Transactions of the American Fisheries Society 129:1060-1065,2000CJ Copyright by the American Fisberies Society 2000

Population Genetic Structure of Nebraska Paddlefish Based onMitochondrial DNA Variation

ALLEN L. SZALANSKI,* RICHARD BISCHOF, AND GERALD MESTL

Nebraska Game and Parks Commission,2200 North 33rd Street, Lincoln, Nebraska 68503-0370, USA

Abstract.-Eighty-three paddlefish Polyodon spathulathat were collected from 1995 to 1999 from the MissouriRiver Galvins Point Dam tailwater were analyzed forgenetic variation in the mitochondrial DNA d-loop re-gion. Additional samples from Montana, South Dakota,and Louisiana were used for comparative purposes. Tofacilitate the efficient analysis of numerous paddlefishsamples, we applied a method that employs polyacryl-amide gel electrophoresis (PAGE) to resolve restrictionfragment length polymorphisms (RFLPs) amplified bypolymerase chain reaction (PCR). DNA sequencing of10 paddlefish revealed 22 polymorphic sites. Polymerasechain reaction-RFLP analysis of 93 paddlefish usingthree restriction enzymes detected six of the polymor-phic sites and revealed six distinct haplotypes. All ofthe observed haplotypes were found in the Missouri Riv-er Galvins Point Dam tail water. No temporal differen-tiation was observed among the 1995, 1998, and 1999samples from the Missouri River Galvins Point Damtail water. Polymerase chain reaction-RFLP, resolvedwith PAGE, provided an efficient method for populationgenetic analysis of paddlefish.

paddlefish population genetic structure is incom-plete. Previous allozyme studies (Carlson et al.1992; Fries and Hutson 1993; Epifanio et al. 1996)and mitochondrial DNA (mtDNA) restriction frag-ment length polymorphism (RFLP) analysis (Epi-fanio et al. 1996) revealed low levels of allozymeallelic and mtDNA haplotype variation within andamong paddlefish populations sampled from up tonine tributaries (Epifanio et al. 1996). The bio-chemical and molecular methods previously em-ployed are not optimal for genetic analysis of nu-merous alcohol-preserved, field-collected sam-ples.

The objective of this study was to characterizemtDNA d-ioop genetic variation from temporallysampled paddlefish collected from the MissouriRiver Galvins Pt. dam tailwater. Additional pop-ulations isolated by geographical range or by damswere included for comparative purposes. To facil-itate the efficient analysis of numerous paddlefishsamples, we have applied a method that employspolyacrylamide gel electrophoresi& (PAGE) to re-solve RFLPs amplified by polymerase chain re-action (PCR).

The paddlefish Polyodon spathula Walbaum isan ancient fish that is native to the Missouri River,the Mississippi River, and several Gulf Coastdrainages of North America (Gengerke 1986; Rus-sell 1986). Paddlefish populations have declinedalong with a reduction in their range because ofimpoundment channel alterations and overhar-vesting (Gengerke 1986; Russell 1986). On theMissouri River, dams have interrupted migrationroutes and flooded or fragmented critical habitats(Unkenholz 1986). The decline of paddlefish pop-ulations has caused them to be listed as a speciesof concern in several states (Johnson 1987); theywere also listed in the Convention on InternationalTrade in Endangered Species in 1992 (Allardyce1992). A report of the National Paddlefish andSturgeon Steering Committee (1993) identified theloss of genetic variation as one of the committee'sprimary concerns. However, information about

Methods

Fin tissue samples (approximately 10 X 30 mm)from individual paddlefish caught and released fromthe Galvins Pt. Dam tailwater on the Missouri Riverwere obtained during 1995, 1998, and 1999; sam-ples were preserved by freezing at -70°C or byimmersion in 95% ethanol (Table 1). Muscle andegg samples that had been preserved by freezingwere also obtained from paddlefish collected in1995 from South Dakota, Louisiana, and Montana(Table 1). DNA was extracted from a 5 X 10-mmsection of tissue or from individual eggs using aphenoVchloroform extraction procedure (Taylor etal. 1996). The mtDNA d-loop region was amplifiedusing the primers Proline-tRNA-2 (5'-ACCCT-TAACTCCCAAAGC-3') and Phenyl-tRNA-l (5'-GTGTTATGCTTTAGTTAAGC-3') (Bematchez etal. 1992) to generate an approximately 875-base

"

* Corresponding author: aszalans@unlserve.unl.edu

Received August 23, 1999; accepted January 27, 2000

1060

NOTES 1061

TABLE 1.-Three-enzyme haplotypes of paddlefish, inthe order Nla 111, Rsa I, and Tsp509 I. Abbreviations areas follows: LA = Mermentau River, Louisiana; SD = Mis-souri River, Lyman County, South Dakota; MT = Yellow-stone River, Dawson County, Montana; NE = MissouriRiver, Galvins Point Dam tailwater, Cedar County, Ne-braska; numerals signify 1995, 1998, 1999, respectively.

tions in order of discovery, with the first patternbeing designated "A," the second "B," and so on.Haplotypes of each paddlefish were then identifiedby the combination of letters representing the re-striction profiles for each restriction enzyme used.Nested analysis of variance from haplotype fre-quencies within and among populations was cal-culated using Analysis of Molecular Variance 1.55(Excoffier et al. 1992).

