likelihood analyses were made for se-
quence alignment corrected (gaps as fifth base for ITS2 and as
missing for cp23S) and for the primary alignment made by Muscle
(Edgar, 2004) with default settings. Phylogenetic trees were made
using Maximum likelihood-ML (PAUPÃ) (Swofford, 2002) with the
best-fit model and parameters according to ModelTest (Posada and
Crandall, 1998) and the Akaike Information Criterion. Above node
branch support was assessed in PAUPÃ for 1000 maximum parsi-
mony bootstrap replicates (Felsenstein, 1985) and 375 and 500
maximum likelihood bootstrap replicates for ITS2 and cp23S,
respectively, according to the estimated sufficient number of repli-
cates using RAxML (Stamatakis et al., 2008). In addition, Bayesian
inference of phylogeny using MrBayes (Huelsenbeck and Ronquist,
2003) was made under different models of sequence evolution:
GTR+G model for ITS2 (3,000,000 generations, sample frequency
300, burn-in 2500) and F81+G model for cp23S (2,000,000 genera-
tions, sample frequency 200, burn-in 2500).
For the secondary structure analysis, the first step was to recog-
nize common structural elements in Symbiodinium ITS2, individual
sequences were superimposed onto the secondary structure of
Symbiodinium clade G (Hunter et al., 2007). Manual alignment
was performed by visual homology for the construction of a Dedi-
cated Comparative Sequence Editor (DCSE) format. Areas of high-
sequence conservation in ITS2 served as reference points. The ac-
quired structures with restrictions and constraints were submitted
in Mfold (Zuker, 2003) and further edited in RnaViz (DeRijk and De
Wachter, 1997).
3. Results
All Cliona spp. specimens collected from the three sites con-
tained Symbiodinium spp. populations. From 31 samples, 18 TaqI
RFLP patterns of 18S rDNA were obtained, which resulted in two
clades of zooxanthellae: 10 samples belonged to clade A and eight
to clade G (Table 1). These banding patterns corresponded to dif-
ferent sponge hosts indicating that the types of zooxanthellae
found were distributed across various hosts.
Chloroplast amplification was positive in only 13 samples, con-
siderably lower than that obtained for 18S rDNA, much of which
belonged to the sponge C. varians. Direct sequencing analysis
showed fragments of about 200–300 bp as expected (Santos
et al., 2003), although some sequences were not efficiently
sequenced in both directions. BLAST search with these sequences,
whether complete or partial, allowed the identification of zooxan-
thellae clades A and G divided in three types of clade G in nine of
the sequences and clade A in the other four sequences.
All samples were run for ITS2 and screened in denaturing gradi-
ent gel electrophoresis (DGGE), obtaining patterns of three differ-
ent clades. No heteroduplexes were found and banding profiles
from genetically distinct Symbiodinium types were highly repeat-
able. Sometimes background bands were observed in the denatur-
ing gel, although most of the samples were characterized by a
single particular band (See electronic supplementary material).
Clade A was the most abundant and was found in 17 samples, fol-
lowed by clade G in 12 and clade B in two. ITS2 sequencing per-
formed from direct cycle sequencing of the amplification product
corroborated the same three clades of Symbiodiniun mentioned
above. Five types of zooxanthellae were depicted according to ITS2.
Sequencing the genes ITS2 and cp23S resulted in similar pat-
terns corresponding of zooxanthellae types (at least 2–3 replicates
in each case) or the discovery of new types. Comparative phyloge-
netic analyses with known sequences from ITS2 and cp23S (e.g. Po-
chon et al., 2007; Santos et al., 2002) corroborated the identity of
zooxanthellae clades in almost all of the samples sequenced suc-
cessful for both genes. ITS2 phylogenies inferred from maximum
parsimony, maximum likelihood and Bayesian inference were
highly congruent both in resolution and support values (Fig. 2A).
Samples grouped with different known types of clades A, B, and
G. The types included one from clade A (Symbiodinium [=Gymniodi-
nium] linuchae, A4), two from clade B (B11, B19–B21: ITS2) and
three from clade G (van Oppen et al., 2005) and types G1 and G3
(Pochon et al., 2007; Pochon et al., 2006). Most sponge species
(three out of five) analyzed had good reliability, in average nine
samples were identified for each species (Table 1). Two of the se-
lected species (C. laticavicola and C. caribbaea) were represented
only by two samples in each case.
