INTRODUCTION Introduction C.R. Robins and H.W. Robison identified nine morphological characteristics for use in distinguishing the Ozark sculpin, C. hypselurus, from the mottled sculpin, C. bairdi. They documented that C. bairdi was restricted to the Niangua River system and a few adjacent streams which feed into the Lake of the Ozarks. They also noted that C. hypselurus distribution was more widespread, encompassing all of the C. bairdi range in Missouri and the Niangua River regions. Since this documentation was published, a disjunct sculpin population in Clear Creek, Marion County, Missouri was discovered and many Missouri sculpin specimens have been found which display morphological characteristics of both C. hypselurus and C. bairdi. Due to divergence and unreliability of morphological data, it is important that methods of genetic analysis be used to establish definitive phylogenetic evidence for distinguishing C. hypselurus from C. bairdi and mapping location of either species` occurrence in Missouri. The use of mitochondrial DNA (mtDNA) to study phylogenetic relationships among natural populations of closely related species has many advantages: many mtDNA copies are present per cell, gene content is highly conserved, increased rate of nucleotide substitution occurs within the molecule, and strict maternal inheritance prevents reduction of genetic divergence. Due to length variation of regions of mtDNA among species, the most widely used method for obtaining phylogenetic data is restriction enzyme fragment analysis. Restriction Fragment Length Polymorphisms (RFLP`s) occur due to different lengths of the same DNA fragment among species. When restriction enzymes cut at their prescribed nucleotide sequences, the fragments may migrate differently (shorter distances or longer distances) on electrophoretic gels due to different fragment lengths.
MATERIALS AND METHODS Materials and Methods Sculpin mtDNA samples from each of the five watersheds studied were previously purified according to Quiagen® molecular isolation protocol. PCR amplification of the 2000 base pair mtDNA fragment from each of the five genomic mtDNA samples was set up in 25 µL PCR reactions. Ready To Go™ PCR beads containing an undisclosed mixture of Thermos aquaticus (Taq) polymerase, 10x PCR buffer, MgCl2, and a mixture of dioxynucleotide triphosphates (dNTP`s) were used to conduct PCR. Other components of the PCR reaction which were added to the PCR beads were 1 µL of HIS forward primer, 1 µL GLY reverse primer, 1 µL of sculpin template mtDNA, and 22 µL of sterile distilled water. All PCR reactions were conducted in an automated thermocycler with a 50 C annealing temperature. Six different restriction enzymes (AccI, AflII, Sau3A, AluI, EcoRI, and SalI) were used to perform restriction enzyme digests. These enzyme digestion mixtures were incubated for 2 hours at 37 C in order to complete the digestion reaction. Products from all enzyme digestion reactions were analyzed for RFLP`s on agarose minigels. All gels were developed in Ethidium bromide (EtBr) and products were viewed on a UV light box for RFLP assessment. RFLP data was used to construct a phylogenetic tree, a haplotype chart, and a polymorphic band distribution chart to represent experimental data.
DATAFigure 1: Polymorphic Band Distribution Chart
Meramec Pearson Clear Little Black Niangua River Creek Creek River River
AccI 1 0 1 1 1 0 1 0 0 0
AflII 1 1 1 1 1 0 1 0 0 0 0 1 0 0 0
AluI - 0 - 1 1 - 1 - 0 0 - 0 - 1 1 - 1 - 1 1
Sau3A - 0 - - 1 - 1 - - 0
EcoRI 1 1 1 1 1
SalI 1 1 1 1 1
1 = band present 0 = band absent - = no band
Figure 2: Haplotype Chart
Watershed 1 2 3 4
Meramec River A A - -Pearson Creek B B B BClear Creek A A - -Little Black River A A A -Niangua River A A A A
Haplotypes are as follows: 1)AccI, 2) AflII, 3) AluI, and 4) Sau3A
DISCUSSION Discussion Results from AccI and AflII enzyme digests were the most clear and useful for establishment of phylogenetic relationships. AccI and AflII results showed identical band patterns among Meramec River, Clear Creek, Little Black River, and Niangua River sculpin species. A clearly differentiable band pattern distinct from these species was observed in results from the Pearson Creek sculpin species. Results from Sau3A and AluI enzyme digests were not completely clear due to failure of some bands to migrate. However, some bands were clearly distinguishable and supported results obtained from AccI and AflII digests. Again, the Clear Creek sculpin species banding patterns were completely distinct from the banding patterns observed in the other four sculpin species. Banding patterns from Little Black River and Niangua River sculpin species were observed to be identical when results of AluI digestion were compared. Electrophoretic results from EcoRI and Sal I enzyme digestion did not yield phylogenetically useful results because both enzymes cut the mtDNA fragment in an identical fashion. It was observed that the restriction enzymes which generated the most bands on a gel after digestion (AflII, AluI, and Sau3A) were those which recognized 4 or 5 nucleotides of base sequence in order to cut the fragment. It was determined that these enzymes were the most useful for comparing very closely related sculpin species due to generation of more bands. Results of this experiment clearly show that the Pearson Creek sculpin species is phylogenetically different from sculpins from the Niangua River, the Little Black River, Clear Creek, and the Meramec River. Based on evidence that sculpin species are more closely related in the Niangua River, the Little Black River, Clear Creek, and the Meramec River and morphological evidence, it was concluded that sculpins from these four watersheds were of the species C. bairdi. It was also concluded that the sculpin species from Pearson Creek was C. hypselurus since banding patterns of Pearson Creek sculpins were very different from banding patterns observed in C. bairdi species from the other four watersheds.
FURTHER INVESTIGATION Further Investigation Further research investigations which will help clarify and more precisely define results of this experiment include the following: 1)The use of more restriction enzymes for further band comparison, 2) Using larger sample sizes from each population studied, 3) Using additional sculpin samples from more Missouri watershed populations and 4) Comparison of RFLP results with results from similar phylogenetic investigative studies such as RAPD analysis.
ACKNOWDEGMENTS Acknowledgments I would like to thank Dr. Todd Eckdahl for helping me design, critique, and complete this experiment. Special thanks to Brian Gasper, Sabrina Schwery, and Elizabeth Nelson for helping me set up lab preparations and answering my questions. I especially would like to thank my parents, Daniel and Dorothy Linebaugh for providing funding for poster materials, transportation to Linda Hall Library for research, and for their continued support and encouragement
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