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Rahim Kamarudin, Noor Faizul Hadry Nordin, 1Ridzwan Hashim and Mohd Nahar Mohammad Department of Biotechnology and 1Department of Biomedical Science, Kulliyyah of Science, International Islamic University Malaysia, P.O. Box 10, 50728 Kuala Lumpur, Malaysia. *Corresponding author. Tel.: +603-61964000 ext. 3349; fax: +603-61964899. Email address: firstname.lastname@example.org ABSTRACT Diverse species of sea cucumber (Echinodermata: Holothuroidea) were documented in Malaysia. Their abundance benefits the Malaysians in traditional and modern medicinal industry as well as in food processing industry. Previously, the identification of sea cucumber species in Malaysia was done mainly based on morphological characters. Among the characters used were the shapes of ossicles, body shapes and colour, shapes of tentacles, the existence and shapes of papillae. However, the morphological approaches were unsuccessfully unravelled the problematic taxonomic status of sea cucumbers as there are unidentified and unknown species present throughout Malaysia to date. As an alternative approach to verify the species status inferred from morphology, molecular ecology analyses were incorporated in this study to examine the phylogenetic relationship between and among selected species of sea cucumbers from Malaysia by using cytochrome c oxidase I (COI) mitochondrial DNA (mtDNA) gene. Three genera were selected namely Holothuria and Stichopus from order Aspidochirotida, and genus Molpadia from order Molpadiida. Polymerase Chain Reaction (PCR) gave the desired products of amplified COI mtDNA region with length of approximately 510 bp. After the multiple sequence alignment, partial sequences of COI mtDNA gene ranging from 488 bp to 489 bp were utilized for further analyses. 22 ingroups of sea cucumbers and one outgroup represented by Diadema setosum were included in the reconstruction of neighbour-joining (NJ) tree and maximum parsimony (MP) tree. Base frequency and genetic distance based on Kimura-2 parameter model were also calculated prior to the reconstruction of phylogenetic tree. Basically, both types of phylogenetic trees suggest the paraphyly of Holothuria and Stichopus due to the clustering of four Stichopus horrens individuals together with Holothuria species. Apart from that, Molpadia species seems genetically close to the main group of genus Holothuria and Stichopus horrens. The phylogenetic results were also supported by the data from genetic distance. Overall, the above findings strongly suggest that the grouping and classification at species and higher level as inferred from COI mtDNA gene are unresolved, requiring further molecular ecology studies to be done in the near future. INTRODUCTION Sea cucumbers (Echinodermata: Holothuriidea) are sometimes known as Holothuroids or Holothurians. The name Holothuria was coined by Aristotle, the famous Greek philosopher whereas Pliny, a taxonomist scientifically named the unique sea invertebrates Cucumaris marimus literally means ‘sea cucumber’ due to their cylindrical and elongated bodily shapes resembling the cucumber (Ridzwan 1993). Sea cucumbers are an abundant and diverse group of marine invertebrate. More than 1400 species have been described and classified into 160 genera (Smiley 1994). Malaysia coastal area possesses about 80 species of sea cucumber (Kamarul 2006). The distribution and the species density vary and change according to topographical conditions of the areas, habitat and surrounding parameters such as temperature ( 29-30 ºC), contamination index such as DO (7.3-7.5 mg/L), salinity (29.0-31.0 ppt) and sea water pH (7.3-7.5)(Ridzwan 1993).
