Genetic heterogeneity analysis and RAPD marker detection among four forms of Atrina pectinata Linnaeus.

ABSTRACT Pen shell (Atrina pectinata Linnaeus) can be distinguished into four forms based on the morphologic characteristics. Genetic similarity and heterogeneity were analyzed among the four forms by random amplified polymorphic DNA (RAPD) technique using 24 10-nucleotide-long primers. Of these primers, 22 primers produced well-identifiable RAPD band patterns. Significant differences in RAPD band patterns were revealed among the four forms. A total of 198 polymorphic fragments were scored from 22 primers, and they are specific for one form, shared by two or three forms. Several primers, such as S451, S453, S463, S464, S470, S473. and S474, produced abundant band patterns and provided sufficient information for reliable discrimination of the four forms. The average genetic distances and phylogenetic relationships were calculated and analyzed according to the distinguishable fragments. The data indicate that pen shells of form G and form Y are similar not only among individuals within the same form, but also between individuals from the two forms, and that shells of form T and form S are highly divergent. The constructed phylogenetic tree matches the average genetic distances. Three clusters were clearly distinguishable, in which two were corresponding to form S and form T respectively and one included forms G and Y. This study will be benefit to further studies on the taxonomy and selective breeding of Pinnid species. It is suggested that the four forms of pen shell should be categorized to at least two species taxonomically.

KEY WORDS: RAPD marker, genetic polymorphism, taxonomy, Atrina pectinata

INTRODUCTION

Marine shells of the family Pinnidae are a popular food source and of high commercial value in a number of Asia-Pacific countries. In China, there are a number of Pinnid species that are presently categorized into three subgenera. The pen shell, Atrina pectinata Linnaeus, is the most commercially prominence of these species and is the only Pinnid species found in both northern and southern China waters (Wang 1997). In the South China Sea, including Baibu Gulf, A. pectinata shows considerable morphologic variation, which has produced considerable taxonomic confusion for more than 40 y (Wang 1964, 1997, Liang et al. 1986, Bernard et al. 1993). In an attempt to characterize its diversity and analyze its population structure, two methods, morphologic comparison and isoenzyme electrophoresis, have been used. These studies have revealed significant variations in not only exterior shell character and size of posterior adductor muscle (Yu et al. 2000), but also in the isozyme electrophoretograms (Wang et al. 2000).

According to their morphologic characteristics, especially the traits of exterior shell, Atrina pectinata in the South China Sea, can be divided into four forms; "green pen shell" (form G), "yellow pen shell" (form Y), "thorny pen shell" (form T), and "scabrous pen shell" (form S), respectively (Fig. 1). In areas such as the Leizhou Peninsula, collections of shells from four sites have shown that the four forms are sympatric. In collections from Donghai Island and Wailuo, all four forms occur in similar numbers. whereas at Shatian and Qintou, forms G and Y are most common, with only a few individuals of form T or S (Fig. 2; Yu's personal observation). Electrophoretic comparisons of isozymes from different tissues have indicated that there are differences in the isozyme phenotypes among the four forms, which are consistent with the morphologic forms. This electrophoretic analysis has shown that forms G and Y are analogous, while form S differs markedly (Yu et al., 2000; Wang et al., 2000).

[FIGURES 1-2 OMITTED]

The random amplified polymorphic DNA (RAPD) technique is a rapid and sensitive polymerase chain reaction (PCR)-based method (Williams et al., 1990) that has been widely used in polymorphism analysis for genetic structures. The RAPD technique had been shown to be a powerful tool for discrimination of different aquatic species or subspecies and for genetic analysis of phylogenetic relationships among strains or populations (Zhou & Gui 2000a, 2000b, 2002, Gui, 2003). In marine bivalves, RAPD techniques have been used to analyze genetic variation in oysters (Liu et al. 1998, Liu & Dai 1998), mussels (Kimura et al. 1997) and scallops (Patwary 1994), and proved an appropriate tool at molecular level for identifying divergence between populations or sibling species.

