Classification of common oysters from North China.
ABSTRACT Oysters are commonly found on rocky shores along China's northern coast, although there is considerable confusion as to what species they are. To determine the taxonomic tax·o·nom·ic also tax·o·nom·i·cal
Of or relating to taxonomy: a taxonomic designation.
tax status of these oysters, we collected specimens from nine locations north of the Yangtze River Yangtze River
Chinese Chang Jiang or Ch'ang Chiang
River, China. Rising in the Tanggula Mountains in west-central China, it flows southeast before turning northeast and then generally east across south-central and east-central China to the East China and conducted genetic identification using DNA sequences. Fragments from three genes, mitochondrial mitochondrial
pertaining to mitochondria.
a unique set of tRNAs, mRNAs, rRNAs, transcribed from mitochondrial DNA by a mitochondrial-specific RNA polymerase, that account for about 4% of the total cell RNA that 16S rRNA, mitochondrial cytochrome oxidase cytochrome oxidase
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 (COI), and nuclear 28S rRNA, were sequenced in six oysters from each of the nine sites. Phylogenetic phy·lo·ge·net·ic
1. Of or relating to phylogeny or phylogenetics.
2. Relating to or based on evolutionary development or history. analysis of all three gene fragments clearly demonstrated that the small oysters commonly found on intertidal in·ter·tid·al
Of or being the region between the high tide mark and the low tide mark.
in rocks in north China are Crassostrea gigas (Thunberg, 1793), not C. plicatula (the zhe oyster) as widely assumed. Their small size and irregular shell characteristics are reflections of the stressful intertidal environment they live in and not reliable characters for classification. Our study confirms that the oysters from Weifang, referred to as Jinjiang oysters or C. rivularis (Gould, 1861), are C. ariakensis (Wakiya, 1929). We found no evidence for the existence of C. talienwhanensis (Crosse, 1862) and other Crassostrea species in north China. Our study highlights the need for reclassifying oysters of China with molecular data.
KEY WORDS: oyster, taxonomy, cytochrome oxidase I, 16S rRNA, 28S rRNA, Crassostrea gigas, C. ariakensis, Alectryonella plicatula, Saccostrea cucullata, Suminoe oyster
There are several unresolved issues in oyster classification. The problems are caused by classification based on shell morphology, which is highly variable in oysters. One of the problems is the taxonomic status of oysters found along China's north coast. Oysters are common and abundant on the rocky shores of North China. They are found on rocks, concrete and other hard surfaces in almost all intertidal zones and harbors. These oysters are typically small in size and highly variable in shell morphology. There is considerable confusion and disagreement about the taxonomic identity of these oysters.
According to according to
1. As stated or indicated by; on the authority of: according to historians.
2. In keeping with: according to instructions.
3. existing literature, eight oyster species occur in North China: the Pacific oyster Pacific oyster
An oyster (Crassostrea gigas) cultured in the United States and Europe, having a scalloped shell and a fruity flavor. Also called Portuguese oyster. (Crassostrea gigas), zhe or folded oyster (C. plicatula [Gmelin, 1791]), Sengmao or monkhat oyster (Saccostrea cucullata [Born, 1778]), Dalianwan oyster (C. talienwhanensis), jinjiang or near-river oyster (C. rivularis, now C. ariakensis), Portuguese oyster Portuguese oyster
Crassostrea angulata, C. pipas. (C. angulata [Lamark, 1819]), milin or dense-scale oyster (Ostrea denselamellosa [Lischke, 1869]), and maozhua or cat's paw cat's paw
a person used by someone else to do unpleasant things for him or her [from the tale of a monkey who used a cat's paw to draw chestnuts out of a fire] oyster (Talonostrea talonata [Hanley, 1864]) (Zhang & Lou 1956, Qi 1989, Li & Qi 1994, Xu & Huang 1993, Guo et al. 1999, Yu et al. 2003, Wang, et al. 2004, Lapegue et al. 2004). The latter two species are well defined, and there is no dispute about their taxonomic status. The dense-scale oysters are characterized by large (up to 15 cm), round and flat shells with densely populated pop·u·late
tr.v. pop·u·lat·ed, pop·u·lat·ing, pop·u·lates
1. To supply with inhabitants, as by colonization; people.
2. layers of scales along the edge. The cat's paw oyster is small in size (2-3 cm), and its left shell has 5-8 ribs protruding pro·trude
v. pro·trud·ed, pro·trud·ing, pro·trudes
To push or thrust outward.
To jut out; project. See Synonyms at bulge. out in the shape of cat's paws. These two species are relatively rare, but they can be easily identified and separated from other species.