SampleHaplo-type LA SD MT NE95 NE98 NE99 N

AAAAADABABACCAACABN

0000022

00

523

8 63880

1843

206

1911

06

000

1047

17

19

05

213693

pair (bp) amplicon. Amplification conditions wereas follows: 35 cycles of 94°C for 45 s, 56°C for45 s, and 72°C for 90 s. For DNA sequencing,PCR products were purified using Micron 50 mi-croconcentrator (Amicon, Inc., Beverly, Massa-chusetts) and resuspended to a volume of 10.0 ILLusing nanopure water. Purified and concentratedDNA, approximately 2.5 ng/IOO bp, was sent tothe DNA Sequencing Facility at Iowa State Uni-versity (Ames, Iowa) for direct sequencing in bothdirections. Consensus sequences for each individ-ual were derived using the Genetics ComputerGroup (GCG; Madison, Wisconsin) GAP program.The GenBank accession numbers for each se-quence are AF176331 to AF176340. Sequenceswere aligned with GCG PILEUP using shovelnosesturgeon Scaphirhynchus platorynchus (Rafin-esque) (GenBank accession number AF176341) asthe outgroup taxon. Parsimony analyses on thealignments were conducted with PHYLIP (version3.2) (Felsenstein 1989). The DNADIST programof PHYLIP was used to calculate genetic distancesaccording to the Kimura two-parameter (Kimura1980) and maximum likelihood models of se-quence evolution. Trees were constructed fromthese distances with the NEIGHBOR and FITCHPHYLIP programs to create neighbor-joining trees(Saitou and Nei 1987). For PCR-RFLP analysis,restriction sites were predicted from the sequencedata using Webcutter 2.0 (Heiman 1997). Ampli-fied DNA was digested according to manufactur-er's recommendations using the restriction en-zymes Nlalll, RsaI, and Tsp509I (New EnglandBiolabs), per Cherry et al. (1997). Polymerasechain reaction-RFLP fragments were separated by10% PAGE (Taylor et al. 1997). Restriction pro-files for each enzyme were given letter designa-

Results and Discussion

Seven paddlefish from Nebraska, two fromMontana, and one from Louisiana were subjectedto DNA sequencing. Relative to the reference se-quence (Figure 1), a total of 22 substitutions oc-curred in the 800 bp of the d-loop region. Ninemitochondrial genotypes were identified amongthese 10 individuals. Up to five nucleotide sitedifferences were found between the most divergenttypes. Genetic distance within P. spathula rangedfrom 0.0 to 1.5%, with a mean of 0.7%. Poly-merase chain reaction-RFLP analysis revealed sixhaplotypes (Figure 2), one of which, AAD, wasnot observed among the 10 individuals sequenced.Polymerase chain reaction-RFLP fragment sizesand restriction enzymes sites are provided in Table2. Nested analysis of haplotype frequencies re-vealed that most of the variation occurred withinpopulations and accounted for 94% of the variancewhen the Galvins Pt. Dam tailwater samples werecontrasted with each other. No temporal differ-entiation (FST = 0.004) was observed among the

1995, 1998, and 1999 Nebraska samples. Thealigned DNA data matrix, including the outgrouptaxon, resulted in a total of 875 characters, in-cluding gaps. Parsimony analysis of the DNA se-quences was unable to reveal any sub structuringamong the non-PCR-RFLP "BAC" haplotypes(Figure 3). The cladogram inferred from the neigh-bor-joining analysis was nearly identical in topol-ogy to the one derived from parsimony analysis.The only differences between the neighbor-joiningand the parsimony analyses were DNA sequencesNE95B and NE95C, which formed a distinct cladefor the neighbor-joining analysis.