In this study, the RAPD technique was used for analyzing genetic diversity in the four forms of Atrina pectinata. We sought to reveal whether the four forms of pen shell distinguished morphologically could also be categorized by RAPD analysis. One of aims was to select suitable strains for farming because several traits differ among the four forms, such as the comparative size of adductor muscle, which is related to consumer popularity and market price in China. Another main aim is to provide more convincing evidence for the taxonomic categorization of the different forms of pen shell.

METHODS

Source of Samples

A total of 85 individuals of the tour forms that caught from coast waters of Zhanjiang and nearby areas, in southern China (Fig. 2), were used for morphologic comparison. Because preliminary experiments demonstrated that most individuals in same forms produced basically identical RAPD band patterns, only eight adult individuals selected from each form were used to analyze genetic similarity and heterogeneity and to seek the specific markers for discriminating different forms.

Morphologic Comparison

Morphologic comparison was performed by observing the shell exterior and partially anatomic characters, such as the shell surface status, the radial sculpture situation, scale, the color of pallial organ and kidney, the ratio of shell height/shell breadth, and so on.

DNA Extraction

Total genomic DNA was isolated from ovarian tissue using standard phenol-chloroform extraction procedures (Sambrook et al., 1989). Briefly, about 1.5 g of tissue from each sample was homogenized into 15 mL of homogenized buffer (10 mM Tris-HCl, pH 8, 75 mM NaCl, 5 mM EDTA, 0.5%SDS) at 4[degrees]C. RNase was added and the suspension was incubated for 1 h at 37[degrees]C. 100 [micro]g x m[L.sup.-1] of proteinase K was then added and the suspension was again incubated for 2 h at 55[degrees]C. Proteins and other impurities were removed by extraction with phenol:chloroform:isoamyl alcohol (25:24:1). The DNA was precipitated with 2 volumes of cold ethanol, 1/10 volume 3 M NaAc and resuspended in TE buffer (10 him Tris-HCl, 1 mM EDTA. pH 8). After quality and concentration, samples were determined by agarose electrophoresis as described (Zhou et al. 1998), the DNA samples were stored at -80[degrees]C.

RAPD-PCR

Because RAPD analysis is particularly sensitive to the concentration of DNA, magnesium, primer, and Taq DNA polymerase, the optimal reaction conditions must be determined before study (Black 1993, Williams et al. 1993). In our system for RAPD amplification of the four forms of pen shells, we adopted the conditions at the raised annealing temperature of 39[degrees]C, which is higher than in other systems and amplified abundant and reproducible band patterns.

Amplification reactions were performed in volume of 25 [micro]L containing approximately 20 ng of template DNA, 15 ng of primer, 0.5 units of Taq polymerase (Promega), 0.1 [micro]M of each dNTP, and 10x Buffer for Taq polymerase (Promega) as described by Zhou et al. (1998). RAPD primers were obtained from Shanghai Biotech (Shanghai, China). A total of 24 10-nucleotide-long primers were used. PCR amplifications were performed in a Perkin-Elmer DNA GeneAmp PCR System 9600, with an initial denaturation at 94[degrees]C for 4 min, followed by 38 cycles of 1 min at 94[degrees]C, 1 s at 39[degrees]C, 50 s at 72[degrees]C, and a final extension at 72[degrees]C for 7 min. Amplification products were separated by electrophoresis on 1% agarose gels. Sizes in kilo base (kb) pairs were inferred by comparisons with a Lambda DNA/EcoRI + HindIII marker (Sino-America Bio). Gels were stained by ethidium bromide and recorded on a white/ ultraviolet transilluminator with computer (UVP, Ultra-Violet Products).

RAPD Data Analysis

Twenty-two primers were found to produce well-amplified and reproducible electrophoretic band patterns. Using these informative bands, all individuals from every form were evaluated by RAPDistance 1.04 program in computer. For each individual, about 104 distinguishable fragments, including universal and specific, were scored with data coded as a vector of 1 or 0 representing band presence or absence respectively. Distance matrices among the 46 individuals of four forms were estimated based Apostol (1993). Phylogenetic trees showing the hierarchical structures of RAPD pattern affinities among individuals and forms were constructed from the distance matrices by NJTREE analysis, as implemented in the program of RAPDistance 1.04 (Apostol 1993).

