Abstract: Morphologically, two species of giant clams Tridacna have been recognized in the Red Sea, T. squamosa and T. maxima. Similar morphology among species makes classification difficult. For proper classification, we sequenced an approximately 450-nucleotide fragment of the mitochondrial 16S rDNA gene from the tissues of Tridacna from eight locations in the Red Sea. Our results suggest that there are three novel species of Tridacna in the Red Sea. Two of these species are related to T. maxima and one is related to T. squamosa. Of the two species related to T. maxima, one species was found in Hurghada, Marsa Ghaleb and Safaga while the other was found in Abu Zenima, Abou Galum, Dahab and Nuweiba. All three novel species were found in Ras Mohamed. Our results do not support the morphological classification that suggests the existence of only two tridacnid species, T. maxima and T. squamosa in the Red Sea.
Introduction
Among the different species of invertebrates colonizing the coral reefs in the Indo-Pacific and Red Sea areas are the giant clams of the genus Tridacna, the largest and fastest growing living members of the class Bivalvia (Yonge, 1980). They are highly morphologically derived cardiids that have evolved an obligate symbiotic association with photosynthetic dinoflagellate algae of the genus Symbodium, living in their enlarged siphonal mantle (Schneider, 1998).
Rosewater (1965) recognized six living species of giant clams: Hippopus hippopus, Tridacna gigas, T. derasa, T. maxima, T. squamosa and T. crocea. Later, Rosewater (1982) recognized Hippopus porcellanus and Benzie and Williams (1998), using electrophoretically detectable allozyme variation, confirmed that the two Hippopus species were genetically differentiated. Lucas et al. (1990) subsequently described T. tevoroa and suggested that it may represent a transitional species between Hippopus and Tridacna (Lucas et al., 1991). Therefore, the number of known giant clam species has increased from six to eight.
Tridacnins, the major protein isolated from Tridacna, has been found to react with various cell types, including red blood cells, lymphocytes, spermatozoa and tumor cells of different origins (Uhlenbruck et al., 1975, 1978). Furthermore, Tridacnin from T. maxima and T. squamosa showed stronger effects than tridacnin from T. derasa and T. gigas (Uhlenbruck et al., 1978). Recently, 24-methylenecholesterol was isolated from the Red Sea Tridacna mantle and was found to possess modest cytotoxic activity (Kilada, 2001).
Along the coasts of the Red Sea, two living tridacnid species have been recognized morphologically: T. maxima and T. squamosa (Mansour,1946a-c, 1949; Hughes, 1977; Mergner and Mastaller, 1980; Kilada et al., 1998). However, classification based on morphological structure makes it difficult to recognize proper species. Therefore, molecular classification based on comparing the sequence of mitochondrial DNA may offer new insights for a more precise classification of the genus Tridacna in the Red Sea.
We sequenced a 450 nucleotide fragment of mitochondrial, large subunit ribosomal gene (16S) from the tissues of the two species of Tridacna from different locations in the northern part of the Red Sea.
Materials and Methods
Collection of Clam Specimens
Tridacnid clams were collected from eight locations in the Egyptian Red
Sea during the spring of 2002; four in the Gulf of Aqaba-Dahab (DH), Nuweiba
(NU), Abou Galum (AG) and Ras Mohamed (RM); three in the main basin of the Red
Sea-Hurghada (HU), Safaga (SF) and Marsa Ghaleb (MG) and one location in the
Gulf of Suez-Abu Zenima (AZ) (Fig. 1). The specimens were
brought alive to the Department of Biochemistry, Faculty of Pharmacy, Suez Canal
University where the animals were removed from their shells and dissected. For
each specimen, a portion of the adductor muscle tissue was excised with a razor
blade and frozen at -80°C. The rest of the organs were separated and frozen
at -80°C and the shells were saved.
Extraction of DNA from the Clams
DNA was extracted exclusively from adductor muscle tissue from the specimens
of all tridacnids. Extraction was limited to the adductor muscle tissue because
symbiotic dinoflagellate zooxanthellae are found within the mantle and internal
organ tissues. Roughly 200 to 300 mg of tissue was diced finally with a razor
blade and the DNA was extracted using a QIAGEN DNeasy™
tissue extraction kit according to the manufacturer’s
instructions (QIAGEN Gmbh, Germany).