Zhang & Lou (1956) classified the common oysters from North China as the following: (1) the monk-hat oyster (O. cucullata), a small sized oyster attached to rocks in shallow water See:
Zhao et al. (1982) named the oyster commonly found on the rocks of Dalian coast as the folded oyster (C. plicatula). Since then, there has been no agreement whether the common intertidal oysters from north China is O. cucullata or C. plicatula, or both (Zhang & Lou 1956, Qi 1989, Guo et al. 1999, Yu et al. 2003, Xu & Huang 1993). Li & Qi (1994) considered that the O. cucullata, C. rivularis, and C. talienwhanensis of Zhang & Lou (1956) and C. plicatula of Zhao et al. (1982) were all C. gigas. They identified the monk-hat and folded oysters as Saccostrea cucullata and Alectryonella plicatula, respectively, two species of different genus found in southern China.
However, after analyzing genetic variation revealed by random amplified polymorphic polymorphic - polymorphism DNA DNA: see nucleic acid.
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. (RAPD RAPD Randomly Amplified Polymorphic DNA
RAPD relative afferent pupillary defect (ophthalmology; aka Marcus-Gunn Pupil) ), Liu & Dai (1998) concluded that C. talienwhanensis, C. plicatula, and C. rivularis were three different species. An analysis of 16S and COI sequences by Yu et al. (2003) indicated that C. gigas and C. talienwhanensis were the same species, and C. plicatula might be a morph morph 1
n. of C. ariakensis. Further, Lapegue et al. (2004) reanalyzed the sequences of Yu et al. (2003) and showed that some of the C. talienwhanensis samples might be C. angulata.
The confusion about the taxonomic status of common oysters from north China calls for further analysis. In this study, we collected and sequenced oysters from 9 locations along China's northern coast for fragments of three genes: the mitochondrial 16S rRNA gene and cytochrome oxidase I (COI), and the nuclear 28S rRNA. Here we provide molecular evidence that the small oysters commonly found on rocky shore belong to one species--C, gigas, and the large oysters (referred to as the near-river oysters) collected from Weifang are the same species as C. ariakensis (Wakiya, 1929).
MATERIALS AND METHODS
Sample Collection and Morphological Analyses
Oysters were collected from nine typical habitats along China's coast north of Yangtze River: Zhuanghe (ZH), Zhangzidao (ZZD ZZD Zeon Zum Deikun (Gundam anime)
ZZD Zig-Zag Diagram ), Dalian (DL) in Liaoning Province; Dongying (DY), Weifang (WF), Rongcheng (RC), Rushan (RS) and Qingdao (QD) in Shandong Province; and Lianyungang (LYG LYG London Youth Games
LYG Louisville Youth Group
LYG Local Youth Group ) in Jiangsu Province (Fig. 1). At Zhuanghe, Zhangzidao, Dongying, Rongcheng, Qingdao, and Lianyungang oysters were collected from rocks in intertidal zones. At Weifang, oysters were collected by divers from a depth of about eight meters. Oysters from Dalian and Rushan were cultured oysters derived from local wild seed.
The following shell characteristics were recorded: shell morphology, shell wall rigidity or thickness, valve lamellae lamellae
n the nearly parallel layers of bone tissue found in compact bone. , radial ribs, shell color, ligament channel, umbonal cavity volume, interior shell color, adductor muscle Noun 1. adductor muscle - a muscle that draws a body part toward the median line
skeletal muscle, striated muscle - a muscle that is connected at either or both ends to a bone and so move parts of the skeleton; a muscle that is characterized by scar color and shape. Shell parameters were identified following definitions of Moore et al. (1971).
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 , PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) Amplification and Sequencing
Six oysters from each site were selected for sequencing (Table 1). Oysters with variable shell morphology were chosen to cover all possible species. DNA was extracted from fresh adductor muscle tissue using phenol/chloroform extraction as described by Moore (1993). Primers (Invitrogen, USA), 16sar and 16sbr, were used to amplify a segment of the mitochondrial 16S ribosomal RNA ribosomal RNA
ribosomal RNA (rī´bōsō´m gene (Palumbi et al. 1991). A segment of cytochrome oxidase subunit sub·u·nit
A subdivision of a larger unit.
Noun 1. subunit - a monetary unit that is valued at a fraction (usually one hundredth) of the basic monetary unit
fractional monetary unit I (COI) was amplified using LCO LCO Lac Courte Oreilles (Wisconsin Native American Tribe)
LCO Levine Communications Office (PR firm; Los Angeles, CA, USA)
LCO Light Cycle Oil
LCO Life Cycle Objectives 1490 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 primers (Folmer et al. 1994). A 28S rRNA fragment was amplified using primers D1F and D6R as described by Park and O Foighil (2000). PCR amplification was performed using a Biometra T1 thermal cycler The Thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus used for PCR. The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. . Reactions were performed in 50 [micro]L with final concentrations of: 2.0 mM Mg[Cl.sub.2], 150 [micro]M of each dNTP, 0.2 [micro]M each primer, 20 ng of template DNA, and 2 units of Taq polymerase Taq polymerase ("Taq Pol," or simply "Taq") is a thermostable polymerase used in polymerase chain reaction to check for the presence or absence of a gene by amplifying a DNA fragment. It replaced E.coli DNA polymerase in PCR because of the temperature conditions of PCR. (Promega, USA) in 5 [micro]L of x 10 buffer. COI and 16S were amplified using the following protocol: initial denature de·na·ture
1. To change the nature or natural qualities of.
2. To render unfit to eat or drink without destroying usefulness in other applications, especially adding methyl alcohol to ethyl alcohol.
