MOLECULAR PHYLOGENY OF MANGROVE OYSTERS
(CRASSOSTREA ) FROM BRAZIL
EDUARDO SOUSA VARELA 1 , COLIN ROBERT BEASLEY 2 ,
HORACIO SCHNEIDER 3 , IRACILDA SAMPAIO 3 ,
NELANE DO SOCORRO MARQUES-SILVA 1 AND CLAUDIA HELENA TAGLIARO 1
1
Laborato´rio de Conservação e Biologia Evolutiva, Campus de Bragança, Universidade Federal do Pará, Alameda Leandro Ribeiro s/n,
Brangança, Pará, Brazil, CEP 68600-000;
2
Laborato´rio de Moluscos, Campus de Bragança, Universidade Federal do Pará, Alameda Leandro Ribeiro s/n, Brangança, Pará, Brazil, CEP 68600-000;
3
Laborato´rio de Gene´tica e Biologia Molecular, Campus de Bragança, Universidade Federal do Pará, Alameda Leandro Ribeiro s/n,
Brangança, Pará, Brazil, CEP 68600-000
(Received 30 May 2006; accepted 12 April 2007)
ABSTRACT
As a result of phenotypic plasticity, the cupped oysters (Crassostrea ) are difficult to identify by means of
their morphology. However, molecular DNA markers are a useful means of discriminating among these
species. Cupped oysters are one of the most widely cultured marine invertebrates and correct species
identification is important in aquaculture. Moreover, the molecular phylogeny of the genus Crassostrea
and the subfamily Crassostreinae is still not clear. In order to identify the Brazilian cupped oysters and
to clarify the phylogenetic relationships of these species, we sequenced a fragment of mitochondrial
DNA (16S rRNA gene) from 120 specimens collected at nine different sites distributed along the
Brazilian coast. The results identified two native species of oyster: Crassostrea gasar, from the Amazon
to the Parnaı́ba delta; and Crassostrea rhizophorae, from the northeast (Fortim) to the south of Brazil.
An exotic Crassostrea species, closely related to Indo-Pacific Crassostrea, was found in one location in
the north of Brazil. Crassostrea showed monophyly and the Atlantic oysters are clearly separated from
the Indo-Pacific cluster.
INTRODUCTION
Mangrove oysters of the genus Crassostrea Sacco, 1897 are widely
distributed along the Brazilian coast. These bivalves live
attached to hard substrates such as mangrove roots and rocks
in the intertidal zone (Nascimento, 1991; Rios, 1994). They
are characterized by extensive phenotypic plasticity such that,
in the course of ontogenetic development, their shells may
reflect the nature of the substrate and/or tidal regime. Thus
morphology alone may be inadequate for the identification of
specimens and for their taxonomy (Korringa, 1952; Stenzel,
1971; Boudry, Heurtebise & Lapègue, 2003).
Harry (1985), based on the morphology of the shell and soft
parts, recognized a single species, Crassostrea virginica Gmelin,
1791, as ocurring in the Western Atlantic, from Brazil northward through the Caribbean, Gulf of Mexico, including the
Antilles, to the St Lawrence river in Canada. In Brazil, Rios
(1994) considered all mangrove oysters to belong to Crassostrea
rhizophorae Guilding, 1828. However, Absher (1989) observed
differences in growth rates and larval morphology among sympatric lineages of Crassostrea from the southern coast of Brazil
and suggested the cooccurrence of two distinct biological species:
C. rhizophorae and Crassostrea brasiliana Lamarck, 1819. Both
species are described as being very similar, but Nascimento
(1991) affirms that C. brasiliana is less cup-like than C. rhizophorae.
The muscle scar in C. brasiliana is generally slightly blue
or brown, whereas in C. rhizophorae it is often unpigmented
(Nascimento, 1991). A study based on allozyme electrophoresis
(Ignacio et al., 2000) also supported the existence of these
two species. Carriker & Gaffney (1996) regarded C. brasiliana
as a synonym of C. virginica, but Singarajah (1980) considers
C. rhizophorae and C. brasiliana as synonymous. Additionally,
Correspondence: C.H. Tagliaro; e-mail: [email protected]
based on morphological and physiological characteristics,
Singarajah (1980) described a new species of oyster, Crassostrea
paraibanensis, from the Paraı́ba river estuary.
