Lack of genetic divergence in nuclear and mitochondrial DNA between subspecies of two Haliotis species.ABSTRACT Haliotis discus and Haliotis diversicolor are economically important abalone abalone (ăbəlō`nē), popular name in the United States for a univalve gastropod mollusk of the genus Haliotis, members of which are also called ear shells, or sea ears, as their shape resembles the human ear. species in northern and southern China
n. See rRNA. ribosomal RNA (rī´bōsō´m (ITS-1) and 18S rRNA) and mitochondrial DNA Mitochondrial DNA (mtDNA) is the DNA located in organelles called mitochondria. Most other DNA present in eukaryotic organisms is found in the cell nucleus. Nuclear and mitochondrial DNA are thought to be of separate evolutionary origin, with the mtDNA being derived from the (16S rRNA and cytochrome oxidase cytochrome oxidase n. An oxidizing enzyme containing iron and a porphyrin, found in mitochondria and important in cell respiration as an agent of electron transfer from certain cytochrome molecules to oxygen molecules. I (CO1) gene). The two species are identical in their 18S rRNA sequences but differ in their ITS-1, 16S rRNA, and COI sequences, with sequence divergence ranging from 9.3% to 18.5%. Sequences of 18S rRNA are identical between the subspecies of H. discus, and those of H. diversicolor. For 16S rRNA and COI, there are 0% to 1.4% divergence between the two H. discus subspecies, and 0.2% to 0.6% divergence between the two H. diversicolor subspecies. Phylogenetic phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history. analyses show that the individual haplotypes of each subspecies are not separated to distinct clades, indicating high genetic similarity between the subspecies of both H. discus and H. diversicolor. KEY WORDS: genetic divergence Genetic divergence is the process of one species diverging over time into more than one species. Passing small random advantages characteristic changes over time from one generation to the next generations. , abalone, Haliotis discus, Haliotis diversicolor, subspecies INTRODUCTION Haliotis discus Reeve, 1846 and Haliotis diversicolor Reeve, 1846 are economically important abalone species in northern and southern China, respectively. Two subspecies have been recognized for each species, namely, H. discus discus Reeve, 1846 and H. discus hannai Ino, 1952 and H. diversicolor diversicolor Reeve, 1846 and H. diversicolor supertexta Lischke, 1870 (= H. diversicolor aquatilis Reeve, 1846) based on morphologic traits (Chen 1984, Nie 1989, Okutani 2001). There have been limited studies on the genetic differences between the two species and their subspecies. Naganuma et al. (1998) reported the differences in partial 18S rRNA sequences between the 2 subspecies of H. discus. No studies have been reported on the genetic differences between these two subspecies based on mitochondrial DNA. In addition, there are no reports on DNA analysis DNA analysis Any technique used to analyze genes and DNA. See Chromosome walking, DNA fingerprinting, Footprinting, In situ hybridization, Jeffries' probe, Jumping libraries, PCR, RFLP analysis, Southern blot hybridization. of the two H. diversicolor subspecies. The taxonomic status of the subspecies of H. discus and H. diversicolor is controversial (Hara & Fijio 1992, Geiger 2000). In particular the genetic differentiation between H. diversicolor diversicolor in mainland China and H. diversicolor supertexta in Taiwan is unknown. Nuclear DNA markers such as 18S ribosomal RNA (18S rRNA) and the first internal transcribed spacer of rRNA (ITS-1) and mitochondrial DNA markers such as 16S ribosomal RNA (16S rRNA) and cytochrome c oxidase The enzyme cytochrome c oxidase or Complex IV (PDB 2OCC, EC 1.9.3.1) is a large transmembrane protein complex found in bacteria and the mitochondrion. Function It is the last protein in the electron transport chain. subunit I gene (COI) have been extensively used in resolving genetic relationships of various taxonomic groups in the species level (Avise 1994, Hillis et al. 1996). This study reports the genetic divergence of the four abalone taxa taxa: see taxon. based on sequence analyses of the above mentioned genes. MATERIALS AND METHODS Sample Collection and DNA Extraction DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis. Outline of a DNA extraction There are three basic steps in a DNA extraction, the details of which may vary depending on the type of sample and any substances that