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Restriction fragment length polymorphisms in the largemouth bass (Micropterus salmoides salmoides) in a small Massachusetts kettlehole.


The natural range of the largemouth bass, Micropterus salmoides, was restricted to the Mississippi River system and its headwaters throughout the midwestern United States and southern Canada (Robbins and MacCrimmon, 1974). The eastern range of this species was blocked by the Appalachian Mountains; the western range was blocked by the Rocky Mountains and the xeric conditions of the Great Plains. Stocking has dramatically extended its range throughout the world. Many early stockings were either poorly documented or unrecorded. Also, no distinction was made between largemouth or smallmouth (Micropterus dolomieui) basses (Robbins and MacCrimmon, 1974). Largemouth bass were stocked into Massachusetts fresh waters in the 1870s by the United States Fisheries Commission and private citizens. The primary source of stocked bass was upstate New York (J. Bergin, Massachusetts Fish and Game Commission, pers. comm.), but fish may have been introduced from the Mississippi River drainage system in Illinois (Robbins and MacCrimmon, 1974), as archival state reports indicate that bass were caught in Massachusetts before the 1870s (J. Bergin, pers, comm.).

Understanding of the population dynamics of many taxonomic groups has been increased by application of molecular techniques. Allozymes and, more recently, mitochondrial (mt) DNA have been studied as molecular markers of evolution. MtDNA has been employed to elucidate many ecological problems not distinguished by allozyme analysis (Avise et al., 1987; Meyer et al., 1990; Meyer and Wilson, 1990). Mt-DNA polymorphisms can be identified between closely related populations, with maternal lineages often distinguished (Avise et al., 1979; Bernatchez et al., 1989). This is due to the mode of inheritance of mitochondria upon fertilization. The sperm does not provide mitochondria to the resultant zygote, hence the offspring is a mitochondrial clone of the mother (Chapman et. al., 1982).

MtDNA has a rapid mutation rate, approximately 10 times that of nuclear DNA (Brown et al., 1979). This is apparently largely due to a lack of repair mechanisms for DNA gamma polymerase within the mitochondrion (Clayton, 1982), a higher rate of nucleotide misincorporation by DNA gamma polymerase in mitochondria than for the DNA alpha polymerase in the nucleus (Kunkel and Loeb, 1981), and a higher turnover of mtDNA than nuclear DNA (Rabinowitz and Swift, 1970). Most mtDNA mutations are point mutations, as compared to deletions or additions (Avise and Lansman, 1983). Despite this high mutation rate, the sequence of mitochondrial genes has been conserved for the past 350 million yr for vertebrates (Brown, 1983). These qualities of mtDNA make it ideal for population studies.

Allozyme variation has been analyzed for the largemouth bass (Whitt et al., 1971; Phillip et al., 1982; Carmichael et al., 1986). Only one mtDNA study concerned largemouth bass that involved restriction fragment length polymorphisms (RFLPs) within a single gene (Whitmore et al., 1992). A study of RFLPs involving the entire mitochondrial genome has not been performed.

The goal of our study was to estimate the size of the mitochondrial genome, and to look for genetic variability within largemouth bass residing in Five Mile Pond of Springfield, Massachusetts. Five Mile Pond is a small natural kettlehole of approximately 19.5 ha, located in western Massachusetts. There is no record of stocking largemouth bass into Five Mile Pond (J. Bergin, pers, comm.).


Micropterus salmoides individuals were collected in the spring and summer of 1990. Liver and gonadal tissues from eight freshly sacrificed fish 25-35 cm long were homogenized with three strokes of a glass teflon homogenizer in TEKS buffer (50 mM Tris-Cl, pH 7.5; 10 mM EDTA; 1.5% potassium chloride; 0.25 M sucrose). Mitochondria were pelleted and mtDNA was isolated using techniques described by Chapman and Powers (1984). Purified mtDNA was digested using 13 restriction endonucleases, under conditions suggested by the supplier (New York Biolabs, Inc.) for 7 h at 37 C. KGB buffer (200 mM potassium glutamate; 50 mM Tris-acetate, pH 7.6; 20 mM magnesium acetate; 100 [[micro]gram]/ml BSA; 1 mM 2-mercaptoethanol) was substituted for the supplied buffers using concentrations recommended by McClelland et al. (1988).
TABLE 1. - Size estimates (base pairs) for fragment patterns
resulting from the restriction analysis of eight largemouth bass
(Micropterus salmoides) mtDNA