RESULTS

Morphologic Difference Among the Four Forms

The data about morphologic traits of the four forms were summarized in Table 1. As shown in Table 1, their morphologic traits can be easily discriminated among the four different forms, especially in the aspects of exterior shell character and the ratio of shell height/shell breadth. Form Y is most analogous to typical parameters of Atrina pectinata described by Wang (1997).

Similarity and Heterogeneity of RAPD Band Patterns Within and Between the Four Forms

The similarity and heterogeneity of RAPD band patterns within and between the four forms were screened using 24 primers, of which, 22 primers reproducibly produced well-identifiable RAPD band patterns (1 to 12 bands ranging from about 0.2 to 2.5 kb; Table 2). No anyone of the RAPD band patterns produced by the 22 primers was identical in all of the four forms. As shown in Figure 3, there are significant differences in RAPD band patterns among the four forms. Some characteristic RAPD band patterns for the different form were produced, and only a few polymorphic bands were observed among individuals in each form. In comparison with form T and form S, forms G and Y exhibit more similarity in RAPD band patterns (Fig. 3).

[FIGURE 3 OMITTED]

Detection of Specific RAPD Markers for Discriminating Different Forms

A total of 198 polymorphic fragments were scored from 22 primers (Table 2), and they are specific for one form, or shared by two or three forms. Several primers, such as S451, S453, S463, S464, S470, S473, and S474, produced abundant band patterns and provided sufficient information for reliable discrimination of the tour forms analyzed. As shown in Figure 3, primer S451 amplified one 2.2-kb fragment shared by all forms, one 0.5-kb fragment specific for form S, one 0.2 fragment specific for form T and another 0.8-kb fragment shared by forms G and Y. Primer S453 gave rise to two bands of 0.9 kb and 1.4 kb shared by forms G, Y and T, one 0.3-kb band shared by forms G and Y, and another 0.4-kb band specific for form S. Primer S463 generated four fragments in all individual of forms G and Y, two fragments in all form S individuals, five fragments in form T. Primer S470 yielded two fragments of 0.7-kb and 1.2-kb band specific for form S, three fragments of 0.2 kb, 0.8 kb and 1.0 kb specific for form T, and one 0.6 kb fragment shared by forms S, G and Y. Most of the polymorphic fragments can be used as specific RAPD markers for discriminating the four different forms in Atrina pectinata.

Average Genetic Distances and Phylogenetic Relationships

The average genetic distances and phylogenetic relationships were calculated and analyzed according to the total of 4788 distinguishable fragments obtained from the all RAPD band patterns. As shown in Table 3, the average genetic distances among individuals in form G (0.1362 [+ or -] 0.02487) and form Y (0.1195 [+ or -] 0.02622) are lower than that in form T (0.1769 [+ or -] 0.05324) and form S (0.1896 [+ or -] 0.02758), and the average genetic distance (0.1447 [+ or -] 0.02309) between form G and form Y is basically equal to that among individuals in the form G or Y. In contrast, the average genetic distances between form S and form T, between form S and form G, between form S and Y, between form T and form G, and between form T and form Y are very much high, and range from 0.4229 [+ or -] 0.02535 to 0.4888 [+ or -] 0.02227 (Table 3). These data indicate that the form G and form Y are closely related not only among individuals within the same form, but also between individuals from the two forms, and form T and form S are highly divergent.

The phylogenetic tree of the 46 individuals from the four forms was obtained by NJTREE analysis in the RAPDistance program, as shown in Figure 4. Three clusters were clearly distinguishable, in which two were corresponding to form S and form T respectively and one included forms G and Y. Any specimen was first clustered with other individuals from the same form. Apparently, forms G and Y are most closely related, while the most divergence occurs between forms S and G or Y. The constructed phylogenetic tree (Fig. 4) matches the average genetic distances among the four forms (Table 3).

[FIGURE 4 OMITTED]

DISCUSSION

In traditional mollusk taxonomy, characteristic shell features and organ structure are the main basis for categorizing different species and subspecies. But, some of these features are malleable and vary in accordance with environmental conditions. These various features and structures resulted in considerable confusion and controversy among taxonomists. For Atrina pectinata, debate as to whether the present species should be divided to two or more species or subspecies have lasted for years. In China, the four forms of pen shell were generally grouped to one species, A. pecinata (Wang 1997). In past decade, some new biochemical and molecular biologic methods, such as allozyme electrophoresis, mitochromzon DNA restriction mapping and RAPD polymorphic analysis, have been applied in comparison of genetic variation and taxonomic studies.