Predicted RNA secondary structure analysis of ITS2 showed a
model with four major helixes designated I–IV (Gottschling and
Plotner, 2004) separated by single-stranded regions of the central
core (Fig. 2B). Helixes showed distinct size classes: helix I (42–
17 bp), helix II (80–53 bp), helix III (174–103), and helix IV (201–
187 bp). Helix III was comprised of a single stem. This model fit
with the one described by Hunter et al. (2007) for clade G including
unique features for this group of Symbiodinium.
Congruence between chloroplast phylogenetic methods was not
as consistent as the ITS2 results. Maximum parsimony presented
better resolution than maximum likelihood and Bayesian infer-
ences, which in some cases, presented low support values
(Fig. 2C). In spite of this, cp23S phylogenetic analysis still corrobo-
rated presence of clade G (Pochon et al., 2006) in C. varians sam-
ples. In addition, most of the samples grouped within
reciprocally monophyletic and formally described zooxanthellae
clades. Probably, the lack of better congruence between the phy-
logenies was due to the failure of obtaining sequences in both
directions.
4. Discussion
This is the first molecular examination to determine the iden-
tity of zooxanthellae associated with boring sponges of the genus
Cliona in the Caribbean. The presence of zooxanthellae sensu lato
had been previously known from histological studies in all sam-
pled species (Pang, 1973; Rützler, 1990; Vicente, 1978; Zea and
Weil, 2003) except in Cliona laticavicola. In its original description,
no zooxanthellae were noted among the sponge cells (Pang, 1973).
However, we were able to see zooxanthellae in macerated tissue
from our samples and this is the first report of symbiotic zooxan-
thellae in C. laticavicola.
556 C. Granados et al. / Molecular Phylogenetics and Evolution 49 (2008) 554–560

The most significant result was the finding of an unexpected
Symbiodinium lineage for the first time in the Atlantic Ocean: clade
G. Despite corroborated results from nuclear and chloroplast
sequences, the ITS2 predicted RNA secondary structure of Cliona-
associated Symbiodinium (Fig. 2B) showed the four-fingered con-
formation that has been previously described for most eukaryotes
Table 1
Information about zooxanthellae clade, host, depth, location, molecular marker used with Genbank Accession number, and reference
Zooxanthella
clade
Host Depth (m) Location Molecular marker Reference
18S P: positive N:
negative
rDNA ITS2 Accession No. cp23S Accession
No.
A C. aprica 14 SM N EF134612 This study
A C. aprica 10 SA P EF134617–EF134618 EU006528–
EU006529
This study
A C. aprica 4 SB N EF134611 This study
A C. caribbaea 11–20 CRI N EF134616 This study
A C. tenuis 11–17 CRI P EF134613–EF134615 EU006526–
EU006267
This study
A C. laticavicola 4 SB ITS2-DGGE (see supplementary
material)
This study
A Aiptasia pallida Cultured Florida keys,
USA
AY035404 Santos et al. (2002)
A1 Cassiopeia xamachana — — AF333505 LaJeunesse (2001)
A2 Zoanthus sociatus — — AF333506 LaJeunesse (2001)
A3 Hippopus hippopus — — AF333507 LaJeunesse (2001)
A4 Linucheunguiculata — — AF333509 LaJeunesse (2001)
B C. aprica 14 SM N EF134620 This study
B C. aprica 4 SB N EF134619 This study
B C. laticavicola 4 SB ITS2-DGGE (see supplementary
material)
This study
B Plexaura flexuosa Cultured Florida keys,
USA
AY035420 Santos et al. (2002)
B Aiptasia pulchella Cultured Hawaii, USA AY035421 Santos et al. (2002)
B11 Anthosigmella varians — — DQ865211 Hunter et al. (2007)
B19 Plexaruella nutans — — DQ865212 Hunter et al. (2007)
B21 Briareum sp. — Florida keys,
USA
DQ865213 Hunter et al. (2007)
B4 Anthopleura
elagantissima
— — AF333510 LaJeunesse (2001)
C Montipora verrucos Cultured Hawaii, USA AY035422 Santos et al. (2002)
C Sinularia sp. Cultured Guam, USA AY035423 Santos et al. (2002)
C22 Turbinaria sp.,
Lobophyllia sp.
1–3 — AY239373 LaJeunesse et al.
(2003)
C31 Montipora sp. 1–6 — AY258496 LaJeunesse et al.