zoogeography and geographic variation (Harrison 1989. the mtDNA gene maps are relatively stable. Although the genetic role of mtDNA appears to be universally conserved. better known as bat or balat are dried into trepang. and also in transcription. Bohadscia argus. Trepang is dried sea cucumbers that had undergone a series of processes. Stichopus chloronotus). maternal inheritance and absence of recombination make mtDNA an ideal marker for tracing maternal genealogies (Harrison 1989). The complete DNA sequences of mitochondrial genomes are available from a variety of different animals. aches in the joints. Amos & Hoelzel 1992. Accordingly. Daniels et al. 2002). transparent body and lives in the gut cavity of the sea cucumber (e. the ATPase complex subunits 6 and 8 (ATPase 6 and ATPase 8). The mouth is equipped with tentacles that are used for feeding. Sea cucumbers host a variety of symbiotic organisms such as crabs. and expression (Gray 1999). The inhabitants of the Peninsular Malaysia recognize sea cucumbers especially gamat in the treatment of wounds. organs that extruded when disturbed and are used as a defense mechanism. The sea cucumber of Malaysia are well known for two reason. The fish leaves and enters (tail first) through the sea cucumber's anus. Encheliophis mourlani or Onuxodon margaritiferae) has a long slender. Simple sequences organization.g. Mitochondrial DNA has the same fundamental role in all eukaryotes that contain it namely. The region of mtDNA containing cytochrome oxidase I and II (COI II) has been used in systematic and population genetic studies of insects. Thelenota ananas. boiling the sea cucumber to soften the tissues and finally drying it for prolonged storage during transport and marketing (Ridzwan 1993). mtDNA consists of 2 ribosomal RNA regions. They probably feed on the gonads and other tissues of the host. Beside morphology. their skeleton is reduced to tiny ossicles in the body wall and many species can be identified by the shapes of these ossicles. the oxidative phosphorylation system cytochrome c oxidase subunits I. shrimps. firstly as a medicinal source and secondly. Most of these genomes are circular DNA molecules that range in size from 14 to 17 kb. worms and even a very unusual fish.a vestigal genome originating from an endosymbiotic αproteobacterial ancestor. RNA maturation and protein import (Burger 2003). conservation. Mitochondria have their own genetic system. Both gamat and other sea cucumbers. arrangement. Among vertebrates. 13 proteincoding regions and 22 tRNAs. Sea cucumber does not have spines. 2006. being involved in a maximum of five mitochondrial processes: invariantly in respiration and/or oxidative phosphorylation and translation. this genome exhibits remarkable variation in conformation and size as well as in actual gene contents. as a source of food in the form of trepang (Kamarul et al. animal mtDNA has been proven useful in addressing questions of population genetic structure. II and III (COI. The animal mitochondrial genome contains genes for 13 proteins. In general. including aphids. They also inhabit some starfish as well as pearl oyster shells. Some species have Cuvierian tubules. COII and COIII) and cytochrome b (Cyt b). Included in the process are the removals of the internal organs. identification of animal based on mitochondrial DNA (mtDNA) has been used since it plays an increasingly important role as a genetic marker in population and evolutionary biology (Harrison 1989). . The number and shape of the tentacles are also the criteria for scientific identification of the species.The classification of sea cucumbers into smaller groups is based on few features. Other species extrude their entire internal organs (evisceration) to distract the predators while they escape. The pearlfish (either Encheliophis homei. Instead. Ridzwan 1993). it encodes a limited number of RNAs and proteins essential for formation of a functional mitochondrion (Gray 1999). white spot disease and high blood pressure (Ridzwan 1993). RNA transcription. A cladistically substantiated phylogeny based upon clearly defined morphological characters can bring insight into the evolution of the sea cucumbers. taxonomic status. and the respiratory chain NADH dehydrogenase subunits 1-6 and 4L (ND1-ND6 and ND4L). The genetic function of mitochondrial DNA is well-conserved.