3. at 95[degrees]C for 2 min, 30 cycles of 95[degrees]C for 1 min, 51[degrees]C (COI) or 57[degrees]C (16S) for 1 min, and 72[degrees]C for 1 min with a final extension at 72[degrees]C for 5 min. The 28S fragment was amplified by an initial denaturing at 94[degrees]C for 4 min, addition of Taq polymerase; 30 cycles of denaturing at 94[degrees]C for 40 s, annealing annealing (ənēl`ĭng), process in which glass, metals, and other materials are treated to render them less brittle and more workable. at 60[degrees]C for 40 s and extension at 72[degrees]C for 1.25 min; plus a final extension at 72[degrees]C for 10 min. A negative control (no template) was included during each PCR run. PCR products were purified using EZ Spin Column PCR Product Purification Kit (BBI BBI Blockbuster Inc. (stock symbol)
BBI Berlin Brandenburg International (Airport)
BBI Browser-Based Interface
BBI Best Brains Inc. , Canada). Sequencing was performed in both directions on an 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 377XL DNA sequencer A DNA sequencer is an instrument used to automate the DNA sequencing process.
DNA sequencers have become more important due to large genomics projects and the need to increase productivity. using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, USA).
[FIGURE 1 OMITTED]
The COI, 16S, and 28S sequences obtained in this study and those of other Crassostrea, Ostrea and Saccostrea species available from GenBank were used for phylogenetic analysis. Initial alignments were performed using CLUSTALW (Thompson et al. 1994). The sequences were trimmed to the same length as other published sequences after alignment. Maximum parsimony Maximum parsimony, often simply referred to as "parsimony," is a non-parametric statistical method commonly used in computational phylogenetics for estimating phylogenies. Under maximum parsimony, the preferred phylogenetic tree is the tree that requires the least number of (MP) and neighbor-joining (NJ) analyses were performed using PHYLIP PHYLIP Phylogeny Inference Package (genetics software) (Vet. 3.56C, Felsenstein 1989). 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. analysis with 1,000 replications was performed using the SEQBOOT and CONSENSE programs. Consensus phylogenetic trees were drawn with DRAWGRAM program in the PHYLIP package. Pair-wise sequence divergences among haplotypes and reference species were calculated using the DNADIST program of PHYLIP according to Kimura's two-parameter model (Kimura 1980).
Distribution and Morphology of Oysters from North China
Oysters are common and abundant at all nine sites sampled in this study (Table 1). Shell morphology varies greatly within and among sites. In general, oysters collected below or at the low tidal line are larger than oysters collected high above the low tidal line, so are the cultured oysters obtained from farms.
[FIGURE 2 OMITTED]
At most sites (ZH, ZZD, DY, RC, QD and LYG), oysters were collected from rocks at or above the low tidal line. These oysters are typically small (30-60 mm), flat and irregular in shape (Fig. 2). The color of the shells is variable ranging from white to yellow to brown. Some oysters especially those from Rongcheng have purple strips on the right valve. The shell's edge is highly variable: some smooth and some with waving plates protruding. Internally, the shells are milky white, sometimes with areas of gray, yellow, or brown. Muscle scars range from pink to dark purple. Ligament channels are mostly short with some exceptions.
Oysters collected from deep waters "Deep Waters" is a short story by P. G. Wodehouse, which first appeared in the United States in the March 25 1910 issue of Collier's Weekly, and in the United Kingdom in the June 1910 issue of the Strand. at Weifang are noticeably different from oysters from other sites. They are large, up to 200 mm, and some are round and horse-shoe shaped. The shells are thick and relatively smooth with multiple layers of overlapping and concentric plates on the right valve. Radiant ribs are absent or weak. Shell color is variable, ranging from yellow to brown (Fig. 2E). Ligament channels are short and shallow. The umbo umbo /um·bo/ (um´bo) pl. umbo´nes [L.]
1. a rounded elevation.
2. the slight projection at the center of the outer surface of the tympanic membrane.
n. cavity is shallow to medium. The internal side of shells is milky white with areas chalky or lacking of the nacre nacre: see mother-of-pearl. layer. The adductor muscle scar has no coloration col·or·a·tion
1. Arrangement of colors.
2. The sum of the beliefs or principles of a person, group, or institution. , which is unique for the subtidal oysters from Weifang.
The cultured oysters from Dalian and Rushan are large (100-200 mm), elongated e·lon·gate
tr. & intr.v. e·lon·gat·ed, e·lon·gat·ing, e·lon·gates
To make or grow longer.
adj. or elongated
1. Made longer; extended.
2. Having more length than width; slender. or ovate. The left valve is convex Convex
Curved, as in the shape of the outside of a circle. Usually referring to the price/required yield relationship for option-free bonds. with obvious radial ribs. The right valve is flat. Shell color ranges from white, yellow to brown, and often with purple strips. The internal sides of the shells are white with pink to purple muscle scars. Ligament channels are shallow, and the umbo cavity is deep (Fig. 2C, G).