(2003)
C39 Mussidae sp. Diploastrea
sp.
2 — AY258484 LaJeunesse (2005)
C49 Mycetophyllia sp. 7–10 — AY589752 LaJeunesse (2005)
C67 Heteractis magnifica 2–3 — AY686647 LaJeunesse et al.
(2003)
C69 Stichodactyla sp. 2 — AY589773 LaJeunesse (2005)
D Litophyton sp. — Red Sea DQ865214 Hunter et al. (2007)
E Heterocaspa sp. Cultured Panama AY035430 Pochon et al. (2006)
E — — — AF334659 LaJeunesse (2001)
F Sorites sp. Shallow
water
Maldives AJ872105 Pochon et al. (2006)
F1 Montipora verrucosa — — AF333517 LaJeunesse (2001)
G C. caribbaea 10 SA P EF134622–EF134623 This study
G C. varians 11–20 SM P EF134621 EU006517–
EU006524
This study
G Marginopora vertebralis Shallow
water
Guam, USA AJ872107 Pochon et al. (2006)
water
water
G1 Soritid foraminifera — Guam, USA AM748597 Pochon et al (2007)
G2 Marginopora vertebralis — Guam, USA AM748598 Pochon et al. (2007)
G2a Marginopora vertebralis — Guam, USA AM748599 Pochon et al. (2007)
G3 Soritid foraminifera — Guam, USA AM748600 Pochon et al. (2007)
G4 Soritid foraminifera — Guam, USA AM748601 Pochon et al. (2007)
G Amphisorus sp. — Guam USA AJ291537 Pawlowski et al.
(2001)
H Sorites sp. — Florida keys,
USA
AJ291513 Pawlowski et al.
(2001)
Locations: SM: Santa Marta; CRI: Cartagena, Rosario islands; SB: Cartagena, San Bernardo islands; SA: San Andrés for Colombian sites or else specified.
All Cliona spp. samples had encrusting growth form.
Symbol ‘‘—” indicates no information available.

(e.g. Coleman, 2003, 2007; Denboh et al., 2003; Joseph et al., 1999;
Mai and Coleman, 1997), which contained a clade G hallmark as an
unusual bifurcated helix unique among all zooxanthellae clades
(Hunter et al., 2007).
Our results showed both similar and contrasting patterns with
respect to what is known for symbiotic cnidarians in the Carib-
bean. Symbiodinium clade A was the most common type found in
all sampling locations. Three of the species (Cliona aprica, C. tenuis,
Fig. 2. (A) Phylogenetic reconstruction of the genus Symbiodinium inferred from ITS2 (best optimum tree). (B) ITS 2 secondary structure obtained from clade G sequences.
Roman numbers (I–IV) represent helixes. (C) Cp23 phylogenetic reconstruction (best optimum tree). Above node numbers indicate congruence from 500 maximum
parsimony bootstrap replicates using branch-and-bound, 375 and 1000 maximum likelihood bootstrap replicates, respectively, using branch-and-bound. Values below
indicate Bayesian posterior probabilities. Ã absence of MP bootstrap values; ÃÃ absence of ML bootstrap values; ÃÃÃ absence of Bayesian posterior probability. Cliona: C. apr
C. aprica; C. car C. caribbaea; C. ten C. tenuis. See legend of Table 1 for abbreviation of localities. (C) Cliona species in the study area.

and C. laticavicola) have symbioses with clade A. This clade of zoo-
xanthellae has been considered as one of the most common in the
Caribbean, which includes symbioses with anemones, zoanthids,
scleractinian corals such as Porites furcata (LaJeunesse et al.,
2003) and octocorals as Pseudoplexaura porosa (Coffroth et al.,
2001 but see: Goulet and Coffroth, 2004). The A4 zooxanthellae
type resolved as sister group with types A1 and A3 which has been
isolated by Zoanthus sansibaricus (Reimer et al., 2007), the reef-
building coral Montastraea franksi (Garren et al., 2006) and Acro-
pora spp. (LaJeunesse, 2002) among others. Consequently, it is
not surprising that an A-type zooxanthella was associated with
more than one species of Cliona.