sample size. Dayang Bunting Island and Pangkor Island within Peninsular Malaysia and Semporna in Sabah. No. Perak Pangkor Island.0 Description of sea cucumber species incorporated in this study. The description of the each sample is as in Table 1. Perak Pangkor Island. Perak Pangkor Island. The samples are preserved in 70% ethanol or stored in -20oC fridge for long term storage. Taxa Abbreviation Locality Order Aspidochirotida Family Holothuriidae 1 Holothuria leucospilota R7 Intan Besar Island. Intan Besar Island. Langkawi 4 Holothuria leucospilota R6 Intan Besar Island. methodology and concentration based on this preliminary study MATERIALS AND METHODS Samples Samples of sea cucumbers were collected from two different parts of Malaysia which are within Peninsular Malaysia and Sabah. Perak Pangkor Island.In general. Langkawi 3 Holothuria leucospilota RH Dayang Bunting Island. Langkawi 5 Holothuria leucospilota R9 Dayang Bunting Island. Langkawi 2 Holothuria leucospilota RG Dayang Bunting Island. this study aims to: 1) obtain partial sequences of COI mtDNA of selected sea cucumbers from Malaysia 2) study the phylogenetic relationship of sea cucumber species by using COI mtDNA gene sequences 3) update the species validity between and among present species of sea cucumbers in Malaysia using COI mtDNA gene as an alternative to morphological studies 4) study the usefulness of COI mtDNA gene in phylogenetic analyses of sea cucumbers 5) suggest further and future recommendation in molecular study of indigenous sea cucumber in terms of sampling sites. Perak Pangkor Island. Perak Tanjung Gemok.0. The study sites are Port Dickson. Perak Pangkor Island. Table 1. Perak Pangkor Island. Perak Pangkor Island. 1 RF R1 R3 R8 RC R10 RE R2 R5 RJ RD RB R4 RA RI Pangkor Island. Perak Pangkor Island. Perak Pangkor Island. Perak Pangkor Island. Perak Pangkor Island. Langkawi 6 Holothuria impatiens R9 Semporna. Perak Pangkor Island. Negeri Sembilan . Perak Pangkor Island. Sabah 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Family Stichopodidae Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Stichopus horrens Order Molpadiida Molpadia sp. Port Dickson.
After the determination of desired PCR product. a purification kit from manufacturer was used for direct purification to get clean and purified PCR products. based on equal base frequencies and unequal ratio of transition to transversion (ti/tv). Rapid thermal ramp is 1oC/s.0 µl of master mix was then added to the prepared solution. 5. Polymerase Chain Reaction (PCR) Two universal primers suggested by Palumbi et al. Prepared PCR reactions were placed in the Eppendorf Thermocycler PCR machine and it was set for 30 cycles based on the following parameters. Phylogenetic analysis Chromas Lite (Version 2. 1 min 30 s at 72oC for extension. 1 min 30 s at optimized temperature for annealing. 60oC: 4 min hold and was proceeded to Ethanol/Sodium Acetate precipitation. Thompson et al. and 7 min at 72oC for final extension.25 µl of 5 u/µL Taq DNA Polymerase. Sequencing was done using the BigDye® Terminator v3. 1997).1 µl of 10% BSA was added with adjustment to the volume of sterilized dH2O to make the total volume of each PCR reaction is 50 µl.0 µl of DNA extract prepared previously was transferred into 0. Sequencing was carried out on ABI 377 automated sequencer (PE Applied Biosystem). Kimura 2-parameter distance (1980) was selected.1) program was used to display fluorescence-based DNA sequencing results. (1993) and also by using DNeasy QIAGEN tissues kit.0 µl of 10X PCR reaction buffer. Multiple sequence alignment for forward reactions was done using ClustalX program (version 1. Approximate yield of amplified DNA was determined by electrophoresis. 45 s at 95oC for denaturation.81. (1991) were used for amplification of partial cytochrome c oxidase I (COI) mitochondrial region:COI (forward) 5’-CCTGCAGGAGGAGGAGAGAGCC-3’ (22 bases) COI (reverse) 5’-CCAGAGATTAGAGGGAATCAGTG-3’ (23 bases) 4.2 ml PCR tube and 46. Cycle sequencing reaction was done for 35 cycles of 96oC: 10 s. 2. 5 min at 96oC for initial denaturation. Basic Local Alignment Search Tool (BLAST) program was used to find corresponding sequences from the GenBank database. Cycle sequencing reaction was done in a programmable cycler (Tpersonal Combi Thermocycler).5 µl of 10 µM reverse universal primer. 1.0 µl of 25 mM MgCl2.5 µl of 10 µM forward universal primer.65 (Felsenstein 2004) was used to reconstruct rectangular cladogram or topology of neighbourjoining tree and maximum parsimony tree. Phylogeny Inference Package (PHYLIP) software version 3. 55oC: 5 s. Sometimes 0. Phylogenetic confidence was estimated by bootstrapping (Felsenstein 1985) with 1000 sequence replications and 100 data sets. were determined by electrophoresis.75 µl of sterilized dH2O. 2. the quantity and quality. .0 µl master mix preparation consists of 31. PAUP* program was also utilized to examine other relevant analyses such as distance matrix and base frequencies. Approximate yields of DNA. 3.DNA Extraction DNA extraction from the muscle tissues of sea cucumbers was conducted according to the modified CTAB method of Grewe et al. DNA Sequencing Purified PCR products in suspension form were prepared prior to DNA sequencing. Each 46. and subsequently aligned by eye. Basic Local Alignment Search Tool (BLAST) All usable sequences obtained after DNA sequencing process were aligned and compared with corresponding sequence(s) from GenBank database to determine the reliability of the sequences.0 Cycle Sequencing kit (ACGT).0 µl of 10 mM dNTP mix and 0.