We observed nine mitochondrial genotypesfrom the DNA sequences of 10 individuals and sixhaplotypes among the 93 paddlefish subjected toPCR-RFLP analysis. All of the observed geno-types and haplotypes were present in the GalvinsPt. Dam tail water population. This finding, in con-junction with the lack of temporal variation for the1995, 1998, and 1999 samples from Galvins Pt.Dam tail water indicates a stable population struc-

1062 SZALANSKI ET AL.

301GAACTATTAC TGGCATCTGG TTCCTATTTCAGGTCCATAA ATGGTTATTTRsa I

351CCACATAACT GAACTATGTC TGGCATCTGATTAATGTTAT AAGTATTATA--

Nla III TspSO9 I401

CAATCCGTGA CCCCACATGC CAAGAATCTT GTCAACATCA GTTAT-TTTTT--Rsa I Nla III Nla III451

T~GGTTACCA TTCACGTACA TGTAAAAACT CCTCGAC=BTG G~AAAATGGANla III TspSO9 I501AGTGGAACAT

GCTATTTI'GT CCTATAAACAGCTGAATGAA TGATTTAATG--

551

ACATAAACAT TATTACACTA CATTAACCTAATACCACGGATspSO9 I

601 AACTCTCATA CTACACCCTG ATACTTCTTA AGCCAATTGT

TTATACCTTC

TCTACATTAC

651 ACATTTATAA CTTTGATGCT TATTCTCGAC AAACCCCCTARsa I

701TTGAACAAGT TCTTGTACfT TCCTGTCAAACCCCAAAAGCNla III

751CGCATCAACA TGTTTTACCA TCCCCGACTACATAAATATT

CCCCCTTACA

AGGACTAACT

TGTATCTATCItRNA-phe

TATAAGCTAG801ATTATATTCG TATACATTAT TACACAATCACACAAAATAAphe-tRNA-l

851TGATGCTTAA CTAAAGCATA ACAC

FIGURE I.-Sequence for 875 base pairs of mtDNA from paddlefish sample NE95B. The 22 variable positionsamong the 10 paddlefish sequences are underlined, and the three restriction enzymes used for PCR-RFLP analysisare listed. Boundaries of the three regions of the genome are indicated, and primer recognition sites are in bold.

concluded that paddlefish have low levels of with-in-population genetic diversity. However, theyalso observed that their sample sizes were not suf-ficient to confidently estimate haplotype frequen-cies within populations. In our study, all of theobserved PCR-RFLP haplotypes were presentwithin the Galvins Pt. Dam tail water population,

ture for this period. Levels of observed geneticvariation were similar to those observed by Epi-fanio et al. (1996). Using total mtDNA RFLP anal-ysis, Epifanio et al. observed four haplotypes from73 paddlefish collected from nine tributaries (av-erage of eight paddlefish per tributary). No singletributary had all four haplotypes present, and they

1063NOTES

acrylamide gels relative to agarose gels allows theuse of less PCR template for each restriction di-gest. Finally, two PAGE apparatuses can resolvethe fragment patterns of up to four restriction en-zymes from 36 samples in 90 min at a significantlylower cost than that associated with agarose gelelectrophoresis. Polymerase chain reaction-RFLPbased on DNA sequence data represents a robustmethod for the analysis of numerous paddlefishsamples. The method can be useful with DNA ex-

a fact that may be attributed to our large samplesize from this location. Because our samples fromLouisiana and South Dakota were minimal, theycan be used only to indicate the presence of theobserved haplotypes, and they cannot provide in-formation concerning the absence of other hap-

lotypes.Polyacrylamide gel electrophoresis allows rou-

tine observation of restriction enzyme fragmentsthat are as small as 10 bp, and the thinness of the

TABLE 2.-Paddlefish PCR-RFLpa restriction sites and fragments.

Rsa IGT/AC

Tsp509 IIAATT

a Polymerase chain reaction-restriction fragment length polymorphism.

1064

SZALANSKI ET AL.

AABABB

BDCBCBD BBCABABFIGURE 3.-Paddlefish cladogram, derived from parsimony analysis and rooted by the outgroup taxon Scaphir-

hynchus platorynchus. Bootstrap values of greater than 50% and PCR-RFLP haplotypes are provided (restrictionenzyme order: NlallI, RsaI, and Tsp509I).

tracted from fin clippings preserved in ethanol,allowing nondestructive sampling. The ability touse individual eggs will be of benefit for the iden-tification of caviar and for the genetic character-ization of stocks before propagation.

12731 of the Nebraska Agricultural Research Di-vision.

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Acknowledgments

We thank Ronald Bromley, Doug Henley, KentKeenlyne, Gina Harris, David Oates, AndrewReinholz, Gloria Seaborn, Phil Stewart, CliftonStone, and Flip Siragusa for providing samples.D. B. Taylor and R. D. Peterson II providedthoughtful suggestions. This is Journal Paper

1065NOTES

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