Zooxanthellae clade B, the most prevalent of the Caribbean
province, were found as symbiont of C. aprica and C. laticavicola
in two different locations and depths, Santa Marta and Cartagena
at 17 and 5 m, respectively. This is consistent with the pattern sug-
gested by LaJeunesse (2002), where clade B shows the highest plas-
ticity in Caribbean reefs. Moreover, zooxanthellae exhibit high
plasticity in their photosynthetic response due to adaptations in
the photosynthetic apparatus (Steindler et al., 2001). Clade B sam-
ples grouped with B11, B19, and B21 types, which have only re-
cently been found in the octocorals Plexaurella nutans and
Briareum sp. (GenBank Accession Nos. DQ865212 and DQ865213,
Hunter et al., 2007; AV239361, LaJeunesse, 2005). Findings of
clades A and B in common boring sponges of the Caribbean fit with
the general pattern of the province, where clade B is almost ende-
mic and types such as A4 engage symbiosis with many species.
A contrasting pattern in the Caribbean, however, was the sur-
prising finding of Symbiodinium clade G (-ITS2- Pochon et al.,
2007; -cp23S- Pochon et al., 2006) in Cliona varians from Santa
Marta and Cliona caribbaea from San Andrés. This zooxanthella
lineage has only been found in diverse western Pacific organisms
such as Junceella fragilis, Euplexaura nuttingi, and Stereonephthya
sp. 1 (Goulet et al., 2008; van Oppen et al., 2005), several foram-
inifera (Pochon et al., 2006), and the Indo-Pacific boring sponge
Cliona orientalis (Schönberg and Loh, 2005). In brief, clade G zoo-
xanthellae are found in a wide range of invertebrates but they
seem restricted to the western Pacific (see Fig. 1 in Goulet
et al., 2008). Particularly intriguing, foraminifera that have clade
G in the Indo-Pacific have Symbiodinium C in the eastern Pacific
and F or H in the Caribbean (Pochon et al., 2004). Additionally,
no free-living zooxanthellae from clade G have been reported in
the Caribbean, as a recent study found the presence of clades A,
B, and C (Porto et al., 2008). Given that this is the first time clade
G has been reported from the Atlantic, the question on the origin
of these Caribbean clade G zooxanthellae arises. One explanation
could be due to differences in the primordial origin of the symbi-
oses between zooxanthellae-sponges and zooxanthellae-corals,
which apparently evolved earlier with sponges than with corals
(ca. 650 mya: Vicente, 1990). However, results did not fit in the
general pattern of the Caribbean province, where most clades
have widely diversified their symbioses in different organisms
(i.e. clades A and B in diverse cnidarians and some Cliona spp.)
(e.g. Goulet et al., 2008). The presence of Symbiodinium G in Cliona
varians and C. caribbaea suggest it may be a phylogenetic relict
through common ancestors of sponges’ clades, because these spe-
cies belong to the C. viridis complex, which have Pacific counter-
parts associated with zooxanthellae as symbionts (Schönberg,
2002). A few cases of hosts changing their symbionts in different
geographic areas exist. For example, Euplexaura nuttingi, a Plexau-
ridae species in the Indo-west Pacific, is the only from this family
that host clade G while congeners host clade B in the Caribbean
(Goulet et al., 2008). Additionally, this is the only gorgonian spe-
cies to harbor symbiotic dinoflagellates in the western Pacific
(van Oppen et al., 2005). Further studies of symbionts of sponges,
together with phylogenetic and phylogeographic studies of both
sponges and their symbionts are needed in order to comprehend
the origin of clade G in the Atlantic.
Acknowledgments
This study was funded by COLCIENCIAS (Grant 120409-16825)
and Facultad de Ciencias, Department of Biological Sciences, Uni-
versidad de los Andes (funding to C. Camargo and J.A. Sánchez).
The Minister of Environment, Household, and Territorial Develop-
ment of Colombia granted access to genetic resources to J.A.S. for
the DNA analyses included in this paper (Contract 007, resolution
634, 14 March 2007). We are very grateful to many colleagues
and friends especially A. Grajales, M. Cardenas, N. Manrique, S. San-
tos, T. LaJeunesse, T. Goulet, M.A. Coffroth and the members from
BIOMMAR, La Tortuga Dive Shop (Christian Martinez), CECIMAR
(UNAL), INVEMAR, and UAESPNN (PNNCR-SB) for their helpful dis-
cussions, cooperation, advice and assistance. Sven Zea’s work is a
contribution of INVEMAR and Centro de Estudios en Ciencias del
Mar—CECIMAR, Marine Biology graduate program, Faculty of Sci-
ences, Universidad Nacional de Colombia.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ympev.2008.07.023.
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