The mean length of the partial DNA sequences is approximately 510 bp. horrens R8 and S. S. Basic Local Alignment Search Tool (BLAST) Basic Local Alignment Search Tool (BLAST) program provided in the GenBank database was used to search for the corresponding sequences to the partial DNA sequences obtained in this study based on the Evalue and Score (S).73%) while G residues show the least mean of 20.41%. horrens RF were clustered with all Holothuria species such as H. Polymerase Chain Reaction (PCR) All positive PCR products showed approximately 500-600 base pairs (bp) of expected length after the amplification of the partial COI mtDNA gene. leucospilota RG and H. GC content averages about 44. leucospilota within the big cluster of genus Stichopus and Holothuria were morphologically identified as H. the neighbour-joining tree (Figure 1. leucospilota RH were not clustered together with the other individuals of the same species that formed a cluster supported by 33% bootstrap value. . DNA Sequencing All the 22 partial DNA sequences of COI mtDNA gene were forwarded to phylogenetic analyses. no corresponding sequence was shown by the BLAST results but the results still indicated that all the 22 DNA sequences of COI mtDNA gene are sequences of sea cucumbers as the sequences are close genetically to sequences of sea cucumbers from family Cucumariidae. Diadema setosum.79% while 24. The aligned partial sequences were used for phylogenetic analyses. Even though all individuals of H. horrens within the big cluster of genus Stichopus and Holothuria.0) shows that both genus Holothuria and genus Stichopus are paraphyletic. was chosen as outgroup. Furthermore. leucospilota. However. with 90% bootstrap value. horrens into the cluster that contains Holothuria species.07% is presented by C residues. Molpadia sp. horrens R3. Base Frequency In terms of base frequencies. a species of sea urchin. few individuals such as H. This condition is due to the grouping of few individuals of S. there is a slight mean excess of T residues (29. Reconstruction of Neighbour-Joining Tree Principally. The range of the original length for the 23 partial sequences of COI mtDNA gene is from 488 bp to 489 bp. S. horrens R1.. In the big cluster of genus Stichopus and Holothuria.48%. 1 is basal to all the other species within the cluster. Genus Molpadia from order Molpadiida is genetically close to Holothuria species and S. impatiens causing genus Holothuria as well as genus Stichopus to be paraphyletic.RESULTS AND DISCUSSION DNA Extraction 22 DNA extracts of selected sea cucumbers from different species were chosen to go through Polymerase Chain Reaction. Multiple Sequence Alignment Multiple sequence alignment of 22 ingroups of sea cucumbers and an outgroup of Diadema setosum using ClustalX program resulted 489 bp of aligned base positions. Four individuals of genus Stichopus namely S. A residues show a mean of 25. Across all species.