Mitochondrial 16S rRNA Sequence
A 497 bp fragment of the mitochondrial 16S rRNA gene was sequenced for 54 individuals collected from nine sites (Table 1). Five haplotypes (Haplotype haplotype /hap·lo·type/ (-tip) the group of alleles of linked genes, e.g., the HLA complex, contributed by either parent; the haploid genetic constitution contributed by either parent.
n. 1, 2, 3, 4, 5) were identified among the 54 sequences. Oysters from Weifang had two haplotypes (Haplotype 4, 5), with five oysters having Haplotype 4 and one oyster having Haplotype 5 (Table 2). Oysters from the other eight sites had 3 haplotypes (Haplotype 1, 2, 3). Haplotype 1 is the common haplotype and shared by 46 of the 48 oysters. One oyster from Dongying has Haplotype 2, and one oyster from Qingdao has Haplotype 3 (Table 2).
[FIGURE 3 OMITTED]
Phylogenetic analysis was conducted with the five 16S haplotypes identified in this study and the following reference sequences from GenBank: C. gigas (S66183), C. ariakensis (Kim AY007427), S. cucullata (AF498507), C. virginica (AF092285), C. rhizophorae (AJ312938), C. gasar (AJ312937), C. hongkongensis (AY160756), and C. sikamea (AY632551). S. cucullata was used as an outgroup. Including the outgroup, 26 nucleotide positions were variable in the 16S data set. Phylogenetic analysis using NJ and MP procedures produced almost identical results (Fig. 3A, B). As expected, all five haplotypes identified in this study were clustered with members of Crassostrea, away from S. cucullata. The two haplotypes from Weifang were closely clustered with C. ariakensis, and formed one 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. . The two haplotypes differed from the reference sequence by only one base. A closer examination indicated that Haplotype 4 was the same sequence as that of C. ariakensis from Korea (Kim et al. 2000). The three haplotypes from all other eight sites were closely clustered with C. gigas. Haplotype 1 is identical with the reference sequence from C. gigas. Haplotype 2 and Haplotype 3 differed from the C. gigas sequence by one base. Sequence divergence between Haplotypes 1-3 and Haplotypes 4-5 was about 4.65% to 5.10%, which was higher than that between C. gigas and C. sikamea (1.73%) (Table 3). No divergence was observed between Haplotype 1 and C. gigas, the divergence between Haplotype 2/3 and C. gigas was only 0.21%. There was no divergence between Haplotype 4 and C. ariakensis from Korea, and the divergence between Haplotype 5 and C. ariakensis was only 0.36%, which is much lower than that between C. gigas and C. sikamea (1.73%). These data show that oysters from Weifang are C. ariakensis, and oysters from all other sites are C. gigas. The haplotypes obtained from this study are neither S. cucullata nor A. plicatula, as evidenced by the high divergences, 17.44% to 21.95% (Table 3).
[FIGURE 4 OMITTED]
Mitochondrial COI DNA Sequence
A 607 bp fragment of the COI sequences was sequenced for the 54 oysters selected in this study, generating 13 haplotypes (Table 4). Three (Haplotype 11, 12, 13) of these haplotypes occurred in Weifang oysters, four oysters had Haplotype 11, and the other two oysters had Haplotype 12 and 13, respectively. The majority of oysters from the other eight sites (38 of 48) shared a common haplotype--Haplotype 1. Haplotype 2 was shared by two oysters from Dalian. Haplotypes 3-10 were represented by one oyster each from variable sites (Table 4).
The partial COI sequences from the 13 haplotypes obtained in this study were subjected to phylogenetic analysis along with the following reference sequences from GenBank: C. gigas (AF152565), C. iredalei (AY038078), C. belcheri (AY038077), C. nippona (AF300616), C. hongkongensis (AY160746), C. virginica (AF152566), S. cucullata (AY038076), and C. sikamea (AY632568). S. cucullata was used as an outgroup. Including the outgroup, 121 nucleotide positions were variable in the COI data set. The NJ and MP trees were nearly identical, and both supported the same general patterns: (1) the oysters in Weifang clustered together with C. ariakensis; (2) the oysters from other sites clustered together with C. gigas (Fig. 4). Haplotype 1 is identical to the C. gigas sequence, and Haplotypel 1 is identical to C. ariakensis.
Sequence divergence between Haplotypes 1-10 and Haplotypes 11-13 ranged from 16% to 16.7%. There was no divergence between Haplotype 1 and C. gigas, and between Haplotype 11 and C. ariakensis. The divergence between Haplotypes 2 ~ 10 and C. gigas was about 0.2% ~ 0.4%. The divergence between Haplotype 12 ~ 13 and C. ariakensis was 0.2%. These levels of divergence were consistent with intraspecific in·tra·spe·cif·ic also in·tra·spe·cies
Arising or occurring within a species: intraspecific competition. variation and considerably smaller than that between C. gigas and C. sikamea (9.74%) and that between C. gigas and C. angulata (2.46%). Divergences between the 13 haplotypes of this study and that of C. sikamea, C. ariakensis, S. cucullata were too high (9.74% to 31.70%) to be conspecific con·spe·cif·ic
Of or belonging to the same species.