A molecular analysis (RFLP-PCR and sequences of 16S
rRNA) allied with karyological data (Lapègue et al., 2002)
revealed the cooccurrence of the mangrove oyster Crassostrea
gasar (Adanson, 1757) along the Western African and
South American Atlantic coasts. The sequence of 16S rRNA of
C. brasiliana deposited in the GenBank (DQ839413) by Pie
et al. (2006) is identical to that of C. gasar (AJ312937) studied
by Lapègue et al. (2002), indicating that both belong to the
same species. In this paper, we shall refer to C. brasiliana as
C. gasar, since the latter name has precedence.
Several studies have shown that mitochondrial DNA
(mtDNA) sequences have been informative for discriminating
among species of oyster and to establish local phylogeny
(Banks, Hedgecock & Waters, 1993; Ó Foighil, Gaffney &
Hilbish, 1995; Boudry et al., 1998; Hedgecock et al., 1999;
Lam & Morton, 2003; Boudry, Heurtebise & Lapègue 2003;
Klinbunga et al., 2005). Although larval dispersion patterns
allow extensive gene flow in cupped oysters (Buroker, 1983),
Reeb & Avise (1990) using molecular markers based on mitochondrial loci, demonstrated a clear phylogeographic division
among populations of the American oyster, C. virginica, from
the north and south of a region of the Atlantic coast of
Florida. They suggested that the present genetic distribution
of C. virginica was caused, over geological time, by reduced precipitation levels in an enlarged Floridian peninsula, creating
discontinuities in suitable estuarine habitat for oysters during
glacial periods. Today, such population divisions are maintained by the combined influence of ecological gradients and
oceanic currents on larval dispersal. Other studies (Ó Foighil
et al., 1998; Huvet et al., 2000) revealed that the Portuguese
oyster, Crassostrea angulata, is derived from Asian oyster stocks,
Journal of Molluscan Studies (2007) 73: 229–234. Advance Access Publication: 28 June 2007
# The Author 2007. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved.
doi:10.1093/mollus/eym018
E.S. VARELA ET AL.
investigating samples from nine different localities along the
Brazilian coast. The aim of our study is to identify the species,
estimate their geographic distribution and establish the phylogenetic relationships among Crassostrea species.
showing great similarity to the Pacific oyster Crassostrea gigas.
Ó Foighil et al. (1998) suggested an explanation through undocumented anthropogenic introductions dating from the earliest
days of circumglobal navigation.
Phylogenetic relationships among seven species of Crassostrea
were inferred from aligned sequences of the 28S rRNA region by
parsimony and maximum likelihood (ML) methods (Littlewood,
1994) and the author suggested the separation of the Atlantic
oysters (C. virginica and C. rhizophorae) from Indo-Pacific
Crassostrea. Moreover, the molecular analyses based on 16S
rRNA sequences developed by Lapègue et al. (2002), Boudry,
Heurtebise & Lapègue (2003) and Lam & Morton (2003) also
indicated a clear division between the Atlantic (C. gasar,
C. rhizophorae and C. virginica ) and the Indo-Pacific Crassostrea.
Brazil has the world’s largest continuous mangrove ecosystem,
estimated at 1.38 million hectares along 6,800 km of coast
(Kjerfve & Lacerda, 1993). Brazilian oysters are distributed
along the entire mangrove coast, but the number of species
and their distribution is not clear. Native mangrove oysters
from Brazil are generally referred to as C. rhizophorae. In the
north and northeast of Brazil, particularly, these oysters are cultivated or harvested from wild populations (Nascimento, 1991)
and correct identification of the species and their distributions
is needed for the effective management of such activities.
The present study is the most extensive molecular analysis,
based on 16S rRNA, of South American cupped oysters,
MATERIAL AND METHODS
Sampling
A total of 120 wild mangrove oysters were collected from nine
locations along the Brazilian coast (Table 1). The sample sizes
varied between 5 and 30 per locality. Crassostrea gasar and
C. rhizophorae were collected from rocks and/or mangrove
roots, and appeared not to have a preference for any particular
substrate (Table 1). Specimens belonging to a third species,
all of which were juveniles, were obtained from artificial
(plastic) spat collectors and a wooden support structure for
the collectors.