may Haliotis discus discus were collected from Putian County, Fujian Province, China. The cultured stock was introduced from Japan. Haliotis discus hannai were collected from an abalone farm in Qingdao, Shandong Province. Wild Haliotis diversicolor diversicolor were collected from Sanya, Hainan Province. Haliotis diversicolor supertexta were collected from an abalone farm in Dongshan County This article is about Dongshan County in Zhangzhou Prefecture Level City, Fujian Province. For the article on Dongshan District in Xinjiang Autonomous Region, see Dongshan District. For other uses, see Dongshan (disambiguation). , Fujian Province. The stock was introduced from Taiwan. Total DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. was extracted from pleopod muscle using QIAamp DNA Mini Kit (QIAGEN) according to according to prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. the tissue protocol. Polymerase Chain Reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is and DNA Sequencing DNA sequencing The determination of the sequence of nucleotides in a sample of DNA. Segments of 18S rRNA, ITS-1, 16S rRNA, and COI were amplified from two individuals of each taxon taxon (pl. taxa), in biology, a term used to denote any group or rank in the classification of organisms, e.g., class, order, family. using polymerase chain reaction (PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) ). Partial 18S rRNA was amplified using primers 21-mer and 19-mer (Medline et al. 1988) following the conditions as described by Naganuma et al. (1998). PCR conditions for ITS-1 amplification using primers sp-1-3 and sp-1-5 followed Chu et al. (2001). 16S rRNA was amplified using the primer pair 16Sar and 16Sbr (Simon et al. 1994). PCR reaction cycling program was as follows: 2 min at 94[degrees]C, 38 cycles of 30 sec at 94[degrees]C, 45 sec at 45[degrees]C, 90 sec at 72[degrees]C, and final extension for 5 min at 72[degrees]C. Two primer pairs COIf and COIa (Palumbi & Benzie 1991) and LCD1490 and HCO HCO Harvard College Observatory HCO Hubbard Communications Office (Scientology) HCO Hearing Carry-Over HCO Health Care Organization HCO Helicopter Control Officer HCO Human Capital Office 2198 (Folmer et al. 1994), were used for amplifying two regions of COI gene. The PCR conditions for amplifying COI were the same as those used in 16S rRNA amplification. Prior to sequencing, PCR products were purified using the QIAquick PCR purification kit. Double-stranded PCR products were sequenced from both directions using both of the primer pairs and ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother. (Application Binary Interface) A specification for a specific hardware platform combined with the operating system. PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit. The products were purified and loaded onto an automatic sequencer See MIDI sequencer. (music) sequencer - Any system for recording and/or playback of music via a programmable memory which stores music not as audio data, but as some representation of notes. (ABI PRISM 3100 Genetic Analyzer) for analysis. Data Analysis Sequences of each gene were aligned by eye with the aid of ABI Sequence Editor 1.0.3. The sequence of each gene in an individual was confirmed by reference to data from both strands except the 18S rRNA. MEGA2 (Kumar et al. 2001) was used in calculating sequence divergence and constructing neighbor-joining phylogenetic trees with 500 bootstrap See boot. (operating system, compiler) bootstrap - To load and initialise the operating system on a computer. Normally abbreviated to "boot". From the curious expression "to pull oneself up by one's bootstraps", one of the legendary feats of Baron von Munchhausen. replicates. RESULTS All sequences determined in this study have been deposited in the GenBank database (accession numbers: AY319420-319447). The size of PCR products from 18S rRNA amplification was approximately 1800 bp, with 620 bp determined from the 5' end and 698 bp determined from the 3' end. The sequences determined in the four abalone taxa were identical. For ITS-1, 16S rRNA, and COI the sequence of each gene was confirmed with reference to data from both strands. The size of PCR product from ITS-1 amplification was approximately 350 bp. While 340 bp of ITS-1 were determined from the two subspecies of H. diversicolor, 307 bp were determined from the two subspecies of H. discus. The sequences from the two individuals in each taxon were