        BamHI     BglI        BglII              DraI
          A        A        A        B        A        C

        Uncut     7571     One    11,225   14,674     7935
                  4846     cut      7487     2485     7050
                  2898                                3754

Total           17,711           18,712    17,159   18,739

        EcoRI         EcoRV       HindIII    MluI     PstI
          A        A        D        A        A         A

        Uncut     8392     8392     8049     8256     Uncut
                  6549     6549     4712     6148
                  4307     2584     1370     2314
                           1879              1676

Total           19,248   19,404   14,131   18,394

          PvuII     SalI      ScaI       XhoI
           A          A         A         A

          8898      Uncut    11,505     11,378
          6835                 3050       8269
          2802                 2446

Total   18,535               17,001     19,647

Products of restriction digests were separated in a 1.0% agarose horizontal gel using TAE (40 mM Tris-acetate; 1 mM EDTA) buffer for 13 h at 30 V (2V/cm). The gel was stained in ethidium bromide (1 [[micro]gram]/ml) after electrophoresis and viewed with a transilluminator. Lambda HindIII ladder served as the sizing standard. The smallest fragment scored was 1370 base pairs. Fragment sizes were determined through the use of a least-squares fit program of Schaeffer and Sederoff (1981).


Thirteen restriction endonucleases generated a total of 28 fragments; four of the restriction endonucleases (BamHI, EcoRI, PstI, SalI) made no cuts, while another restriction endonuclease (BglII) resulted in a single cut (Table 1). Each restriction endonuclease possessed a unique recognition site.

The mean mitochondrial genome size was calculated by averaging the sum of the individual fragments for each restriction digest (Table 1). The mean mitochondrial genome size was 18,450 base pairs (95% confidence interval = 17,800-19,800). This is suspected to be a low estimate since lower end fragments were not scored. The value for the HindIII restriction digest was 14,131 base pairs, well below the confidence limits for the mean, and therefore was not included in our determination of the mean. It is possible that a second restriction fragment of ca. 4700 base pairs was present in the HindIII digest but not discriminated, resulting in the low size estimate. The study sampled approximately 1% of the largemouth bass mitochondrial genome (180 base pairs recognized as restriction sites relative to a mean genome size of 18,450 base pairs).

Five of the eight (62.5%) individuals had the same haplotype and were designated as haplotype A; three variant genotypes were designated as haplotypes B, C and D, respectively, All three polymorphisms were apparently a result of single point mutations, with the addition of restriction sites for BglII and EcoRV, and the loss of a restriction site for DraI (Table 1).


The size of the mitochondrial genome for the largemouth bass, 18,450 base pairs, is at the upper end of the range of other fish species. Mitochondrial genomes for fish range from 15,800 for the lake whitefish, Coregonus clupeaformis (Shields et al., 1992), to 19,500 for the marine toadfish, Opsanus tau (Arise et al., 1987) and the sculpin, Scorpaena guttata (Beckwitt and Petruska, 1985).

The polymorphism exhibited by the eight individuals within this small pond poses two questions worthy of further study. First, is the high diversity of the individuals (four haplotypes from eight individuals) studied representative of the lake population? Considerably lower diversities have been demonstrated for other fish species (e.g., Advise et al., 1987; Shields et al., 1992; Smith et al., 1989; Ward et al., 1989; Wirgin et al., 1989). Second, as stocking records are lacking, is it possible to identify the source(s) of the Micropterus parent stock based upon the genotype of a resident population? The question of ancestry and the relatedness of populations has been investigated for various fishes, such as cod, Gadus morhua (Smith et al., 1989), lake whitefish, Coregonus clupeaformis (Shields et. al., 1992), salmonids, Onchorhyncus and Salmo sp. (Thomas and Beckenbach, 1989), striped bass, Morone saxatilis (Wirgin et al., 1989) and the walleye, Stizostedion vitreum (Ward et al., 1989). This same approach can be informative in the identification of parental bass stock.

Acknowledgments. - We thank D. Vieira and R. Stanton for their assistance in the laboratory and B. Falcetti for his technical assistance.


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RONALD L. JOHNSON, Department of Zoology, Arkansas State University, State University 72467, AND JUDITH PIGNATARE, 685 Suffield Ave., Agawam, Massachusetts 01001. Submitted 21 February 1994; accepted 15 September 1994
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Title Annotation:Notes and Discussion
Author:Johnson, Ronald L.; Pignatare, Judith
Publication:The American Midland Naturalist
Date:Apr 1, 1995
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