Thorp (1982) deduced from studies of classification of populations that the genetic similarity (I) below 0.85 between two populations should be on inter-species level, I value between 0.2 and 0.8 on level of inter-species in same genus, between 0.8 and 0.97 on level of populations of same species. In other words, while the genetic distance (1 - I) over 0.20 between two populations, the two populations should be categorized into different species.

In several mollusk species, the genetic distances (D) were estimated from data of isozyme electrophoresis and morphologic differences. Krause et al. (1994) surveyed the genetic variation among three geographic populations of calico scallops Argopecten gibbus. The genetic distance between populations was between 0.009 and 0.070. The nearer genetic distances were agreed with lower level of morphologic variation, both on the level of geographic population within same species. Three mussel species (Mytilus edulis, M. galloprovincialis, and M. trossulus) were analyzed by Skibinski et al. (1980), Grant and Cherry (1985), and Vainola and Hvilsom (1991), the Nei's (1972) distance (D) among these three mussels was in the range of 0.16 to 0.28. Gosling (1992) regarded the above three mussels as a subspecies status. The swan mussel Anodonta woodiana living in same pond in Japan has been known to exhibit two forms. Tabe et al. (1994) made clear that D distance between the two forms was 0.707 and no hybrid was observed, they suggested that these two forms should be regarded as distinct species. Buroke (1979a, 1979b) studied the intraspecific and interspecific genetic divergence of the family Ostreidea, and found that the D value of 0.0118-0.0408 was on the level of intraspecific populations, whereas above 0.165 was beyond sibling species. The reports on RAPD analysis in mollusc were not as much as that of isozyme electrophoresis. Patwary (1994) first used RAPD technique in bivalve mollusc for analyzing genetic divergence in scallop Placopecten magellanicus Gmelin and distinguished different allele frequencies in different populations. Wilding et al. (1998) analyzed genetic differentiation of morphotypes of periwinkle Littorina saxatilis from two locations of England. They considered the diverse RAPD patterns as evidence of separate gene pools. Liu et al. (1998a, 1998b) analyzed RAPD polymorphism of oyster species in northern China, and found that genetic distances between three species Crassostrea talienwhanesis, C. plicatula and C. gigas were among 0.333-0.400, whereas the intraspecific distances between four geographic populations of C. talienwhanesis were below 0.2004. These researches were almost conformed to inferences about D distance suggested by Thorp (1982)

In Pinnadea species, very few data about their genetic background could be obtained. Koji Yokogawa (1996) had examined and calculated genetic divergence of nonscaly and scaly pen shell Atrina pectinata distributing in partial Japanese marine region by isozyme electrophoresis. Based on the high D value (0.469) and low frequency of hybrids, Koji Yokogawa (1996) suggested that the two forms would be taxonomically distinguished. In this study, we demonstrated that different forms of Atrina pectinata amplified diverse RAPD band patterns, and that specific markers created by some primers can be efficiently used to discriminate the four different forms of pen shell. Twenty-two primers produced a total of 4,788 distinguishable bands from 46 individuals of the four forms, and 87 markers specific for some forms (Table 2). The dendrogram and average genetic distances clearly indicate phylogenetic relationships among the four forms. Among the four different forms, form G was clustered together with form Y, and their average genetic distance is the smallest ([less than or equal to] 0.15), even smaller than the intra-form distance of form T ([greater than or equal to] 0.17) and S ([greater than or equal to] 0.18); The most divergence occurred between form S and forms G (or Y), their genetic distance is more than 0.48. The data imply that form G has a very high genetic homogeneity to form Y, and form S is distinctly contrasted to form G (or Y) in the genetic background. It should be reliable to say that the pen shell, previously regarded as one species, Atrina pectinata, should be a conspecific complex composed of at least two species. Form G and Y are same species, and form S is another species different from them. Form T remains undecided, and it might belong to either group or be the third species or subspecies.