identical (Table 1). There was 0.6% (2 bp) sequence divergence between H. discus discus and H. discus hannai, whereas the sequences from H. diversicolor diversicolor and H. diversicolor supertexta were identical. The sequence divergence of ITS-1 between H. discus and H. diversicolor was 15.9%. The size of PCR product from 16S rRNA amplification was approximately 560 bp. While 526 bp of 16S rRNA were determined from the two subspecies of H. discus, 531 bp were determined from the two subspecies H. diversicolor. There was 9.4% to 9.6% sequence divergence between H. discus and H. diversicolor. The sequences of 16S rRNA between the two H. discus discus individuals were identical, whereas for H. discus hannai, the sequence of one of the individuals was identical to that of H. discus discus, and exhibited 0.4% (2 bp) divergence from the other sequence. There was 0.4% (2 bp) sequence divergence between the two individuals of H. diversicolor diversicolor, and also between individuals of H. diversicolor supertexta. Between H. diversicolor diversicolor and H. diversicolor supertexta, the sequence divergence was 0.2% to 0.6% (1-3 bp). The size of PCR products from COI was approximately 680 bp using the primers COIf/COIa and 700 bp using LCD1490/ HCO2198. In the four taxa, 639 bp of COI were determined from the COIf/COIa products and 709 bp were determined from the LCD1490/HCO2198 products. The two overlapping segments were combined to a length of 1,272 bp for analysis. There was 17.5% to 18.5% sequence divergence between the two species. Between the two H. discus discus individuals, there was 0.6% (7 bp) sequence divergence whereas there was 0.5% (6 bp) sequence divergence between the two H. discus hannai individuals. There was 0.6% to 1.4% (7-18 bp) sequence divergence between two subspecies H. discus. The sequence divergence between the two subspecies (H. discus discus and H. discus hannai) was smaller than that of the sequence divergence between each individual of H. discus discus. The sequence divergence was 0.6% (8 bp) between the two H. diversicolor diversicolor individuals, and 0.6% (7 bp) between the two H. diversicolor supertexta individuals. There was 0.3% to 0.6% (4-8 bp) sequence divergence between the two subspecies of H. diversicolor. Figure 1 shows the neighbor-joining (NJ) phylogenetic trees of the four abalone taxa based on ITS-1, 16S rRNA, and COI analyses. Trees constructed based on each of the three genes clearly separated the two species (Fig. 1A to C). In the tree based on ITS-1 sequence (Fig. 1A), the two subspecies of H. discus was clustered into two different clades with over 60 bootstrap support but the two H. diversicolor subspecies did not group separately. In the trees based on 16S rRNA and COI (Fig. 1B and C), each of the four taxa did not cluster together, except in the COI tree the two H. discus discus individuals clustered together. To enhance the phylogenetic signals, the data sets of the three genes were combined for analysis (Fig. 1D). In this tree, the two H. discus discus individuals clustered into a single clade clade Cladus, subtype Genetics A branch of biological taxa or species that share features inherited from a common ancestor; a single phylogenetic group or line. See Inheritance, Species. with 97 bootstrap support, whereas individuals of the other taxa did not cluster together. [FIGURE 1 OMITTED] DISCUSSION 18S rRNA is one of the nuclear DNA markers often used for investigating the phylogenetic relationship and evolutionary history of various species. In this study, the partial 18S rRNA sequences (1308 bp) determined in H. discus and H. diversicolor are identical. The 3' sequence of the 18S rRNA segment obtained is identical to that of H. discus hannai (390 bp compared, Naganuma et al. 1998; GenBank Accession No. D88571). However, the sequence exhibits 0.5% (2 bp) divergence when compared with the sequence of H. diversicolor diversicolor (390 bp compared; D88572) reported by Naganuma et al. (1998). It is unclear why the sequences of H. discus discus are different between the two studies. Yet it should be pointed out that not only the 18S rRNA sequence determined in the present study is identical in the four taxa studied, but it is also identical to that of H. tuberculata Linnaeus, 1758 (390 bp compared, AF120511) and exhibits 99.0% homology homology (hōmŏl`əjē), in biology, the correspondence between structures of different species that is attributable to their evolutionary