DNA extraction, PCR amplification and sequencing
The adductor muscle of all samples was preserved in 100%
ethanol and kept at 2 208C until DNA extraction. Total
genomic DNA was extracted from the tissue according to the
phenol-chloroform protocol of Sambrook, Fritsch & Maniatis
(1989). DNA amplification of partial mitochondrial 16S
Table 1. Details of oyster samples used in the present study, including location, type of substrate from which oysters were collected and GenBank accession numbers.
Samples (species or population)
n
H
Sampling location coordinates
Type of substrate
Tutóia
12
2
28370 3000 S, 428220 3000 W
Intertidal rocks
GenBank accession number
EF473274 (H1)
EF473270 (H2)
Nova Olinda (Augusto Correa)
12
2
018050 27,200 S, 468280 28,600 W
Intertidal rocks
EF473274 (H1)
EF473271 (H3)
São João de Pirabas
10
2
008500 41,200 S, 478070 27,500 W
Mangrove roots and intertidal rocks
EF473274 (H1)
EF473272 (H4)
Parnaı́ba
10
1
28480 4500 S, 418480 4500 W
Mangrove roots
EF473273 (H5)
Canela Island (Bragança)
30
3
008470 0200 S, 468430 32,900 W
Artificial collectors (plastic) and wooden
EF473278 (H6)
support
EF473280 (H7)
EF473281 (H8)
Fortim
12
2
48260 1500 S, 378480 4500 W
Mangrove roots
EF473282 (H9)
EF473279 (H15)
Maceió
10
1
98410 1500 S, 358410 1500 W
Mangrove roots
EF473275 (H10)
Vitória
19
3
208180 4500 S, 408180 4500 W
Mangrove roots
EF473283 (H11)
EF473276 (H12)
EF473284 (H13)
278330 4500 S, 488330 4500 W
Intertidal rocks
EF473277 (H14)
–
–
AJ312938
1
–
–
AJ312937
1
–
–
AF092285
Crassostrea angulata †
1
–
–
CGI553902
Crassostrea iredalei †
1
–
–
CIR553913
Crassostrea gigas †
1
–
–
AF280611
Crassostrea belcheri †
1
–
–
AY160759
Crassostrea ariakensis †
1
–
–
AY160757
Crassostrea rivularis †
1
–
–
AY510450
Crassostrea hongkongensis †
1
–
–
AY160756
Saccostrea cuccullata †
1
–
–
AF458909
Ostrea edulis †
1
–
–
Florianópolis
5
1
Crassostrea rhizophorae †
1
Crassostrea gasar †
Crassostrea virginica †
AF540597
†
The GenBank acession number is repeated when the same haplotype is present in more than one locality. Sequences obtained from GenBank; n is the number
of sequences; H is the number of haplotypes.
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MOLECULAR PHYLOGENY OF BRAZILIAN OYSTERS
rRNA was obtained using the primers 16SL1987 50 GCC TCG
CCT GTT TAC CAA AAA C 30 (this study) and 16Sbr 50 CCG
GTC TGA ACT CAG ATC ACG T 30 (Palumbi et al., 1989).
The amplification reaction was performed with a cycling
profile of 948C for 3 min, followed by 30 cycles of 948C for
1 min, 508C for 1 min, 728C for 1 min, with an additional extension period of 728C for 5 min during the last cycle. The PCR
products were purified using enzymes ExoSap IT (AmershamPharmacia Biotech. Inc., Piscataway, NJ, USA). Sequencing
was performed on a MegaBace 750 automated sequencing platform according to the manufacturer’s protocols.