An organism belonging to the same species as another.
Noun 1. (Table 5).
Nuclear 28S rRNA Sequence
A 945 bp fragment of the nuclear 28S rRNA gene was sequenced for all 54 oysters collected in this study. Only two haplotypes were identified: one (Haplotype 1) for the 48 oysters from ZH, ZZD, DL, DY, RC, RS, QD, and LYG and the other (Haplotype 2) for the six oysters from Weifang.
Phylogenetic analysis was conducted with the two haplotypes identified in this study and reference sequences from GenBank: C. gigas (AF 137051), C. ariakensis (AF137052), C. virginica (AF137050), C. rhizophorae (AF137049), C. belcheri (Z29545), S. cucullata (Z29553), S. commercialis (Z29552), and C. sikamea (AY632554). S. cucullata was used as an outgroup. Including the outgroup, 33 nucleotide positions were variable in the 28S data set. In NJ and MP trees, oysters from Weifang formed one clade with C. ariakensis, and oysters from other sites were identified with C. gigas (Fig. 5).
No divergence was observed between Haplotype 1 and C. gigas and between Haplotype 2 and C. ariakensis, and the oysters collected in this study were clearly different from C. sikamea, S. cucullata, and A. plicatula (Table 6).
Classification of the Oysters in North China
The taxonomic status of the oysters in north China has been the subject of much confusion. Because the oysters are typically found on intertidal rocks, their morphological characteristics vary greatly because of the stressful and variable environment they live in. Consequently, classification based on shell characteristics has been difficult, and different studies have reached different conclusions (Zhang & Lou 1956, Qi 1989, Li & Qi 1994, Xu & Huang 1993). The existence of C. gigas and C. rivularis in north China is well accepted, although the two species have often been misidentified by different authors (Zhang & Lou 1956, Li & Qi 1994). It has been shown that the C. rivularis from Bohai Sea Bohai Sea (Chinese: ; Pinyin: Bó Hăi), also known as Bohai Bay or Bohai Gulf, is the innermost gulf of the Yellow Sea on the coast of northeastern China. in north China is the same species as C. ariakensis (Wang et al. 2004). Two questions remain unresolved: (1) what species are the small oysters that are commonly found in intertidal zones along the northern coast? and (2) is C. talienwhanensis an independent species?
[FIGURE 5 OMITTED]
The Small Intertidal Oysters
The small intertidal oysters from north China were first named the monk-hat oyster (O. cucullata) by Zhang & Lou (1956) and then the folded oyster C. plicatula by Zhao et al. (1982). After analyzing anatomic characteristics, Li & Qi (1994) concluded that neither classification was correct, and the small intertidal oysters were actually C. gigas. On the other hand, a genetic analysis suggested that C. plicatula was an independent species (Liu & Dai 1998).
In this study, we collected the common oysters from nine sites and sequenced three gene fragments for phylogenetic analysis. Our data support Li & Qi's (1994) classification that the small intertidal oysters from all sites (except Weifang) are C. gigas. This is clear from all three genes studied. At the mtl6S gene, the common haplotype shared by 96% of the oysters from the eight sites (excluding Weifang) are identical to that of C. gigas. The remaining two haplotypes are minor variants of C. gigas haplotypes, as shown by the small sequence divergences (Table 3). For the mtCOI gene, although many haplotypes are observed, the common haplotypes are identical with that of C. gigas, and all other haplotypes are minor variants and closely clustered with C. gigas (Fig. 4). The highly conserved 28S rRNA genes show that all intertidal oysters have the same haplotypes as C. gigas.
The small intertidal oysters are clearly not O. cucullata or C. plicatula, two species now known as S. cucullata and A. plicatula, respectively (Li & Qi 1994). This is evident from both morphological and molecular data. The molecular data show that the divergence between the oysters sampled in this study and S. cucullata is larger than that between any sister-species within Crassostrea. Similarly, oysters from this study are clearly not the folded oyster (A. plicatula) as indicated by large divergences in mtl6S and 28S sequences (Table 3 and 6).
Except for the small size, the intertidal oysters collected in this study have similar characteristics as C. gigas (Li & Qi 1994, Torigoe 1981, Okutani 2000), albeit more variable. The small size is probably caused by limitations in food as a consequence of living in the intertidal zone. The strong wave action may also force oysters to stick close to the substrate, causing the shells to be flat and irregular in shape.