descent from a common ancestor. with the sequence from H. madaka (Habe, 1977) (390 bp compared, D88573). Moreover, the 5' sequence of the 18S rRNA segment obtained from the present study shows 99.8% and 99.3% homology to those of H. tuberculata (466 bp compared, AF120511) and H. rufescens Swainson, 1822 (445 bp compared, L78885) respectively. The small genetic differences (<1%) between these taxa in 18S rRNA indicate that this gene is quite conserved among abalone taxa. Therefore, 18S rRNA is not an appropriate marker in discriminating abalone taxa at the species level, although it may serve as a good marker for phylogenetic studies at higher systematic levels. Haliotis discus discus and H. discus hannai are recognized to be two subspecies because the ridges on the shell are thin in H. discus discus and relatively thicker in H. discus hannai. However, a survey on wild abalone, hatchery-reared abalone seed, and adult abalone from sea ranching (Kobayashi et al. 1992) and growth experiments on the two subspecies under different temperature regimes (Hara 1992), show that the morphologies of the two abalone taxa vary with environmental conditions, particularly temperature. Under low temperature or unstable culture conditions, the morphologic features of H. discus discus become similar to those of H. discus hannai. Under high temperature or relatively stable growth conditions, the morphologic features of H. discus hannai become similar to those of H. discus discus. Based on the analysis of 12 allozymes, Hara and Fijio (1992) suggested that the two subspecies could not be separated and their morphologic differences are related to the environmental factors in different localities. In the present study, the two subspecies cannot be distinguished from each other based on 18S rRNA, 16S rRNA, and COI sequence analyses. Their ITS-1 sequences only differ by 0.6%, indicating low genetic differentiation between these two abalone taxa. In addition, most of the phylogenetic trees do not clearly separate the two subspecies, and thus fail to support the subspecies status of these two abalone taxa. Similarly, there are only minor morphologic differences between H. diversicolor diversicolor and H. diversicolor supertexta with the ridges on the shell thicker in H. diversicolor diversicolor but thinner in H. diversicolor supertexta. Based on our observations, morphologic variations occur among individuals of a population and growth conditions and diet could affect shell morphology of H. diversicolor (Wang et al. unpublished data). This is consistent with the observation made by Lu (1978) who concluded that it is difficult to separate the two subspecies. Geiger (2000) indicated H. diversicolor diversicolor and H. diversicolor supertexta are synonyms. In the present study, H. diversicolor diversicolor and H. diversicolor supertexta did not show any distinct differences in their nuclear and mitochondrial DNA sequences. The sequences of ITS-1, 16S rRNA and COI of these two subspecies have also recently become available in the GenBank database (Accession Nos. AF296868, AY146396, AY146401, AY146406, AY146397, and AY146402). Comparison with the corresponding sequences obtained from the present study show that the differences are within 0% to 0.34% from each other. The ITS-1 sequences are identical to those from the present study. There was 0.2% to 0.9% (532 bp compared) and 0.4% to 0.6% (544 bp compared) divergence in 16S rRNA and COI, respectively. Our phylogenetic analyses based on the three genes show that individuals of each of the two subspecies never cluster together. In addition, a parallel study in our laboratories based on AFLP showed there are no diagnostic bands between the two subspecies (Wang et al. 2004, this issue). These results suggest that H. diversicolor diversicolor and H. diversicolor supertexta are possibly not genetically distinct, thus putting their subspecies status in doubt. Results from the present study show a lack of genetic divergence between the subspecies of Haliotis discus or Haliotis diversicolor based on sequences analyses of the four commonly used nuclear/mitochondrial DNA markers. Analysis of other more variable markers such as mitochondrial DNA control region (Chu et al. 2003) should be performed to investigate the genetic differentiation of the four abalone taxa over their geographical range (see, for example, Sites & Crandall 1997) to elucidate their evolutionary relationship and taxonomic status.