Data analysis and phylogenetic reconstruction
The nucleotide sequence data for the haplotypes used in this
paper were deposited in GenBank under accession numbers
EF473270 –EF473284. One representative of Saccostrea (Saccostrea cuccullata ) was used to verify if this genus is sister to or
derived from Crassostrea. One species of Ostrea (Ostrea edulis)
that belongs to the same subfamily (Ostreinae), was used as an
outgroup. Sequence alignments were produced using the
BioEdit (Hall, 1999) and ClustalX 1.81 (Thompson et al.,
1997) programs. Nucleotide frequencies and transition/transversion ratio were obtained using Mega 3 software (Kumar,
Tamura & Nei, 2004). A saturation test was performed using
the DAMBE program (Xia & Xie, 2001). Sequences of other
species of Crassostrea were obtained from the GenBank and
included in the analysis (Table 1). Haplotypes were identified
by DnaSP 4.0 (Rozas et al., 2003).
Phylogenetic analyses were performed with PAUP 4.0
(Swofford, 2002) using the neighbour-joining (NJ), maximum
parsimony (MP) and ML methods. MODELTEST 3.07
(Posada & Crandall, 1998) was used to choose the best model
for use in the NJ and ML analysis by Hierarchical Likelihood
Ratio Tests (HLRTs). Evaluation of statistical confidence was
based on bootstrapping with 2,000 pseudo-replicates for NJ
and MP, and 1,000 for ML (Felsenstein, 1985). The criterion
adopted to evaluate robustness was to consider nodes with bootstrap values equal or superior to 90% as well supported. Bremer
decay values (Bremer, 1994) were calculated using PRAP
(Müller, 2004). The monophyly of the clades was tested according to Shimodaira & Hasegawa (1999) and Templeton (1983),
comparing the likelihoods and length differences, respectively,
between constrained and unconstrained topologies.
Figure 1. Map of Brazil indicating the sampling locations of mangrove
oysters (Crassostrea ).
bases. The settings for the best-fit model selected were: base
frequencies (A ¼ 0.2895, C ¼ 0.1801, G ¼ 0.2252, T ¼ 0.3052),
gamma distribution shape parameter (a ¼ 0.2690) and substitution model rate matrix (Rmat; A-C ¼ 0.4671, A-G ¼ 5.1045,
A-T ¼ 2.9464, C-G ¼ 0.8834, C-T ¼ 5.1045, G-T ¼ 1.0000).
The distance (d ) matrix measure using the GTR method with
the above evolutionary model parameters also showed the existence of three groups. The specimens of each group were closely
related (d ¼ 0–0.005). One of the sequences from the NC haplogroup was identical to the C. gasar sequence (GenBank
AJ312937) and the maximum divergence observed between C.
gasar and the haplotypes from NC was 0.002. The EC haplogroup
was found to belong to C. rhizophorae (d ¼ 0.014–0.017) and was
closely related to the other two Atlantic species: C. virginica
(d ¼ 0.099–0.102) and C. gasar AJ312937 (d ¼ 0.118–0.120).
The distances between Indo-Pacific (C. angulata, C. iredalei,
C. gigas, C. belcheri, C. ariakensis, C. rivularis and C. hongkongensis)
and Atlantic species (C. gasar, C. rhizophorae and C. virginica )
ranged from 0.149 to 0.215 and CA was found to be more
closely related to the Indo-Pacific group (d ¼ 0.092–0.112). The
distances between the haplogroup CA (Crassostrea sp.) and the
Brazilian native oysters (NC and EC) ranged from 0.197 to 0.208.
Forty most-parsimonious trees were obtained (300 steps,
CI ¼ 0.684, RI ¼ 0.889). The topologies of the trees obtained
by MP (consensus tree), ML and neighbour joining were similar
(Fig. 2). All trees clearly showed that the Atlantic cupped
oysters are monophyletic (bootstrap values: ML ¼ 100%,
RESULTS
The alignment of 120 Brazilian mangrove oyster sequences
resulted in 15 different haplotypes that were used in the analysis
with other sequences from GenBank (Table 1). The 16S rRNA
fragments were composed of 438 sites after alignment. There
were 28 indels, none of which were included in the analysis. Polymorphism was detected at 155 sites and 112 of these were informative for parsimony. The transition/transversion rate was 1.1.
The saturation test using the 16S rRNA sequences detected
little saturation (Iss ¼ 0.5136; Issc ¼ 0.6993; P ¼ 0.0010). The
average base frequencies were 0.292 for adenine, 0.175 for cytosine, 0.242 for guanine and 0.291 for thymine. From the 15 haplotypes, we recognized three Crassostrea haplogroups: Canela
Island (CA) (Bragança municipality), northern Brazilian
Coast (NC) from São João de Pirabas to Parnaı́ba, and the
eastern and northeastern Brazilian Coast from Fortim to Florianópolis (EC; see Fig. 1 and Table 1).