The Large Subtidal Oysters from Weifang
In shell morphology and DNA sequences, oysters collected from deep waters at Weifang are different from oysters from other sites. In morphology, the Weifang oysters have distinctive smooth shells with layered concentric plates and a white adductor muscle scar. In DNA sequence, all three genes show that the Weifang oysters are different from oysters from other sites (shared no haplotypes). Phylogenetic analysis clearly indicates that the Weifang oysters, which are referred to as jinjiang or near-river oyster C. rivularis locally, are indeed C. ariakensis. This finding is in agreement with our previous studies (Wang et al. 2004, Wang 2004). The shell morphology of the Weifang oyster is almost identical to that of C. ariakensis from Ariake Bay in Japan (Okutani 2000).
Status of the Dalianwan Oyster (C. talenwhanesis)
Some believe C. talienwhanensis is an independent species (Zhang & Lou 1956, Liu & Dai 1988), whereas others consider it a synonym synonym (sĭn`ənĭm) [Gr.,=having the same name], word having a meaning that is the same as or very similar to the meaning of another word of the same language. Some are alike in some meanings only, as live and dwell. of C. gigas (Li & Qi 1994). We consciously collected oysters from three sites in and around Dalian. Some of the oysters we collected, especially those from Dalian and Dongying had obvious radial ribs and purple strips that are characteristic of C. talienwhanesis. However, the molecular data indicate that they are the same species as C. gigas. It is possible that our sampling missed true C talienwhanensis, but available data from this study do not support the existence of the Dalianwan oyster (C. talienwhanensis) as an independent species.
Lapegue et al. (2004) reanalyzed the sequences of Yu et al. (2003) and showed that some of the C. talienwhanensis sequences are possibly C. angulata. Our analysis finds no C. angulata from Dalian or any of the nine sites in north China. The COI haplotypes obtained from this study are clearly different from that of C. angulata (Table 5). In another analysis of thousands of oysters from China, we observed C. angulata in southern China but never north of Yangtze River (Guo et al. 2006). It is possible that our sampling missed C. angulata from north China. It is also possible that the C. angulata samples of Yu et al. (2003) may not be native to Dalian as there is considerable cross-regional transportation of oyster seed in China. Anyway, further research is needed to determine if there is C. angulata in north China.
In summary, this study provides clear molecular evidence that the common oysters from north China belong to two species: the small intertidal oysters are C. gigas and the large subtidal oysters from Weifang are C. ariakensis. We see no evidence for the existence of C. angulata and C. talienwhanensis in Dalian, and the latter is probably synonymous with synonymous with
adjective equivalent to, the same as, identical to, similar to, identified with, equal to, tantamount to, interchangeable with, one and the same as C. gigas.
The authors thank Fengshan Xu, Suping Zhang, and Xiaoxu Li for advice on oyster taxonomy. This work is supported by grants from National Science Foundation of China (No.40406032 to Wang, No. 39825121 to Guo and No. 40730845 to Zhang), SEPAC SEPAC Special Education Parent Advisory Council
SEPAC Space Experiments with Particle Accelerators
SEPAC Space Experiment with Particle Accelerators (species 07-2-9), and the US NOAA NOAA
National Oceanic and Atmospheric Administration
Noun 1. NOAA - an agency in the Department of Commerce that maps the oceans and conserves their living resources; predicts changes to the earth's environment; CBO CBO
