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MITOCHONDRIAL-DNA VARIATION IN PALLID AND SHOVELNOSE STURGEONS, SCAPHIRHYNCHUS (PISCES: ACIPENSERIDAE).

ABSTRACT

Sequencing analysis was used to characterize a mtDNA D-loop amplicon of 838 bp. Four pallid sturgeons, Scaphirhynchusalbus, and four shovelnose sturgeons, S. platorynchus, from four states (Illinois, Louisiana, Montana, North Dakota) were used for this analysis. DNA sequencing of the nine fish revealed eleven polymorphic sites and six distinct haplotypes. No fixed nucleotide substitutions were observed between pallid and shovelnose sturgeons.

Pallid (Scaphirhynchusalbus Forbes & Richardson), shovelnose (S. platorynchus Rafinesque), and Alabama (S. suttkusi Williams & Olemmer) sturgeons represent a morphologically distinct genus indigenous to the Mississippi and Mobile river drainages of North America (Bailey and Cross 1954). The shovelnose sturgeon is relatively common and inhabits most major rivers of the Mississippi River drainage, while the pallid sturgeon is endangered and restricted to the Missouri and the lower Mississippi River (Birstein 1993, Dryer and Sandoval 1993). The Alabama sturgeon is very rare and is restricted to the Mobile River basin of Alabama and Mississippi (Williams and Clemmer 1991). While the pallid and shovelnose sturgeons are morphologically distinct (Keenlyne and Henry 1994), hybrids are known to occur (Carlson et al. 1985, Keenlyne et al. 1994). The issue of the genetic distinction of pallid and shovelnose sturgeons has been controversial and was recently reviewed by Wirgin et al (1997). While several studies (Phelps and Allendorf 1983, Genetic Analysis, Inc. 1994) found no genetic differentiation between the species, others (Campton et al. 1995) suggest they are distinct species. The purpose of this study was to determine if pallid and shovelnose sturgeons sampled from four US states can be differentiated using DNA sequencing of the mitochondrial D-Loop region.

METHODS AND MATERIALS

Fin tissue from five pallid and four shovelnose sturgeons from four US states was obtained during 1995 (Table 1). Tissue samples were preserved by freezing at -70[degrees]C. Voucher specimens are maintained at the Genetic and Forensic Analysis Laboratory of the Nebraska Game and Parks Commission, Lincoln, Nebraska, USA. DNA was isolated from a 5x5 x 5 mm portion of each sample using the Puregene DNA isolation kit D-5000A(Gentra, Minneapolis, MN). The extracted DNA was stored at -20[degrees]C. PCR was conducted using the primers Proline-tRNA-2 (5'-ACCCTTAACTCCCAAAGC-3') and PhenyltRNA-1 (5'-GTGTTATGCTTTAGTTAAGC-3') (Bernatchez et al. 1992) per Szalanski et al. (in press). Amplified DNA was purified using Micron 50 microconcentrator (Amicon Inc, Beverly, MA) and resuspended to a volume of 10.0 [micro]l using nanopure water.

Purified and concentrated DNA, approximately 2.5 ng/100 bp, was sent to the DNA Sequencing Facility, Iowa State University (Ames, IA) for direct sequencing in both directions. Consensus sequences for each sequenced individual were derived using GCG (Genetics Computer Group, Madison, WI) GAP program. The GenBank accession numbers for each sequence are AF176341 and AF224873 to AF224880. Sequences were aligned with GCG PILEUP program using paddle-fish, Polyodon spathula (GenBank accession number AF176340), as the outgroup taxon. Parsimony analyses on the alignments were conducted with PHYLIP v3.57c (Felsenstein 1993) on phylogenetically informative characters only, with gaps being treated as a fifth character state. Bootstrapping was performed by generating 1000 data sets with the SEQBOOT program. Most-parsimonious trees were constructed using DNAPARS, and a majority rule and combinable competent consensus of these trees were constructed using the CONSENSE program in PHYLIP. The DNADIST program of PHYLIP was us ed to calculate genetic distances according to the Kimura 2-parameter (Kimura 1980) and maximum likelihood models of sequence evolution. Trees were constructed from these distances with the NEIGHBOR program to create neighbor-joining (Saitou and Nei, 1987) trees.

RESULTS

Polymerase chain reaction resulted in an 838 bp amplicon for the nine sturgeons sequenced. Among the nine individuals, 11 nucleotide positions are polymorphic, of which eight are transitions and three are transversions. Six mitochondrial haplotypes were observed. Genetic distance among all sturgeons sequenced ranged from 0.0 to 0.84%, with a mean of 0.26%. Alignment of the Scaphirhynchus and Polyodon mtDNA sequences resulted in a total of 941 characters, including gaps. Distance analysis resulted in a cladogram similar to the one derived from parsimony analysis (Fig. 1). Neither analysis resulted in a distinct clade for pallid and shovelnose sturgeons.