Of course, RAPD data in our study could only be a proof for classification of these four forms, further investigations should be conducted to provide more and convincing evidences. Two experiments are going to be carried out: 1) by crossing between the form G and form S, the most genetically distant forms, to observe the survival status of their hybrids, 2) by cloning and sequencing specific RAPD fragments to develop diagnostic PCR SCAR primers (Zhou et al. 2001) for reliable discrimination of the four forms. These new evidences will be benefit to further reclassification of A. pecinata.

TABLE 1.
Observed growth of oyster populations at Horsehead.

                      Population #1      Population #2

             Temp            Mean                Mean
   Date      (C)     n    Length (mm)    n    Length (mm)

10/15/1992   19.8   196      15.8
11/11/1992   13.8   202      16.7       100      15.4
 12/9/1982   7.6    140      16.2        79      16.4
 1/28/1993   7      201      16.7        98      15.3
 3/31/1993   12.4                       175      14.1
 4/14/1993          211      16.2
  5/3/1993   18.5   210      16.6       120      17.3
  6/2/1993   22     239      16.7        94      17.9
 6/28/1993   27.2   212      20.2        77      21.1
 7/27/1993   28.8   208      25.3        76      26.6
 8/24/1993   28.1   251      28.1        75      30.7
10/25/1993   19     251      33.2        75      36.8
11/16/1993   14.8   251      33.8        72      37.3
12/13/1993    8     243      33.9        72      37.6
  4/4/1994   14     243      33.6        72      37
  5/9/1994   18.5   235      33.2        72      36.7
 6/10/1994   24.2   232      33.9        71      37.4
 7/11/1994   28.6   234      37.1        70      41.4
  8/8/1994   25.8   234      39.3        69      42.8
 9/13/1994   24.5   230      41.4        69      43.9
10/18/1994   18     230      44.3        68      47.2
11/15/1994   17     230      45.3        68      49
 2/20/1995    5     227      45.8        67      49
 4/18/1995   16.9   229      45.4        65      49.1
 5/24/1995   23.1   228      46.2        65      49.7
 7/14/1995   28.9   160      47.2        38      50.7
 8/29/1995   26.4   131      51          32      52.7
 9/27/1995   20.7   135      52.2        30      52.9
10/17/1995   10.5   122      56.5        28      54.7

TABLE 2.
Estimation of size specific growth rate incorporating
temperature effects.

                     Estimated Monthly
                       Growth Rate
                  (mm/month) = (mx + c)
                     where x is Mean
Month   Temp (C)      Temperature

  J        4.8
  F        5.1
  M        7.9
  A       13.7               m       c
  M       19.0     year 0   0.27   -2.80
  J       24.1     year 1   0.20   -1.74
  J       27.0     year 2   0.17   -1.42
  A       27.2     year 3   0.14   -1.16
  S       24.6     year 4   0.11   -0.94
  0       19.2     year 5   0.09   -0.77
  N       13.7     year 6   0.07   -0.64
  D        8.3

TABLE 3. Average genetic distances among the four forms
of Atrina pectinata.

Form              G                         Y

 G     0.1362 [+ or -] 0.02487
 Y     0.1447 [+ or -] 0.02309   O.1195 [+ or -] 0.02622
 T     0.4319 [+ or -] 0.02469   0.4229 [+ or -] 0.02535
 S     0.4888 [+ or -] 0.02227   0.4686 [+ or -] 0.02178

Form              T                         S

 G
 Y
 T     0.1769 [+ or -] 0.05324
 S     0.4458 [+ or -] 0.02881   0.1896 [+ or -] 0.02758

ACKNOWLEDGMENTS

These studies were supported by the Chinese Academy of Sciences (KSCX2-SW-303) and the State Key Laboratory of Freshwater Ecology and Biotechnology.

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XIANGYONG YU, (1,2) YONG MAO, (2) MEIFANG WANG, (2) LI ZHOU (1) AND JIANFANG GUI (1), *

(1) State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China and (2) Fisheries College, Zhanjiang Ocean University, Zhanjiang 524025, China

* Corresponding author. E-mail: [e-mail]