| Fig. 1: | Map of Egypt indicating sample collection sites of Tridacna used in this study |
Amplification of 16S rDNA and Sequencing
An approximately 450 nucleotide portion of mitochondrial large subunit (16S)
rRNA gene was amplified from the genomic DNA using the primers 16Sar 5'-CGCCTGTTTATCAAAAACAT-3'
and 16Sbr 5'-CCGGTCTGAACTCAGATCACGT-3' (Kessing et al., 1989). Polymerase
chain reaction (PCR) amplification was performed using an Eppendorf Master Cycler®
gradient and a reaction mixture of 100 μL containing 60-120 ng of genomic
DNA, 2 units of Taq polymerase enzyme, 0.5 μM of each primer, 25
mM of each dNTP, 2.5 mM MgCl2 and 1 X Taq buffer (100 mM Tris
HCl and 500 mM Kcl). Cycle parameters were 1 min 94°C initial denaturation,
35 cycles of the pattern (30 sec 94°C denaturation, 30 sec 47°C annealing,
1 min 72°C extension) and 10 min 75°C final extension. Double-stranded
products were isolated on 2% agarose gels, excised using a QIAquick gel extraction
kit (QIAGEN, Germany). A single strand of the amplified partial 16S rDNA gene
fragments was directly sequenced with the forward (16Sar) amplification primer
using the protocols and reagents for cycle sequencing in the ABI Prism Dye Terminator
Cycle sequencing ready reaction kit version 3 (PE Applied Biosystems). Extension
products were sequenced on an ABI Prism 3700 automated DNA sequencer (PE Applied
Biosystems).
Phylogenetic Analysis
The 16S rDNA sequences were aligned with available Tridacna 16S rDNA
sequence data (Schneider and Foighil, 1999) from the GenBank database using
PHYDIT 3.1 phylogenetic software (Chun, 1995). The computer-generated sequence
alignments were adjusted manually and 387 bp was used in the final analysis.
Phylogenetic reconstruction was done using Neighbor Joining (NJ) (Saitou and
Nei, 1987), Maximum Parsimony (MP) and Fitch-Margoliash (F) (Fitch and Margoliash,
1967) methods. NJ analysis was performed using the tree construction algorithms
available with PHYDIT V. 3.1 (Chun, 1995). Evolutionary distance matrices for
the NJ and F methods were generated as described by Jukes and Cantor (1969).
Tree topologies were evaluated by 1000 bootstrap re-samplings of the NJ data
(Felsenstein, 1985). Phylogenetic trees were drawn with Tree View V. 1.6.6 (Page,
1996) and Canvas™ V. 5 on a Macintosh computer in Center of Marine Biotechnology,
University of Maryland Biotechnology Institute, Baltimore, Maryland, USA.
Results and Discussion
In the present study, the length of the amplified fragment of mitochondrial 16S rDNA gene was approximately 450 bp. The amplified region contained sufficient conserved residues to permit unambiguous alignment to be made among tridacnid species. Of the 387 sites, 115 were variable in our data. The neighbor joining tree in Fig. 2 shows a group supported by a high bootstrap value (97) made up of T. crocea, T. squamosa, T. maxima and tridacnid clams from different locations in the northern Red Sea (Fig. 1). The bootstrap value at the node T. squamosa and sample RM1 was 92. This indicates that they are closely related. The rest of the tridacnid samples are related to T. maxima (bootstrap value of 51). The genetic distance between the samples collected from Safaga, Hurghada, Marsa Ghaleb and Ras Mohamed (RM2) is negligible. Moreover, the genetic distance between samples colleted from Abu Zenima, Nuweiba, Dahab, Abou Galum and Ras Mohamed (RM3) is also insignificant.
Our phylogenetic analyses of 16S rDNA sequences suggest that there are three novel species of Tridacna in the Red Sea. Two of these species are related to T. maxima and one is related to T. squamosa. Of the two species related to T. maxima, one species was found solely in the main basin of the Red Sea (Hurghada, Marsa Ghaleb and Safaga) whereas the other was confined to the Gulfs of Aqaba and Suez (Abu Zenima, Abou Galum, Dahab and Nuweiba). Ras Mohamed, a site at the point where the two gulfs join the main basin, was found to harbor all three novel species. Our results do not support the morphological classification (Mansour, 1946b, c, 1949; Hughes, 1977; Mergner and Mastaller, 1980; Kilada et al., 1998) that suggests the existence of only two tridacnid species, T. maxima and T. squamosa in the northern Red Sea.
| Fig. 2: | Neighbor-joining phylogenetic tree from analysis of 387 bp of 16S rDNA gene sequences of Tridacna species and Tridacnid clams from the Red Sea. f and p indicate branches which were also found using Fitch-Margoliash and maximum parsimony methods, respectively. The numbers at the nodes are percentages indicating the levels of bootstrap support, based on neighbor-joining analysis of 1,000 re-sampled data sets. The scale bar represents 0.1 substitutions per nucleotide position |
In conclusion, this study suggests that the Red Sea Tridacna population is not exclusive to the two species T. maxima and T. squamosa as was previously believed. Three novel species were discovered. More rapidly evolving sequences will be useful for future additional DNA-based genetic studies of these species.
Acknowledgments
This study was funded in part by the Egyptian Academy of Scientific Research Program of the National Strategy for Biotechnology and Genetic Engineering (project code: 31). We thank Mr. Osama M. Wahba for his technical assistance. This is contribution No. 03-590 from the Center of Marine Biotechnology.