The ML best-fit model for the 29 sequences was the General
Time-Reversible model (GTR; Lanave et al., 1984; Rodriguez
et al., 1990), which takes into account gamma distributed rate variation across sites and allows for six different rates of change between
231
E.S. VARELA ET AL.
MP ¼ 96%, NJ ¼ 99%; decay ¼ 9). The Brazilian samples
from Florianópolis to Fortim grouped with C. rhizophorae in MP
and NJ analyses (bootstrap values: MP ¼ 92%, NJ ¼ 89%;
decay ¼ 4), whereas the samples from Parnaı́ba to São João
de Pirabas joined with C. gasar (bootstrap values: ML, MP
and NJ ¼ 100%; decay ¼ 17). The trees joined C. rhizophorae,
C. virginica and the Brazilian samples from Florianópolis to
Fortim in the same clade (bootstrap values: ML ¼ 87%,
MP ¼ 79, NJ ¼ 99%; decay ¼ 2). Although the trees tended to
group together all the Indo-Pacific oysters, only the MP boostrap
value was well supported (ML ¼ 58%, MP ¼ 97%, NJ ¼ 81%;
decay ¼ 7). The exotic Crassostrea sp. (Melo et al., 2005) from
Canela Island joined the Indo-Pacific species. The phylogenetic
trees generated with the tests of comparisons of monophyly
suggested that Crassostrea is monophyletic.
The topology of the trees obtained in our results show that
C. rhizophorae, C. virginica (AF092285) and C. gasar are closely
related, agreeing with the trees generated by Lapègue et al.
(2002) and Boudry, Heurtebise & Lapègue (2003) who also
used the 16S rRNA gene.
Our results disagree with Rios (1994) and Singarajah (1980)
who recognized a single species of native cupped oyster
from Brazil: C. rhizophorae. There is no report of sequences of
C. paraibanensis described by Singarajah (1980) to compare
with our results, and we did not find any other native species
besides C. gasar (¼C. brasiliana ) and C. rhizophorae.
Ignacio et al. (2000), using allozyme data, identified two
different Crassostrea species (C. gasar, reported as C. brasiliana,
and C. rhizophorae ) from the southern Brazilian coast (258300 S,
488300 W). We found no C. gasar in our samples from Fortim to
the South of Brazil and this may have been the result of
a lack of samples between Vitória (208180 S, 408180 W) and
Florianópolis (278330 S, 488330 W).
A third species, Crassostrea sp. – Canela, is closely related to
the Indo-Pacific oysters. The MP analysis offers strong evidence
to support the monophyly of the Indo-Pacific group including
Crassostrea sp. – Canela. The samples of this species are the
same as those used in another study with cytochrome c oxidase
subunit I (COI; Melo et al., 2005) that also shows this species
to be a non-Atlantic oyster. It may have dispersed by itself but
it also may represent an isolated case of accidental introduction
by trans-oceanic navigation since this species was observed at
only one sampling location where there is an intense transit of
international shipping entering and leaving the Amazon
River. More sampling in different areas and ecological studies
are necessary to clarify the distribution and ecological characteristics of Crassostrea from Canela Island.
The present study showed that the distribution of C. rhizophorae (EC group) occurs along the greater part of the Brazilian