6b (Felsenstein 2004). Numbers at nodes indicate the bootstrap values in percentage (%).0. impatiens causing genus Holothuria as well as Stichopus to be paraphyletic. horrens with 100% bootstrap value. Four individuals of genus Stichopus namely S. horrens R8 and S.0).0) is considered identical to neighbour-joining tree (Figure 1. S. a sea urchin (GenBank accession number: AY012733). However. horrens RF were clustered with all Holothuria species such as H. The tree is rooted with a sequence of Diadema setosum. horrens in particular due the paraphyly of genus Holothuria. S.Order Molpadiida Figure 1. Overall. Each partial sequence detail is described in Table 1.0 Neighbour-joining tree (rectangular cladogram) of sea cucumber species inferred from COI mtDNA gene using PHYLIP version 3. Another big cluster is a group of S. horrens R3. Reconstruction of Maximum Parsimony Tree The clustering pattern shown by maximum parsimony tree (Figure 2. The maximum parsimony tree also shows that both genus Holothuria and genus Stichopus are paraphyletic. Kimura 2-parameter distance (1980) with 1000 sequence replications and 100 data sets was used. the neighbour-joining tree does not support the species classification of sea cucumbers by morphology thus showing the low and weak resolution shown by COI mtDNA gene in phylogenetic analysis of sea cucumbers using neighbour-joining method. the strong bootstrap value still does not support the monophyly of Stichopus in general and S. . horrens R1.
Thus. genus Molpadia from order Molpadiida is genetically close to the members of the cluster.Within the big cluster of genus Stichopus and Holothuria. Although molecular approaches are promising to illustrate the genetic relationship and status of sea cucumbers. they were not clustered together. the maximum parsimony tree also does not support the species classification of sea cucumbers by morphology thus showing the low and weak resolution shown by COI mtDNA gene in phylogenetic analysis of sea cucumbers using character-based method of maximum parsimony. The second big cluster is a group of S. Likewise the neighbour-joining tree. other molecular approaches such as microsatellites analyses must be considered. In general. with 97% bootstrap value. . CONCLUSION In conclusion. Both neighbour-joining tree and maximum parsimony tree indicate the paraphyly of genus Holothuria and genus Stichopus. the findings also did not conform to the morphology or taxonomic classification of sea cucumbers. Molpadia sp. horrens with 100% bootstrap value. the strong bootstrap value still does not support the monophyly of Stichopus in general and S. Only H. However. leucospilota RG and H. leucospilota R7 formed a small cluster within the big cluster of genus Stichopus and Holothuria supported by 43% bootstrap value. 1 representing genus Molpadia is basal to all the other species within the cluster. morphologically and genetically. horrens in particular due the paraphyly of genus Holothuria. further and more morphological studies are also needed in order to support and verify the subsequent phylogenetic analyses of sea cucumbers. More samples of sea cucumber species from various genera and families are also required in the future in order to get more reliable and up-to-date data. Apart from that. more intensive studies are needed to get better view of the phylogenetic relationship and also to validate the species records of sea cucumbers especially in Malaysia. this preliminary molecular study shows that the phylogenetic relationship of sea cucumbers at the species level and higher level was not resolved by using partial sequences of COI mtDNA gene. even though all individuals of H. leucospilota. leucospilota within the big cluster of genus Stichopus and Holothuria were morphologically identified as H. Furthermore.
0 Maximum parsimony tree (rectangular cladogram) of sea cucumber species inferred from COI mtDNA gene using PHYLIP version 3. Department of Museums and Antiquities Malaysia (Mrs.6b (Felsenstein 2004). Each partial sequence detail is described in Table 1.0. Mr.Order Molpadiida Figure 2. Numbers at nodes indicate the bootstrap values in percentage (%). International Islamic University Malaysia (IIUM) Gombak for the great assistance and valuable advice. Farizawati Sabri. a sea urchin (GenBank accession number: AY012733). . Marine Park Section. Mohd Khairill Jemangin and Mr. Ardi Asmera Saeman) and Institute of Oceanography and Maritime Studies (INOCEM) of Kulliyyah of Science. ACKNOWLEDGEMENTS Special thanks to Department of Fisheries (DOF) Malaysia. The tree is rooted with a sequence of Diadema setosum.
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