TABLE 1.
Percentage divergence of ITS-1, 16S rRNA and COI among the four abalone
taxa
ITS-1 16S rRNA
Taxon Hdh Hdd Hdc Hds Hdh Hdd Hdc Hds
Hdh 0 0.4
Hdd 0.6 0 0-0.4 0
Hdc 15.9 15.9 0 9.4-9.6 9.4 0.4
Hds 15.9 15.9 0 0 9.3-9.4 9.4 0.2-0.6 0.4
COI
Taxon Hdh Hdd Hdc Hds
Hdh 0.5
Hdd 0.6-1.4 0.6
Hdc 17.5-18.0 18.2-18.5 0.6
Hds 17.5-17.8 18.0-18.2 0.3-0.6 0.6
ACKNOWLEDGMENTS The authors thank Dr. Fuhua Li (Institute of Oceanology, Chinese Academy of Sciences The Chinese Academy of Sciences (CAS) (Simplified Chinese: 中国科学院; Pinyin: Zhōngguó Kēxuéyuàn), formerly known as Academia Sinica ) for collecting specimens of Haliotis discus hannai used in this study. The work described in this article was fully supported by grants from Hi-Tech Research and Development (863) Program of China (Project nos. 2001AA620108 & 2003AA603240), and an AoE Fund from The Chinese University of Hong Kong The motto of the university is "博文約禮" in Chinese, meaning "to broaden one's intellectual horizon and keep within the bounds of propriety". . LITERATURE CITED Avise, J. C. 1994. Molecular markers, natural history and evolution. New York New York, state, United States New York, Middle Atlantic state of the United States. 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Beckenbach, B. Crespi, H. Liu & P. Flook. 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87:652-701. Sites, J. W., Jr. & K. A. Crandall. 1997. Testing species boundaries in biodiversity studies. Conserv. Biol. 11:1289-1297. Wang, Z. Y., C. H. Ke, Y. L. Wang, Z. Q. Xiao, K. C. Ho & K. H. Chu. 2004. Genetic variations and divergence of two Haliotis species as revealed by AFLP analysis. J. Shellfish Res. (in this issue) ZHI YONG WANG, (1,2) KA CHAI chai n. A beverage made from spiced black tea, honey, and milk. [Ultimately from Chinese (Mandarin) chá, tea.] HO, (1) DA HUI YU, (1) CAI (1) (Computer-Assisted Instruction) Same as CBT. (2) See CA. CAI - Computer-Aided Instruction HUAN KE, (3) WAI WAI Web Accessibility Initiative (W3C) WAI Where Am I? WAI Wales Arts International (UK) WAI Women in Aviation, International WAI Warm Air Intake WAI Web Application Interface YAN YAN Youth Action Network YAN Yangambi, Zaire (airport code) YAN You Are Nice YAN Yancey Railroad Company MAK Mak Falstaffian figure; categorically maintains his innocence. [Br. Lit.: The Second Shepherds’ Play] See : Deceit Mak sheep stealer succeeds by waiting till the shepherds fall asleep. [Br. Lit. , (4) AND KA HOU CHU (1) * (1) Department of Biology, The Chinese University of Hong Kong, Hong Kong, China; (2) Fisheries College, Jimei University, Xiamen, Fujian, 361021, China; (3) Department of Oceanography oceanography, study of the seas and oceans. The major divisions of oceanography include the geological study of the ocean floor (see plate tectonics) and features; physical oceanography, which is concerned with the physical attributes of the ocean water, such as , Xiamen University, Xiamen, Fujian, 361005, China; (4) Molecular Biotechnology Program, The Chinese University of Hong Kong, Hong Kong, China * Corresponding author. E-mail.kahouchu@cuhk.edu.hk |
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