See: Collateralized Bond Obligation. Non-native Oyster Research Program (NA04NMF NMF
An abbreviation for "no meaningful figure". You'll often see this when comparing financial data among companies where a certain ratio or figure isn't applicable.
For example, if company has negative earnings, it cannot have a P/E ratio. 4570424). This is contribution No. IMCS-2008-7 and NJSG-08-688.
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HAIYAN WANG, (1,2) GUOFAN ZHANG, (1) XIAO LIU (1) AND XIMING GUO (2) *
(1) Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, Shandong 266071, People's Republic People's Republic
A political organization founded and controlled by a national Communist party. of China," (2) Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Sciences The Institute of Marine and Coastal Sciences (IMCS) focuses on marine science-related education and research. IMCS was founded in 1993 on the Cook Campus at Rutgers University in New Brunswick, New Jersey. , Rutgers University Rutgers University, main campus at New Brunswick, N.J.; land-grant and state supported; coeducational except for Douglass College; chartered 1766 as Queen's College, opened 1771. Campuses and Facilities
Rutgers maintains three campuses. , 6959 Miller Avenue, New Jersey 08349
* Corresponding author. E-mail: firstname.lastname@example.org
TABLE 1. Location and number of oysters sequenced. Location Code No. Sequenced Zhuanghe, Liaoning Province ZH 6 Zhangzidao, Liaoning Province ZZD 6 Dalian, Liaoning Province DL 6 Dongying, Shandong Province DY 6 Weifang, Shandong Province WF 6 Rongcheng, Shandong Province RC 6 Rushan, Shandong Province RS 6 Qingdao, Shandong Province QD 6 Lianyungang, Jiangsu Province LYG 6 TABLE 2. Distribution of the five 16S rRNA haplotypes obtained in this study. Haplotype Site 1 2 3 4 5 ZH 6 0 0 0 0 ZZD 6 0 0 0 0 DL 6 0 0 0 0 DY 5 1 0 0 0 WF 0 0 0 5 1 RC 6 0 0 0 0 RS 6 0 0 0 0 QD 5 0 1 0 0 LYG 6 0 0 0 0 TABLE 3. Pair-wise divergence among five mt16S rRNA haplotypes obtained in this study (1-5) and reference species. 1 2 3 4 5 1 (all) 0 2 (DY) 0.0021 0 3 (QD) 0.0021 0.0043 0 C. gigas 0.0000 0.0021 0.0021 0 C. sikamea 0.0173 0.0195 0.0195 0.0173 0 4 (WF) 0.0488 0.0510 0.0510 0.0488 0.0442 5 (WF) 0.0465 0.0488 0.0488 0.0465 0.0419 C. ariakensis 0.0465 0.0488 0.0488 0.0465 0.0419 C. hongkongensis 0.0239 0.0261 0.0261 0.0239 0.0239 C. gasar 0.1967 0.1967 0.1995 0.1967 0.1908 C. rhizophorae 0.1859 0.1859 0.1831 0.1859 0.1884 C. virginica 0.2027 0.206 0.1993 0.2027 0.2156 S. cucullata 0.1772 0.1799 0.1744 0.1772 0.1747 Alectryonella plicatula 0.2165 0.2195 0.2135 0.2165 0.2139 6 7 8 9 10 1 (all) 2 (DY) 3 (QD) C. gigas C. sikamea 4 (WF) 0 5 (WF) 0.0021 0 C. ariakensis 0.0000 0.0036 0 C. hongkongensis 0.0285 0.0263 0.0307 0 C. gasar 0.1995 0.1995 0.1967 0.1967 0 C. rhizophorae 0.2034 0.2034 0.2005 0.1943 0.1181 C. virginica 0.2305 0.2271 0.2271 0.2194 0.2349 S. cucullata 0.1744 0.1717 0.1717 0.1690 0.2567 Alectryonella plicatula 0.1980 0.1980 0.1951 0.2046 0.2960 11 12 13 14 1 (all) 2 (DY) 3 (QD) C. gigas C. sikamea 4 (WF) 5 (WF) C. ariakensis C. hongkongensis C. gasar C. rhizophorae 0 C. virginica 0.1446 0 S. cucullata 0.2335 0.2913 0 Alectryonella plicatula 0.2576 0.3308 0.1744 0 TABLE 4. Distribution of 13 mt COI haplotypes obtained in this study. Haplotype Site 1 2 3 4 5 6 7 ZH 4 0 0 0 1 1 0 ZZD 4 0 0 0 0 0 0 DL 4 2 0 0 0 0 0 DY 5 0 1 0 0 0 0 WF 0 0 0 0 0 0 0 RC 5 0 0 0 0 0 1 RS 5 0 0 0 0 0 0 QD 5 0 0 1 0 0 0 LYG 6 0 0 0 0 0 0 Haplotype Site 8 9 10 11 12 13 ZH 0 0 0 0 0 0 ZZD 0 1 1 0 0 0 DL 0 0 0 0 0 0 DY 0 0 0 0 0 0 WF 0 0 0 4 1 1 RC 0 0 0 0 0 0 RS 1 0 0 0 0 0 QD 0 0 0 0 0 0 LYG 0 0 0 0 0 0 TABLE 5. Pair-wise divergence among 13 COI sequences obtained in this study and reference species. 