DISCUSSION

Although the examined sturgeons had nucleotide polymorphisms at 11 nucleotide sites of the mitochondrial D-Loop region, shovelnose and pallid sturgeons are indistinguishable by our results. Because of the limited sample size of each population in our study, no conclusions could be drawn regarding the extent of the variation and possible interpopulational differences. This lack of genetic differentiation between pallid and shovelnose sturgeons has been observed in several other studies. Phelps and Allendorf (1983), using allozyme analysis, observed a lack of genetic evidence supporting pallid and shovelnose sturgeons as different species. PCR-RFLP analysis of nuclear DNA amplicons by Genetic Analysis, Inc. (1994) showed no significant difference between species for the only variable locus. Campton et al. (1995) sequenced approximately 435 bp of mitochondrial DNA of pallid and shovelnose sturgeons from the upper Missouri River and found a difference in haplotype frequencies between shovelnose and pallid sturge ons, but the haplotypes overlapped such that maximum parsimony analysis did not result in two species-congruent clades. Avise (1994) suggested that divergence between genetic and phenotypic data may represent introgressive hybridization or recent divergence of pallid and shovelnose sturgeons. While other nuclear and mitochondrial markers that are commonly used as phylogenetic tools showed low levels of variation when applied to Acipenserifomes (Birstein et al. 1997), the examination of nuclear ribosomal DNA internal transcribed spacer regions or other nuclear markers may yield different results and should be a goal of future research.

ACKNOWLEDGMENTS

We thank the National Fish and Wildlife Laboratory in Ashland, Oregon, for providing samples. D. B. Taylor and R. D. Peterson II provided thoughtful suggestions. This is Journal Paper No. 12837 of the Nebraska Agricultural Research Division.

LITERATURE CITED

Avise, J. C. 1994. Molecular Markers, Natural History and Evolution. New York, Chapman and Hall: 511 pp.

Bailey, R. M., and F. B. Cross. 1954. River Sturgeons of the American genus Scaphirhynchus: characters, distribution, and synonymy. Papers of the Michigan Academy of Sciences, Arts, and Letters 39: 169-208.

Bernatchez, L., R. Guyomard, and F. Bonhomme. 1992. DNA sequence variation of the mitochondrial control region among geographically and morphologically remote European brown trout, Salmo trutta, populations. Molecular Ecology 1: 161-173.

Birstein, V. J. 1993. Sturgeon and paddlefishes: threatened fishes in need of conservation. Conservation Biology 7: 773-787.

-----, R. Hanner, and R. DeSalle. 1997. Phylogeny of the Acipenseriformes: cytogenetic and molecular approaches. Environmental Biology of Fishes 48: 127-155

Campton, D. E., A. I. Garcia, B. W. Bowen, and F. A. Chapman. 1995. Genetic evaluation of pallid, shovelnose and Alabama sturgeon (Scaphirhynchus albus, S. platorynchus, and S. suttkusi) based on control region (D-loop) mitochondrial DNA. Final Report to the U.S. sequences of Fish and Wildlife Service, Bismarck, North Dakota. 35 pp.

Carlson, D. M., W. L. Pflieger, L. Trial, and P. S. Haverland. 1985. Distribution, biology and hybridization of Scaphirhynchus albus and S. platorynchus in the Missouri and Mississippi rivers. Environmental Biology of Fishes 14: 51-59.

Dryer, M. P., and A. J. Sandoval. 1993. Recovery plan for the pallid sturgeon (Scaphirhynchus albus). U.S. Fish and Wildlife Service, Denver, 55 pp.

Felsenstein, J. 1989. PHYLIP -- Phylogeny Inference Package (Version 3.2). Cladistics 5: 164-166.

Genetic Analysis, Inc. (1994) Genetic studies of Scaphirhynchus spp. Report to the U.S. Army Corps of Engineers, Omaha District; U.S. Fish and Wildlife Service, Bismarck, North Dakota; U.S. Army Corps of Engineers, Mobile District. 41 pp

Keenlyne K. D., and C. J. Henry. 1994. Morphometric comparisons of Upper Missouri River sturgeons. Transactions of the American Fisheries Society 123: 779-785.

-----, L. K. Graham, and B. C. Reed. 1994. Hybridization between the pallid and shovelnose sturgeons. Proceedings of the South Dakota Academy of Science 73: 133-148.

Kimura, M. 1980. A simple method for estimating evolutionary rate of base substitution through comparative study of nucleotide sequences. Journal of Molecular Evolution 16: 111-120.

Phelps, S. R., and F. W. Allendorf. 1983. Genetic identity of pallid and shovelnose sturgeon (Scaphirhynchus albus and S. platorynchus). Copeia 1983: 696-700.

Saitou, N., and M. Nei. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4:406-425.

Szalanski, A. L., R. Bischof, and R. Holland. 2000. Population genetic structure of Nebraska paddlefish based on mtDNA variation. Transactions of the American Fisheries Society 129, (in press).

Williams, J. D., and G. H. Clemmer. 1991. Scaphirhynchus suttkusi, a new sturgeon (Pisces: Acipenseridae) from the Mobile Basin of Alabama and Mississippi. Bulletin of the Alabama Museum of Natural History 10: 17-31.

Wirgin, I. I., J. E. Stabile, and J. R. Waldman. 1997. Molecular analysis in the conservation of sturgeons and paddlefish. Environmental Biology of Fishes 48: 385-398.
Table 1. Samples of Scaphirhynchus platorynchus and S. albus used in
this study.
Sample Species State River
ID
SS635 S. platorynchus MT Yellowstone
SS638 S. platorynchus MT Yellowstone
SS641 S. platorynchus MT Yellowstone
SS644 S. platorynchus MT Yellowstone
PS109 S. albus ND Missouri
PS115 S. albus ND Missouri
PS129 S. albus IL Mississippi
PS560 S. albus IL Mississippi
PS572 S. albus LA Atchafalaya
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Author:Bischof, Richard; Szalanski, Allen L.
Publication:Transactions of the Nebraska Academy of Sciences
Article Type:Statistical Data Included
Geographic Code:1USA
Date:Jun 1, 2000
Words:1507
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