coast, from Florianópolis to Fortim. According to Rios (1994),
C. rhizophorae occurs from the southern Caribbean to Uruguay.
Crassostrea gasar (NC group) is a trans-Atlantic species (South
America and Africa) and we found it to occur from Parnaı́ba
to São João de Pirabas. Lapègue et al. (2002) using molecular
data (16S rRNA), identified C. rhizophorae from Salvador
(128520 S, 388370 W) and Paranaguá Bay (258300 S, 488300 W) in
Brazil, and from Martinica Island (Caribbean). The same
authors found C. gasar in Paranaguá Bay (258300 S, 488300 W)
and Cananéia Bay (258070 S, 478520 W) in Brazil, and in
French Guiana. We did not find C. gasar between Fortim and
Florianópolis. Most studies show that larval development,
survival (Lemos et al., 1994) and settlement (Sandison, 1966;
Nascimento, 1991; Marques-Silva et al., 2006) in Crassostrea
are associated with high salinities (27 – 37‰). However, experiments by Sandison (1966) indicated that adults of C. gasar
(as Gryphaea gasar; see Stenzel, 1971) survive better in lower
(0 – 15%) rather than higher salinities (18 – 30‰). In Brazil,
both C. gasar and C. rhizophorae grow and develop faster in
lower salinites but C. gasar tends to tolerate a wider variation
in salinity (8 –34%) than C. rhizophorae (7 – 28‰) (Nascimento,
1991). The northern Brazilian coast from Cape Orange (48300 N,
51.58W) to the Parnaı́ba River delta (28520 S, 41.58W) has an
overall mean annual fresh water input of 137,000 m3s21,
whereas from Parnaı́ba to Chuı́ (southern Brazil; 338480 S,
538220 W) the input is only about 11,120 m3s21 (Ekau & Knoppers, 1999). Therefore, the greater input of fresh water from the
large rivers of the north generates changes in coastal waters,
decreasing salinity and increasing suspended sediment loads
and turbidity (Müller-Karger, McClain & Richardson, 1988).
Differences in the tolerance of mangrove oysters to salinity
may explain the presence of C. gasar in the north and the predominance of C. rhizophorae in the east.
DISCUSSION
The present study identifies the existence of three Crassostrea
species from the Brazilian coast (two native and one exotic).
Figure 2. Consensus tree of ML, MP and NJ methods for genus Crassostrea and Saccostrea cuccullata based on 16S rRNA. Ostrea edulis was used as
an outgroup. EC is from Fortim to Florianópolis, NC is from Parnaı́ba to
São João de Pirabas, CA is Canela Island. Numbers above the branches
are the percentage of 1000 (ML, top), 2000 (MP, middle) and 2000 (NJ,
bottom) bootstrap pseudo-replicates. Bremer’s decay values are presented below the branches.
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phylogenetic trees that separated Crassostrea from Saccostrea,
which agrees with our data. The main morphological character
that identifies the genus Saccostrea is the presence of chomata in
the shell (Stenzel, 1971). This feature is absent in living Crassostrea but it can be observed in the fossil record of the North American Crassostrea gigantissima (Lawrence, 1995). Assuming our
molecular tree topologies are correct, this character may be
ancestral to all Crassostreinae but was lost from the lineage of
the living Crassostrea.
Besides taxonomic and phylogenetic considerations, the
results have important implications for aquaculture. Oyster
culture has rapidly expanded in Brazil during the past decade.
The exotic C. gigas is the most commonly cultured species,
mainly between Santa Catarina and São Paulo (Streit et al.,
2002). However, in the north and northeast, where C. gigas
cannot grow due to higher water temperatures, native mangrove
oysters are cultivated. These oysters have long been regarded as
being C. rhizophorae but, as our results show, this may not always
be the case. The correct identification of species being cultivated
is essential for the success of the culture, as well as that of selective
breeding programs and marketing.
The export of oyster spat to other parts of Brazil is common
(personal observation) and it is possible that C. rhizophorae and
C. gasar spat have been introduced to areas outside their
ranges. Samples of spat and adults being cultured should be
screened as part of a program for monitoring genetic and sanitary impacts of oyster culture. The presence of an, as yet, unidentified exotic Crassostrea species at Canela Island, northern Brazil,
increases the need for such monitoring.
ACKNOWLEDGEMENTS
Eduardo S. Varela was supported by the Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico (CNPq) and the
Mangrove Dynamics and Management Project (Brazilian –
German Scientific Cooperation). This research was made possible by grants from the CNPq (62.0052/2001-5 – Institutes of
the Millennium Program). We would like to thank Daniele
Pequeno Lopes, Cleovonsostenes Varela, Danilo César Lima
Gardunho and Francisco Arimatéia dos Santos Alves for collecting some of the samples used in this paper. A license (No 109/
2004) to collect oysters was obtained from the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renovavéis.
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