1 2 3 4 5 1 (all) 0 2(DL) 0.0017 0 3(DY) 0.0017 0.0035 0 4(QD) 0.0035 0.0052 0.0052 0 5(ZH) 0.0017 0.0035 0.0035 0.0052 0 6(ZH) 0.0035 0.0052 0.0052 0.0070 0.0052 7(RC) 0.0017 0.0035 0.0035 0.0052 0.0035 8(RS) 0.0017 0.0035 0.0035 0.0052 0.0035 9(ZZD) 0.0017 0.0035 0.0035 0.0017 0.0035 10(ZZD) 0.0017 0.0035 0.0035 0.0052 0.0035 C. gigas 0.0000 0.0017 0.0017 0.0035 0.0017 C. angulata 0.0246 0.0246 0.0263 0.0281 0.0263 C. sikamea 0.0974 0.0995 0.0994 0.0974 0.0994 11(WF) 0.1622 0.162 0.1643 0.1622 0.1601 12(WF) 0.1643 0.1641 0.1664 0.1643 0.1622 13(WF) 0.1645 0.1643 0.1666 0.1645 0.1624 C. ariakens 0.1622 0.162 0.1643 0.1622 0.1601 C. hongkong 0.1327 0.1349 0.1347 0.1327 0.1347 C. nippona 0.1618 0.1616 0.1639 0.1639 0.1639 C. belcheri 0.1777 0.1775 0.1799 0.1777 0.1756 C. iredalei 0.1817 0.1793 0.1839 0.1861 0.1795 C. virginica 0.2677 0.2705 0.2702 0.2726 0.2652 S. cucullata 0.3143 0.3139 0.3170 0.3117 0.3170 6 7 8 9 10 1 (all) 2(DL) 3(DY) 4(QD) 5(ZH) 6(ZH) 0 7(RC) 0.0052 0 8(RS) 0.0052 0.0035 0 9(ZZD) 0.0052 0.0035 0.0035 0 10(ZZD) 0.0052 0.0035 0.0035 0.0035 0 C. gigas 0.0035 0.0017 0.0017 0.0017 0.0017 C. angulata 0.0281 0.0263 0.0263 0.0263 0.0228 C. sikamea 0.1013 0.0994 0.0994 0.0955 0.0955 11(WF) 0.1622 0.1643 0.1643 0.1601 0.1622 12(WF) 0.1643 0.1664 0.1664 0.1622 0.1643 13(WF) 0.1645 0.1666 0.1666 0.1624 0.1645 C. ariakens 0.1622 0.1643 0.1643 0.1601 0.1622 C. hongkong 0.1368 0.1347 0.1347 0.1306 0.1347 C. nippona 0.1660 0.1639 0.1618 0.1618 0.1639 C. belcheri 0.1777 0.1756 0.1777 0.1799 0.1756 C. iredalei 0.1817 0.1839 0.1839 0.1839 0.1795 C. virginica 0.2677 0.2652 0.2677 0.2702 0.2677 S. cucullata 0.3143 0.3170 0.3117 0.3117 0.3143 11 12 13 14 15 1 (all) 2(DL) 3(DY) 4(QD) 5(ZH) 6(ZH) 7(RC) 8(RS) 9(ZZD) 10(ZZD) C. gigas 0 C. angulata 0.0246 0 C. sikamea 0.0974 0.0960 0 11(WF) 0.1622 0.1680 0.1660 0 12(WF) 0.1643 0.170 0.1680 0.0020 0 13(WF) 0.1645 0.1710 0.1660 0.0020 0.0040 C. ariakens 0.1622 0.1680 0.1660 0.0000 0.0020 C. hongkong 0.1327 0.1390 0.1480 0.1400 0.1380 C. nippona 0.1618 0.170 0.1630 0.1500 0.1480 C. belcheri 0.1777 0.1750 0.1660 0.1740 0.1760 C. iredalei 0.1817 0.1790 0.1900 0.1780 0.1800 C. virginica 0.2677 0.2670 0.2740 0.2970 0.2990 S. cucullata 0.3143 0.3140 0.3350 0.3350 0.3380 16 17 18 19 20 1 (all) 2(DL) 3(DY) 4(QD) 5(ZH) 6(ZH) 7(RC) 8(RS) 9(ZZD) 10(ZZD) C. gigas C. angulata C. sikamea 11(WF) 12(WF) 13(WF) 0 C. ariakens 0.0020 0 C. hongkong 0.1420 0.1400 0 C. nippona 0.1520 0.1500 0.1280 0 C. belcheri 0.1740 0.1740 0.1790 0.1790 0 C. iredalei 0.1780 0.1780 0.1750 0.1650 0.1770 C. virginica 0.2940 0.2970 0.2820 0.270 0.2760 S. cucullata 0.3350 0.3350 0.3320 0.3170 0.3280 21 22 23 1 (all) 2(DL) 3(DY) 4(QD) 5(ZH) 6(ZH) 7(RC) 8(RS) 9(ZZD) 10(ZZD) C. gigas C. angulata C. sikamea 11(WF) 12(WF) 13(WF) C. ariakens C. hongkong C. nippona C. belcheri C. iredalei 0 C. virginica 0.2810 0 S. cucullata 0.2970 0.4000 0 TABLE 6. Pair-wise divergence among two 28S rRNA sequences obtained from this study and reference species. 1 2 3 4 1 (all) 0 C. gigas 0.0000 0 2 (WF) 0.0111 0.0111 0 C. ariakensis 0.0111 0.0111 0.0000 0 C. sikameal 0.0033 0.0033 0.0100 0.0100 C.sikamea2 0.0022 0.0022 0.0089 0.0089 C. belcheri 0.0271 0.0271 0.0294 0.0294 C. rhizophorae 0.0798 0.0798 0.0797 0.0797 C. virginica 0.0684 0.0684 0.0707 0.0707 S. cucullata 0.0685 0.0685 0.0709 0.0709 Alectryone plicatula 0.0957 0.0957 0.0983 0.0983 5 6 7 8 1 (all) C. gigas 2 (WF) C. ariakensis C. sikameal 0 C.sikamea2 0.0000 0 C. belcheri 0.0260 0.0249 0 C. rhizophorae 0.0835 0.0824 0.0853 0 C. virginica 0.0709 0.0697 0.0774 0.0848 S. cucullata 0.0711 0.0699 0.0776 0.0876 Alectryone plicatula 0.0970 0.0959 0.1130 0.1222 9 10 11 1 (all) C. gigas 2 (WF) C. ariakensis C. sikameal C.sikamea2 C. belcheri C. rhizophorae C. virginica 0 S. cucullata 0.0090 0 Alectryone plicatula 0.0807 0.0835 0